FERN GAZ. 20(1):1-13. 2015 1



H. A. KELLER1 & G. T. PRANCE2 1 Consejo Nacional de Investigaciones Científicas y Tecnológicas, Instituto de Botánica del Nordeste, Sargento Cabral 2131, Corrientes, . 2 Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3AB, UK, corresponding author: [email protected]

Keywords: ethnobotany, ethnopteridology, archaeobotany, medicinal , ornamental plants

ABSTRACT A summary is presented of the most important ways in which ferns have been important to humanity. Many of these categories are positive such as the use of ferns for subsistence. On the negative side is their role as weeds and as bearers of substances harmful to human health. Many of the traditional uses such as for medicines have been transferred to modern life as societies have modernized. Some uses have even become important in industrial society, for example in the assay of new medicines.

INTRODUCTION Ferns are distributed in all climate zones of the planet, but have a greater diversity in the tropics (Smith et al., 2006; Strasburger et al., 2003). The discipline that studies the relationship between the uses of ferns and humans has been termed ethnopteridology, and it was well explained and amplified by Boom (1985). These reciprocal interactions may or may not be related to a particular use category in or regional cultures, but the concept of ethnobotany goes beyond the utilitarian spheres of economics and uses to include symbolic values, nomenclature, religion and also the place that particular plants occupy in the cosmology of peoples. Strictly economic aspects of uses are addressed by the discipline of economic . It is not easy to separate economic botany and ethnobotany and indeed economic botany implies a of relationship between human groups and plants. The object of this work is to present a global panorama of the state of the art in ethnobotany of . We will show the great diversity of relationships between these plants and humans. Pteridophytes feature in some way in many papers about ethnobotany and economic botany and here we will concentrate mainly on papers whose object was specifically about the study of ferns or lycophytes. A few previous papers have addressed similar topics at least locally. For example, May (1978) made a summary of the economic and folkloric uses of ferns. Díaz de León et al. (2007) reviewed the many uses of ferns and lycophytes in Mexico and to some extent the rest of the world. Murillo (1983) produced a major work on the uses of ferns in with a special emphasis on the ferns of . Mannar Mannan et al. (2008) gave a short review of the potential uses of ferns. We have used the names as cited in the various papers rather than trying to update the nomenclature in any way. The literature about the reciprocal relationships between humans and pteridophytes is so extensive that here we cannot possibly cover it all, but we hope to show the variety of possible relationships through presenting a number of wide-ranging examples. 2 FERN GAZ. 20(1):1-13. 2015

ARCHAEOBOTANY Archaeobotany is the study of ancient remains found in archaeological contexts. The name is obviously a parallel to, and a derivative from, ethnobotany. In both archaeobotany and ethnobotany the focus of attention is upon the uses of plants by, and their association with people (Wiley, 1995). Since remote times human populations in many parts of the world knew and made use of ferns and lycophytes. The archaeological literature offers much evidence of the close relationship between humans and these plants over many generations. Anderson & White (2001) suggested that was used for consumption by humans at least 888 years ago in Norfolk Island. Dental evidence from human remains in show ancient use of the of Pteridium esculentum (G.Forst.) Cockayne (Houghton, 1978). In China there was use of edible ferns at least 3000 years ago (Zhang, 2007). There is evidence of the prehistoric use of the of Marattia fraxinea Sm. in ritual involving the incipient use of iron in (Schmidt & Avery, 1983).

FERNS IN PRE-LINNEAN LITERATURE Our study has shown that fern ethnobotany is nothing new and that there is much about ferns and local cultures in ancient literature. We have mainly concentrated here on the more recent literature, but a good example of early studies of fern ethnobotany is that of Georgius Everhardus Rumphius (1627-1702) in Ambon, now in (Rumphius, 2011). Rumphius devoted 44 pages to descriptions of ferns, lycophytes and their uses. The pre-Linnean names are often hard to identify to , but the good drawings and the interpretation by E. D. Merrill (1917) make it possible to relate to current species names. These pages are full of ethnobotanical information about fern uses by the Ambonese and natives of other islands in the seventeenth century. Many of these uses come under the subheadings we have used below. For example one chapter is entitled “The Edible Fern” and is about Athyrium esculentum (Retz.) Copel. Rumphius said that this fern and related species “are a renowned potherb of all of these islanders. One can make a good salad from its leaves and shoots….it cools moderately and loosens the bowels, especially if one drinks some tree-wine after it.” Rumphius describes the culinary use of several different species of ferns together with great details about the effects of eating them. The Balinese stick the tops of Tectaria crenata Cav. behind their ears when they go to war, because this plant will keep them from getting hurt by dart poison, rendering it powerless. They also rub the dry leaves over their bodies when bathing to get rid of sweat and odours. The lower stems of Lygodium circinnatum (Burm. f.) Sw. are split into four strips and used for seams around the edges of baskets by the Ambonese. An interesting use of the leaves of Drynaria sparsisora (Desv.) Moore is to tie them to a baited fishhook and use them as sails to carry the hooks out to sea until fish bites. The leaves of the same species are suspended over little children to keep them safe from evil spirits. The pages of Rumphius contain much interesting fern ethnobotany and many similar uses are reported in the more recent literature on the subject.

ETHNOPTERIDOLOGY In the literature surveyed we found a number of articles that were specifically about ethnopteridology of human groups and also ethnobotany about individual species of pteridophytes, and we highlight examples of recent works from different . From the Americas, Navarrete et al. (2006) presented information on the uses attributed to more than 200 species of pteridophytes of , and . Boom (1985) KELLER & PRANCE: THE ETHNOBOTANY OF FERNS AND LYCOPHYTES 3 treated the use of ferns by the Chácobo tribe of Amazonian Bolivia and Macía (2004) compared the ethnopteridology of the Tacana of Bolivia with that of the Huaorani of Ecuador. The only species in common was Cyathea pungens (Willd.) Domin. Hernández Cibrián & Sutherland (2007) carried out an ethnobotanical study of the ferns of a national park in Honduras and found only eight species that were used by the local population. For Argentina Keller et al. (2011) treated the various uses of 50 species of ferns and lycophytes by the Guaranies of Misiones Province (Figures 1-3) and Hurrell & De La Sota (1996) did the same for the villagers of Santa Victoria in Salta Province. In Christensen (1997) studied the ethnopteridology of ethnic groups in Malaysia. A study of the ethnobotanical uses of ferns in the Indian States of Jammu and Kashmir (Kirn & Kapahi, 2001) listed 17 species of which 11 were medicinal, four used for thatching roofs and three as foods. Joshi (1997) listed ethnobotanical uses of 44 species of ferns in Uttar Pradesh State of . For Africa the ethnopteridological study of ethnic groups by Nwosu (2002) mentioned 36 species in 23 families. In addition to fern uses for food and medicines, many of these studies mention uses in rituals of love, for magic ceremonies, as indicators of cardinal points and the presence of and also as material to make crafts and weapons. The demands of today’s markets have led various indigenous groups to commercialize ornamental ferns and flowerpots made out of erect rhizomes and the trunks of tree ferns.

FERNS IN TRADITIONAL MEDICINE Studies of uses of ferns in ethnomedicine are abundant on all inhabited continents. In Córdoba (Argentina) the study of ferns used in traditional medicine has developed to

Figure 1. Microgramma squamulosa (Kaulf.) de la Sota, a multipurpose species for the Guarani, used for slimming, menstrual analgesic, post partum washing, and treatment of lumbago. 4 FERN GAZ. 20(1):1-13. 2015

such an extent that anatomical and morphological evidence is used to detect adulterants of the products (Luján et al., 2007; 2011). However, the adulteration of medicinal ferns is nothing new. Hipólito Ruiz (1805) described the species Polypodium calguala Ruiz with the intention of clearly differentiating this medicinal ethnospecies of the indigenous peoples of Peru from other fern species that were being imported into from the New World as adulterants of the legitimate “calaguala.” In a comparison between the ethnopteridology of the Tacana of Bolivia and the Huaorani of Peru, Macía (2004) found that 76% of the recorded uses for ferns were medicinal either for people or for animals to heal wounds or expel parasites. Most of the uses by the Tacana are external, whereas the Huaorani uses are mainly internal. This study cites uses for 24 species of ferns and lycophytes. There are many medicinal uses of ferns in India. For example, Sharma and Vyas (1985) described the use of six species in Rajastan. Srivastava (2007) emphasised the importance of ferns in tribal medicine from a study made in various places throughout India. Benniamin (2011) reported on the use of 51 species of ferns in the east of India and Kumari et al. (2011) gave information about the use of 66 species in ethnomedicine in India. Dixit (1982) is an example of the ethnobotanical use of a single species, bryopteris (L.) Bak. This species is much revered in local medicine and commands a high market price. The medicinal use of Helminthostachys zeylanica (L.) Hook. has had serious effects on its state of conservation in Himalaya (Joshi, 2011). regalis L. is used as a medicine in the north of Spain (Molina et al., 2009). Of the 36 species of ferns reported in the ethnobotany study of ferns of Southern Nigeria by Nwosu (2002), 34 have medicinal uses. The paper reads like a complete

Figure 2. Alsophylla setosa Kaulf. The base of the rhizomes are used as stands for ornamental plants. The Guaranis and local farmers agree that the presence of this fern is an indicator that the soil is not suitable for agriculture. The Guarani use a soup of the petioles for the treatment of herpes. KELLER & PRANCE: THE ETHNOBOTANY OF FERNS AND LYCOPHYTES 5 pharmacy to treat many different ailments and all from pteridophytes. Ferns supply treatment for external injuries and wounds and many are taken internally to treat such diseases as malaria, ulcers, intestinal worms, liver disease etc. Many of the species listed have multiple uses, for example, a decoction of the and leaves of Polypodium microrhizoma Clarke ex Bak. is used for the relief of gastrointestinal disorders, backache and jaundice; a paste from the dried leaves (dried over an open fire) is applied externally for fissures on hands and wound healing; and a paste mixed with palm-kernel oil is applied externally to domestic animals such as sheep and . The whole plant of Osmunda regalis is taken internally for psychosis as it is believed that the tonic can chase away evil spirits, and an infusion of the roots is used to treat malaria and jaundice. The importance of medicinal ferns is evidenced by the growing interest in methods for their reproduction. An example is the achievement of in vitro propagation of the Asiatic fern Drynaria quercifolia (L.) J.Sm. that is much used in traditional phytotherapy (Mazumder et al., 2011).

VETERINARY The literature has many examples of the use of ferns for treating animals. According to Nwosu (1922), the leaves of Tectaria macrodonta (Fée) C.Chr. are powdered and mixed with castor oil and given to goats and sheep to stop a running stomach; young are chewed by cows after delivery of a calf to accelerate the expulsion of the afterbirth.

EDIBLE FERNS Fern rhizomes were an important source of food for Native Americans in western North

Figure 3. The rhizome of Dicksonia sellowiana Hook. in a Guarani basket offered for sale at a roadside stand. 6 FERN GAZ. 20(1):1-13. 2015

America. Turner et al. (1992) produced a summary of this in a detailed paper that listed at least 15 species of ferns together with their native nomenclature. Ferns are much eaten in India. For example, Pandy and Pangtey (1987) list seven different species of ferns consumed in Western Himalaya and Joshi (1997) lists 10 edible species used in Uttar Pradesh State. For China, Liu et al. (2012) listed a total of 42 edible pteridophytes, but they estimated that the potential total could be as high as 144 species. Some ferns are eaten as though they were sweets, as in the case of Pecluma pectinatiformis (Lindm.) M.G.Price, where the sweet leaves are commonly chewed by Guaraní children in Misiones, Argentina (Keller et al., 2011). Some species of Polypodium are known for their property of sweetness. A variety of Polypodium vulgare L. was used to flavour tobacco for its liquorice taste and it contains small amounts of ostadin, a steroid saponin 3000 times as sweet as sucrose. In former times the fronds of this species were used in Ireland to treat coughs, colds and asthma. Polypodium glycyrrhiza D.Eaton also has a liquorice flavour and was eaten by Native American peoples (Mabberley, 2008).

FERNS AS BUILDING MATERIALS The Guaranies of southeastern use the stems of Dicksonia sellowiana Hook. to support the walls of their traditional houses (Prudente, 2007). Joshi (1997) lists Cyathea spinulosa Wall. ex Hook. and Dicranopteris liniaris (Burm. f.) Underw. as used for thatching roofs in India. Kirn and Kapahi (2001) mention Pteridium aquilinum (L.) Kuhn. var. wightianum (Ag.) Tryon, Pteris vittata L., erubescens (Wall. ex Hook.) Ching and Woodwardia unigemmata (Makino) Nakai for the same purpose.

FERNS IN ORNAMENTATION AND ART Ferns are often used for body ornamentation. The stipes of farinosa (L.) Brogn. and lunulatum Burm. are used as nose and ear studs by children and poor women in Uttar Pradesh (Pande & Pangey, 1987) and Adiantum venustum D. Don is used as ear studs by girls in Kashmir (Kirn & Kapahi, 2001). The tree ferns Cyathea divergens var. tuerckenheimii R.M.Tryon and C. fulva (Martens & Galeotti) Fée are harvested to produce handicrafts for garden ornamentation by artisans of the mountains of Cuetzalan, Mexico (Elutério, 2006). In the Philippines the and rachis of Lygodium japonicum (Thunb.) Sw. are used to decorate baskets (Novellino, 2006). In Argentina the Guaranies use the petioles of various ferns to make necklaces (Keller et al., 2011).

USES OF FERN Frye (1934) reported that the spores of Lycopodium clavatum L. were used for dusting on open raw wounds and chafed infants by natives of northwestern . The spores are fine and light and so repel water and prevent stickiness. Lycopodium powder has also been used as a lubricating dust on latex gloves and condoms, though the latter use is not recommended (see Balick & Beitel, 1989), because these spores have been known to cause allergic reactions, ranging from hay-fever to more serious giant cell granulomas. May (1978) reported that the easily flammable spores of species of Lycopodium have been used in theatre as a flash powder. KELLER & PRANCE: THE ETHNOBOTANY OF FERNS AND LYCOPHYTES 7

FERNS AS PLACE NAMES The importance of ferns to local communities has often led to the names of places, topographic formations, watercourses and political divisions. In Misiones Province (Argentina), the most tropical and wettest province of Argentina, there are various places based on plant names. The ‘Diccionario geográfico toponímico’ of Stefañuk (2009) gives several examples. “Los helechos” is the name of a stream and a municipality in this province. In the town of Oberá there is a place and a stream named “Samambaya” the common name for ferns derived from the Guaraní word for them “amambái” which translated into English is a generic name of ferns. Locally amambái refers to the large populations of Pteridium arachnoideum (Kaulf.) Maxon. In the Department of L.N. Alem there is a stream called “Chachi” which is the Guaraní name for the tree fern Cyathea atrovirens (Langsd. & Fisch.) Domin (). In the Department of San Martín there is stream called “Culandrillo”, a term that refers to species of the Adiantum (). In the derivation of the name of the political Amambay Department is derived from the Guaraní word for fern. There are many place names in the United associated with ferns, for example, Ferndown in Dorset, Fernilee in Derbyshire and Ferness in the Highlands. Fern is a town in Tayside and Fernie a stream and a castle in Fife. Bracken (Pteridium aquilinum) features in Brackenfield in Derbyshire and Brackenthwaite in Cumbria.

FERNS IN PHARMACOLOGY Substances with antioxidant activity are now used in medicine to reduce the effects of oxidation stress. Antioxidant activity has been reported in Adiantum capillus-veneris L., a widely distributed fern (Kumar, 2009). A recent study of the lateral branches of giganteum L. of South and Central America showed that they can be used as a source of antioxidant compounds (Ricco et al., 2011). In Malaysia studies have shown similar properties in various ferns: Blechnum orientale L., Cibotium barometz (L.) J.Sm., Cyathea latebrosa (Wall. ex Hook.) Copel., Dicranopteris linearis Burm., Drynaria quercifolia (L.) J.Sm. and Stenochlaena palustris (Burm. f.) Bedd. (Chai et al., 2012; Lai & Lim, 2011, Lai et. al., 2010). In China several fern rhizomes have been shown to have antioxidant properties: Drynaria fortunei (Kze.) J.Sm., Pseudodrynaria coronans (Wall. ex Mett.) Ching, divaricata Bl., D. mariesii Moore ex Bak., D. solida (Forst.) Sw., and Humata griffithiana (Hook.) C.Chr. (Chang et al., 2007). Ferns also contain substances with antibacterial activity as was shown in the study by Thomas (2011) of Osmunda regalis. India has made important advances in this area and Kumarpal (2013) showed that there was good antimicrobial activity in three species of ferns from three different families used in traditional medicines in Darjeeling, Athyrium filix-femina (L.) Roth (Woodsiaceae), Dicranopteris linearis (Burm. f.) Underw. (Gleicheniaceae) and Pleopeltis macromarpa (Bory ex Willd.) Kaulf. (). Patric Raja et al. (2012) found antibacterial and antifungal activity in Cyathea nilgiriensis Holttum, C. crinita (Hook.) Copel., Leptochilus lanceolatus Fée and in Osmunda hugeliana Presl. Studies made in Romania by Soare et al. (2012) showed that the bladder fern fragilis (L.) Bernh. and Polypodium vulgare L. strongly inhibited various bacteria, especially Escherichia coli. Most of the species that have been studied for their pharmaceutical properties have notable antecedents in traditional medicine. The development of pharmaceuticals from plants used in the pharmacopeia of local peoples ought to ensure that the benefits are shared with the traditional communities from where the original information came (Prance, 1991). 8 FERN GAZ. 20(1):1-13. 2015

FERNS IN TOXICOLOGY The relationship between plants and people is not always a positive one This includes those species that have strong substances that are damaging either to human health or to that of domesticated animals. Pteridium aquilinum has been studied for the relationship between the various uses and its toxicity (Alonso Amelot, 1999; Franca et al., 2002; Ortega, 1993; Vetter, 2011). Recent studies show that Blechnum orientale is able to absorb heavy metals from the environment and it is therefore a potentially toxic source of food and medicine. The accumulation is mostly in the rhizomes and leaves, the very parts of the plant that are eaten (Zhu et al., 2013). A positive side for the environment of this absorptive capacity of ferns is the accumulation of arsenic by Pteris vittata L., which could be used as a method of decontaminating toxic sites. Phytoremediation of arsenic contaminated environments will involve growing the arsenic hyperaccumulator ferns in the contaminated environment, harvesting the arsenic-rich biomass and the safe disposal of the biomass (Gumaelius, 2004; Rathinasabapathi et al., 2006).

FERNS AS ORNAMENTAL PLANTS A large number of fern species are used as ornamentals in the gardens around the world, and this dates back to ancient times and often to domestication for other uses. Studies of the ornamental potential of pteridophytes of regional floras has led to an increase of their use as cultivated plants. Abraham et al. (2012) listed a total of 153 ferns and 18 lycophytes from Nilgiris, India with ornamental potential. Macaya (2004) mentioned 20 species of ferns native to that are cultivated as ornamentals and in Macaya (2008) he expanded this to a list of 75 species. An example of the relevance of ethnobotany for making strategies for use of populations of native vegetation is that of Baldauf et al. (2007), who made an ethnobotanical study of the management systems of Rumohra adiantiformis (G. Forst.) Ching. This species is used as an ornamental in southern Brazil. Another fern of the southern cone region of South America with high ornamental potential is Blechnum tabulare (Thunb.) Kuhn, a species that resembles the Cycadaceae in appearance. A population study of this species was made in southern Brazil (Rechenmacher et al., 2007). The stems of some tree ferns are used extensively as a substrate for the cultivation of epiphytes. However, the indiscriminate use of this resource is threatening populations of these plants in some places. An example of this is the use of Dicksonia sellowiana Hook., the conservation of which has led to studies of growth, phenology and germination of spores (Filippini et al., 1999; Schmitt et al., 2009).

FERNS AND SOIL QUALITY Azolla Lam. has long been used as a fertilizer in rice paddies (Jones, 1987). In Misiones, Argentina farmers identify compacted and degraded soils by means of observations on the presence of Pteridium aquilinum. In the central Andean region of Peru indigenous peoples of the Quechua language group use the fronds of Dennstaedtia glauca (Cav.) Looser to fertilize the soils where they cultivate potatoes (Camino & Jhons, 1988), which is similar to the use of bracken peat as a fertilizer in some places in the .

CONCLUSIONS This brief summary has shown the diversity of important uses to which humans have put ferns and lycophytes, and some of these are illustrated in Figures 1-3. Many of these use categories originated in traditional societies (the white area of Figure 4) such as use in rituals, the construction of houses and to make weapons. Other uses are typical of KELLER & PRANCE: THE ETHNOBOTANY OF FERNS AND LYCOPHYTES 9 contemporary society (the dark area of Figure 4) such as the large scale commercialization of ornamental plants and for drug prospecting. However, the division between traditional and modern uses is not precise because many uses fall into both social categories (the grey area of Figure 4). For example many medicinal uses arose in traditional societies that have gradually modernized, still using ferns in their medicine but also leading to prospecting and use in pharmacology. This much more intensive use of ferns in modern societies has led to the destruction of the where they grow. This has increased the importance of conservation of ferns and the development of commercial production rather than harvesting from the wild.

Figure 4. Examples of the importance of ferns in traditional and modern contexts. 10 FERN GAZ. 20(1):1-13. 2015

ACKNOWLEDGMENTS We thank Blanca León for helpful suggestions when reviewing the manuscript.

REFERENCES ABRAHAM, S., RAMACHANDRAN, V.S. & SOFIA, C. 2012. Potential ornamental Ferns from Nilgiris, Tamil Nadu. Advances in Applied Science Research 3 (4):2388-239. ALONSO AMELOT, M. E. 1999. Helecho macho, salud y salud humana. Rev.Fac. Agron. (LUZ) 16: 528-541. ANDERSON, A. & WHITE, P. 2001. Approaching the Prehistory of Norfolk Island. Records of the Australian Museum, Supplement 27: 1–9. BALDAUF, C., HANAZAKI, N. & SEDREZ DOS REIS, M. 2007. Caracterização etnobotânica dos sistemas de manejo de samambaia-preta (Rumohra adiantiformis (G. Forst) Ching - ) utilizados no sul do Brasil. Acta bot. bras. 21(4): 823-834. BALICK, M. J. & BEITEL, J. M. 1989. Lycopodium spores used in condom manufacture: associated health hazards. Economic Botany 43: 373-377. BENNIAMIN, A. 2011. Medicinal ferns of North Eastern India with special reference to Arunachal Pradesh. Indian Journal of Traditional Knowledge 10(3): 516-522. BOOM, B. M. 1985. Ethnopteridology of the Chácobo Indians in Amazonian Bolivia. Amer. Fern J. 75:19–21. CAMINO, A. & JOHNS. T. 1988. Laki-Laki (Dennstaedtia glauca, Polypodiaceae): A Green Manure Used in Traditional Andean Agriculture. Economic Botany 42(1):45-53. CHRISTENSEN, H. 1997. Uses of ferns in two indigenous communities in Sarawak, Malaysia. In JOHNS, R. J. (Ed.) Holttum Memorial Volume, pp. 177-192. Royal Botanic Gardens, Kew. CHAI, T.-T., PANIRCHELLVUM, E., ONG, H.-C. & WONG, F.-C. 2012. Phenolic contents and antioxidant properties of Stenochlaena palustris, an edible medicinal fern. Botanical Studies 53: 439-446. CHANG, H.C., HUANG, G.J., AGRAWAL, D.C., KUO, C.L., WU, C.R. & TSAY, H.S. 2007. Antioxidant activities and polyphenol contents of six folk medicinal ferns used as “Gusuibu”. Bot. Stud. 48: 397-406. CLINE, W. 1937. Mining and Metallurgy in Negro Africa. George Banta Pub. Co.: Menasha, Wisconsin. DÍAZ DE LEÓN, M. E. M., MENDOZA-RUÍZ, A. & PÉREZ-GARCÍA, B. 2007. Usos de los helechos y plantas afines. Etnobiología 5: 117-125. DIXIT, R.D. 1982. Selaginella bryopteris (L.) Bak. – An ethnobotanical study IV. J. Econ. Tax. Bot. 3: 309-312. ELUTÉRIO, A.A. 2006. Management of tree fern (Cyathea spp.) for handicraft production in Cuetzalan, Mexico. Economic Botany 60: 182-191. FILIPPINI, E.C.P., DUZ, S.R. & RANDI, Á.M. 1999. Light and storage on the germination of spores of Dicksonia sellowiana (Presl) Hook., Dicksoniaceae. Revta. Bras. Bot. 22: 21-26. FRANCA, T. N., TOKARNIA, C. H. & PEIXOTO, P. V. 2002. Enfermidades determinadas pelo princípio radiomimético de Pteridium aquilinum (Polypodiaceae). Pesq. Vet. Brass. 22(3): 85-96. FRYE, T. C. 1934. Ferns of the Northwest. Metropolitan Press, Portland. GUMAELIUS, L., LAHNER, B., SALT, D. E. & BANKS, J. A. 2004. Arsenic KELLER & PRANCE: THE ETHNOBOTANY OF FERNS AND LYCOPHYTES 11

hyperaccumulation in of Pteris vittata. A new model system for analysis of arsenic hyperaccumulation. Plant Physiol. 136:3198–3208. HERNÁNDEZ CIBRIÁN, R. K. & SUTHERLAND, C. H. N. 2007. Etnobotánica de los Helechos de Honduras. Ceiba 48(1-2): 1-10. HOUGHTON, P. 1978. Dental evidence for dietary variation in prehistoric New Zealand. Journal of the Polynesian Society 87(3): 257-263. HURREL, J. A. & DE LA SOTA, E. R. 1996. Etnobotánica de las Pteridofitas de los Pastizales de altura de Santa Victoria (Salta, Argentina). Revista Mus La Plata (NS). Botánica 15(105): 353–364. JONES, D. L. 1987. Encyclopaedia of Ferns. An Introduction to ferns. Their structure, biology, importance, cultivation and propagation. Lothian Publishing Company PTY LTD. Melbourne, Sydney, Auckland. JOSHI, P. 1997. Ethnobotany of Pteridophytes of hilly districts of Uttar Pradesh, India. Indian Fern J. 14: 14-18. JOSHI, P. 2011. Ecology and Medicinal Uses of Helminthostachys zeylanica (L.) Hook. “An endangered flora of India” reported at Foothills of Kumaun Himalaya (Kashipur), Uttarakhand. Researcher 3(4): 51-54. KELLER, H.A., MEZA-TORRES, E.I. & PRANCE, G.T. 2011. Ethnopteridology of the Guaranis of Misiones Province, Argentina. Amer. Fern J. 101(3): 193-204. KIRN, H.S. & KAPAHI, B.K. 2001. Ethnobotanical notes on some ferns and fern-allies of Jammu and Kashmir State, India. Indian Fern J. 18: 35-38. KUMAR A. 2009. Antioxidant effect of Adiantum capillus veneris Linn. on human lymphocyte: an in vitro study. Journal of Cell and Tissue Research 9 (2): 1899-1902. KUMARI, P., OTAGHVARI, A. M., GOVINDAPYARI, H., BAHUGUNA, Y. M. & UNIYAL, P. L. 2011. Some ethno-medicinally important Pteridophytes of India. Int. J. Med. Arom. Plants 1(1): 18-22. KUMARPAL, S. 2013. Study of Activity of Some Medicinal Ferns of Darjeeling. International Journal of Scientific and Research Publications 3(8): 1-4. LAI, H.Y. & LIM, Y.Y. 2011. Evaluation of Antioxidant Activities of the Methanolic Extracts of Selected Ferns in Malaysia. International Journal of Environmental Science and Development 2(6): 442-447. LAI, H.Y., LIM, Y. Y. & KIM, K.H. 2010. Blechnum Orientale Linn - a fern with potential as antioxidant, anticancer and antibacterial agent. BMC Complementary and. Alternative Medicine, vol.10: 15. LIU, Y., WUJISGULENG, W. & LONG, C. 2012. Food uses of ferns in China: a review. Acta Soc. Bot. Poloniae 81: 263-270. (52 species used) LOOSER, G. & RODRÍGUEZ, R. 2004. Los helechos medicinales de Chile y sus nombres vulgares. Gayana Bot. 61(1): 1–5. LUJÁN, M. C., MORERO, R., BONZANI, N. E. & BARBOZA, G. E. 2007. Sobre la identidad de algunos helechos medicinales que se comercializan en Córdoba (Argentina). Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 6(6): 376-377. LUJÁN, M. C., MORERO, R. & BARBOZA, G. E. 2011. Estudios epidérmicos en helechos y licófitas medicinales de la Provincia de Córdoba, Argentina. Hoehnea 38(4): 609-659, 2 tab., 28 fig. MABBERLEY, D.J. 2008. Mabberley’s Plant-book, ed. 3: 690. Cambridge Univ. Press MACAYA, J. 2004. Helechos nativos de Chile cultivados con fines ornamentales. Chloris Chilensis 7(1) URL: http://www.chlorischile.cl. MACAYA, J. 2008. Helechos introducidos en Chile con fines ornamentales (parte I). Chloris Chilensis Año 11, N°2. URL: www.chlorischile.cl. 12 FERN GAZ. 20(1):1-13. 2015

MACÍA, M.J. 2004. A comparison of useful Pteridophytes between two Amerindian Groups from amazonian Bolivia and Ecuador. Amer. Fern J. 94: 39-46. MANNAR MANNAN, M., MARIDASS, M. & VICTOR, B. 2008. A Review on the Potential Uses of Ferns. Ethnobotanical Leaflets 12: 281-285. MAY, L. W. 1978. The economic uses as associated folklore of ferns and fern allies. Bot. Rev. 44: 491-528. MAZUMDER, P. B., MAZUMDER, B., CHOUDHURY, M D. & SHARMA, G.D. 2011. In Vitro Propagation of Drynaria quercifolia (L.) J. Sm., a Medicinal Fern. Assam University Journal of Science & Technology, Biological and Environmental Sciences 7(1): 79-83. MERRILL, E. D. 1917. An interpretation of Rumphius’s “Herbarium Amboinense”. Manila Bureau of Printing. MOLINA, M., REYES-GARCIA V. & PARDO-DE-SANTAYANA, M. 2009. Local knowledge and management of the Royal Fern (Osmunda regalis L.) in Northern Spain: Implications for Conservation. Amer. Fern J. 99 (1): 45–55. MURILLO, M.T. 1983. Usos de los helechos en Suramérica con especial referencia a Colombia. Ed. Instituto de Ciencias Naturales, Biblioteca José Jerónimo Triana, Nº5, 156 pp. Bogotá. NAVARRETE, H., LEÓN, B., GONZALES, J., AVILES, D. K., SALAZAR LECARO, J., MELLADO, F., ALBAN, J. & ØLLGAARD, B. 2006. Helechos. In: MORAES R., M., ØLLGAARD, B., KVIST, L. P., BORCHSENIUS, F. & BALSLEV, H. (Eds.): Botánica Económica de los Centrales. Universidad Mayor de San Andrés, La Paz, p: 385-411. NOVELLINO, D. 2006. An introduction to basketry in Island Southeast Asia. Proceedings of the IVth International Congress of Ethnobotany (ICEB 2005), 2006: 621-625. NWOSU, M. O. 2002. Ethnobotanical studies on some Pteridophytes of southern Nigeria. Econ. Bot. 56: 255–259. ORTEGA, F. 1993. La etnobotánica de Pteridium aquilinum (L.) Kuhn en y sus posibles riesgos asociados a la carcinogénesis. Medula 2 (3-4): 51-56. PANDE, P.C. & PANGTEY, Y.P.S. 1987. Studies on ethnobotany –I. On some less known edible and economic ferns of Kumaun region of Western Himalaya. J. Econ. Tax. Bot. 11: 81-85. PATRIC RAJA, D., JOHNSON, M., IRUDAYARAJ, V. & JANAKIRAMAN, N. 2012. Antimicrobial efficacy of selected ferns of Western Ghats, South India. International Journal of Current Pharmaceutical Research 4 (2): 58-60. PRANCE, G. T. 1991. What is ethnobotany today? J. Ethno-pharmacol. 32: 209-216. PRUDENTE, L. T. 2007. Arquitetura Mbyá-Guaraní na Mata Atlântica do Rio Grande do Sul. Estudo do caso do Tekoá Nhüu Porã. Tese (Mestrado) em engenharia. Universidades Federal do Rio Grande do Sul. Escola de Engenharia. Porto Alegre, 164 pp. RATHINASABAPATHI, B., MA, L. Q. & SRIVASTAVA, M. 2006. Arsenic Hyperaccumulating Ferns and their Application to Phytoremediation of Arsenic Contaminated Sites. Floriculture, Ornamental and Plant Biotechnology 3: 304-311. RECHENMACHER, C., SCHMITT, J. L. & BUDKE, J. C. 2007. Estrutura e distribuição espacial de uma população de Blechnum tabulare (Thunb.) Kuhn (Pteridophyta, ) em um mosaico floresta-campo no sul do Brasil. Pesquisas, Botânica 58: 177-186. RICCO, R. A., AGUDELO, I., GARCÉS, M., EVELSON, P., WAGNER, M. L. & GURNI, A. A. 2011. Polifenoles y actividad antioxidante en KELLER & PRANCE: THE ETHNOBOTANY OF FERNS AND LYCOPHYTES 13

L. (). Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas 10 (4): 325 – 332. RUIZ, H. 1805. Memoria sobre la legítima calaguala y otras dos raíces que nos vienen de la América meridional. Imprenta de D. José del Collado, Madrid, 60 pp. RUMPHIUS, G. E. 2011. The Ambonese Herbal vol 5: 121-165. English translation by E.M. BEEKMAN of The Ambonese herbal first published in 1741, Yale University Press. SHARMA, B.D & VYAS, M.S. 1985. Ethnobotanical studies on the ferns and fern allies of Rajastan. Bull. Bot. Survey India 27: 1-4. SCHMITT, J. L., SCHNEIDER, P. H. & WINDISCH, P. G. 2009. Crescimento do cáudice e fenologia de Dicksonia sellowiana Hook. (Dicksoniaceae) no sul do Brasil. Acta bot. bras. 23(1): 282-291. SCHMIDT, P. R. & AVERY, D. H. 1983. More Evidence for an Advanced Prehistoric Iron Technology in Africa. Journal of Field Archaeology 10(4): 421-434. SMITH, A. R., PRYER, K. M., SCHUETTPELZ, E., KORALL, P., SCHNEIDER, H. & WOLF, P. G. 2006. A classification for extant ferns. Taxon 55(3): 705-731. SOARE, L. C., FERDEŞ, M., DELIU, I. & GIBEA, A. 2012. Studies regarding the antibacterial activity of some extracts of native pteridophytes. U.P.B. Sci. Bull., Series B 74(1): 21-26. SRIVASTAVA, K. 2007. Importance of Ferns in Human Medicine. Ethnobotanical Leaflets 11: 231-234. STEFAÑUK, M. Á. 2009. Diccionario geográfico toponímico de Misiones. Contratiempo Ediciones. Buenos Aires, 817 pp. STRASBURGER, E., NOLL, F., SCHENCK, H. & SCHIMPER, A. F. W. 2003. Tratado de Botánica, 35ª edición, actualizada por SITTE, P., WEILER, E. W., KADEREIT, J. W., BRESINSKY, A. & KÖRNER, C. Ediciones Omega, Barcelona, 1134 pp. THOMAS, T. 2011. Preliminary Antibacterial and Phytochemical Assessment of Osmunda regalis L. International Journal of Pharmaceutical & Biological Archives; 2(1): 559-562. TURNER, N.J, JOHNSON GOTTESFELD, L.M., KUHNLEIN, H.V. & CESKA, A. 1992. Edible wood fern rootstocks of western North America: solving an ethnobotanical puzzle. J. Ethnobiol. 12: 1-34. VETTER, J. 2011. Toxicological and medicinal aspects of the most frequent fern species, Pteridium aquilinum (L.) Kuhn. Chapter 25. In H. Fernández et al., (eds.): Working with ferns: Issues and applications. Springer Sciences & Business Applications. P. 361. WILEY, G. R. 2003. Archaeobotany: Scope and Significance . In: Richard Evans Schultes & Siri Von Reis (Eds.): Ethnobotany. Timber Press, Portland. P: 400-406. ZHANG, Y. M. 2007. A preliminary investigation of the history of eating in China. Journal of Southwest Agricultural University (Social Science Edition) 5(4): 103–106. ZHU, X., KUANG, Y., XI, D., LI, J. & WANG, F. 2013. Absorption of Hazardous Pollutants by a Medicinal Fern Blechnum orientale L., BioMed Research International Vol. 2013: 1-6. FERN GAZ. 20(1). 2015 14


Héctor Alejandro Keller, holds a Forestry Engineer’s degree from the Universidad Nacional of Misiones, Argentina (2000), and a Ph.D. in Natural Resources from the Universidad Nacional del Nordeste (2008). He is currently lecturer in botany and vegetation systematics at the National University of Misiones. Technical officer responsible for the Multiple-use Guaraní Reserve, 2006-2007. Since 2010 Scientific Researcher of the National Council for Scientific and Technological Research of Argentina. He has collected 12,300 herbarium specimens, mainly from Misiones Province, Argentina including ten new species and one new beetle. Author of one book chapter and 87 articles in scientific journals on ethnobotany, ethnography, ethnobiology and floristic studies. Epononyms: : Lasiodactylus kelleri Cline (Nitidulidae); Gaya kelleri Krapov. (Malvaceae), Phaeostemma kelleri Morillo (Asclepiadaceae).

Professor Sir Ghillean Prance, University of Oxford BA in Botany (1960) M.A., D.Phil. (1963); research assistant The New York Botanical Garden 1963, B A Krukoff Curator of Amazonian Botany (1966), Director and Vice-President of Research (1976-1982), Senior Vice President for Science (1982-1988). 39 expeditions to Amazonia, collected 350 new species of plants. Director of the Royal Botanic Gardens, Kew 1988 -1999; McBryde Professor, National Tropical Botanical Garden Hawaii 2001-02, McBryde Senior Fellow (2003-). Visiting Professor at Reading University; author of 20 books and 529 scientific and general papers in , ethnobotany, economic botany, conservation and ecology; International COSMOS Prize 1993; Fellow of the Royal Society; Knight Bachelor; 1995; Victoria Medal of Honour of Royal Horticultural Society, 1999; David Fairchild Medal for plant exploration, 2000; Allerton Award in 2005; Commander of the of the Southern Cross Brazil, 2000; Order of the Rising Sun from , 2012.

The authors in a Guarani village in Misiones, Héctor Keller at left, Ghillean Prance second from left. FERN GAZ. 20(1):15-18. 2015 15



Department of Biological Sciences, Burdwan Town School, Burdwan-713101, India Email: [email protected]

Key words: Angiopteris indica, Herb. Desvaux, India, Marattiaceae, type.

ABSTRACT The type of the tree fern Angiopteris indica Desv. (Marattiaceae) was rediscovered in Herb. Desvaux at P and its status is discussed.

INTRODUCTION Three species of the marattioid fern genus Angiopteris Hoffm. (Marattiaceae) are generally accepted to occur in India (Fraser-Jenkins, 2008; Fraser-Jenkins & Benniamin 2010), namely Angiopteris indica Desv., A. helferiana C.Presl, and A. palmiformis (Cav.) C.Chr. Fraser-Jenkins (2008) accepted A. indica as the oldest available name for plants characterized by the combination of the following characters: the soral lines are located close or at the margin, the lamina segments possess prominent teeth near their tips, the lamina colour is darker than in other Indian species, and with the false (recurrent) veins reaching the soral line or just passing beyond it. In contrast, A. helferiana is distinguishable from A. indica by its inframarginal sori, whereas A. palmiformis has long false veins extended up to the pinnule-midrib. Angiopteris indica was described by Desvaux in 1813 (Desvaux 1813: 267) and not in 1811 (Desvaux 1811: 207), as misquoted by Moore (1857: 75) and Christensen (1906: 57), but see Hooker & Greville (1831) for the correct citation of the name. In the protologue, Desvaux (1813: 267) described the plants as “frondibus pinnatis, pinnis lanceolatis utrinque attenuates” and mentioned the area of origin as “Habitat in India orientali”. He further considered the non-cordiform base on the segments as the main feature distinguishing this species from previously described taxa. Desvaux in his protologue mentioned that he saw a specimen of the species at Herb. de Jussieu. Later authors (Vriese & Harting, 1853; Fraser-Jenkins, 2008) concluded that no specimens of this species were found in the Herb. de Jussieu, which is located in the herbarium at the “Museum national d’Histoire naturelle” at Paris (P). Subsequently, Fraser-Jenkins (2008) designated a neotype: Herb. Wight e Nilghiry [“S. India”], alt. 5000 ped. Wallich List No. 187.8. (K-W). This designation is based on the crucial assumption that no specimen seen by Desvaux can be found.

RESULTS Here, I present the discovery of a single specimen located at “Herbier de A.N. Desvaux Donné par Mme Vve Lavallée en 1896” in the herbarium P (Fig. 1A) with the barcode P01646217 (http://sonneratphoto.mnhn.fr/2010/08/09/4/P01646217.jpg). The specimen carries two labels on the left side that were interpreted as written by Desvaux showing the name “Angiopteris indica Desv., journ. bot. appl, p. 267, Habitat in India orientali” (Fig. 1D). The second label mentions (in French) “was not described in my memoir given at Berlin” (Fig. 1D). By this annotation Desvaux indicated that the name A. indica was 16 FERN GAZ. 20(1):15-18. 2015 not published in “Berlin Magazine” in 1811 along with many new taxa he described there. A third label below the pinna fragment shows the name “Angiopteris glauca Desv.” (Fig. 1B). This name does not correspond to Angiopteris glauca Alderw. Mal. Ferns, Suppl. I, Corr. 61 (1917) and it is interpreted here as an unpublished name considered by Desvaux.

Figure 1. Specimens of Angiopteris indica Desv. A. Herbarium sheet (P01646217) with two pinnule-segments, B. fragmented pinnule (Type), C. second pinnule with long false veins, D. labels written by Desvaux. Images © Muséum national d’Histoire naturelle, Paris Herbarium (P), reproduced with permission. MAZUMDAR: THE REDISCOVERY OF ANGIOPTERIS INDICA 17

The putative type is a fragment of pinnule showing prominent marginal teeth, inconspicuous false veins and sori inframarginal (Fig. 1B). A morphologically distinct second pinnule pasted above it (see Fig. 1A) has long false veins extended up to pinnule- midrib, rounded base and nearly median sori (Fig. 1C).

DISCUSSION One of these pinnules should be considered as type of A. indica as it was definitely studied by Desvaux, supported by the evidence of his own handwritten labels, and which can be assumed to be the lost type of A. indica from Herb. de Jussieu.. The second pinnule (Fig. 1C) is not a separate portion of the same plant but was pasted from a different specimen (pinna): Java, Leschenault 642 (barcode P01646218, http://sonneratphoto.mnhn.fr/2010/08/09/4/P01646218.jpg); Jean-Baptiste Leschenault was a French botanist and his plant collections from Java were subsequently used by A.L. de Jussieu and others. But there is no evidence to support the view that Desvaux selected this second pinnule (Fig. 1C) as type and pasted it himself. Actually the second pinnule was originally cut and pasted in an inverted position in P01646218. As a result it was easily detached and later erroneously pasted in P01646217. Desvaux coined the name A. glauca Desv. probably due to glaucous lamina of the pinnule (Fig. 1B), but later revised it as A. indica considering the distribution “India Orientali,” which includes modern India, Sri Lanka, Indonesia, Philippines and Marianas (Cook 2009).

CONCLUSION The fragment of pinnule (Fig. 1B) is chosen here as the type of A. indica from the two pinnules in Figure 1A. This type, actually lectotype (designated here), refutes the application of the name A. indica to plants from India with marginal sori by Fraser- Jenkins (2008) in displaying inframarginal sori (Fig. 1B), similar to A. helferiana (having sori about 1 mm distant from margin). But due to wide range of morphological variability of species of Angiopteris further additional data are required to confirm the identity and correct number of species of Angiopteris in India.

ACKNOWLEDGEMENTS I thank Mr C.R. Fraser-Jenkins, Nepal for discussing the present topic and translation of Desvaux’s French annotation; Muséum national d’Histoire naturelle (MNHN), Paris Herbarium (P) and Dr G. Rouhan, Curator of Ferns & Lycophytes, MNHN for granting permission for publication of the image; and an anonymous reviewer for improving the manuscript.

REFERENCES CHRISTENSEN, C. 1906. Index Filicum. H. Hagerup, Copenhagen. 744 pp. (http://www.biodiversitylibrary.org/item/9241) COOK, A. 2009. Linnaeus and Chinese plants: A test of the linguistic imperialism thesis. Notes Rec. R. Soc. (doi:10.1098/rsnr.2009.0051) DESVAUX, N.A. 1811. Observations sur quelques nouveaux generes de fougères et sur plusieures espéces nouvelles de la meme famille. Berl. Mag. 5: 297-330. (http://babel.hathitrust.org/cgi/pt?id=hvd.32044106317993;view=1up;seq=218) DESVAUX, N.A. 1813. Description de Fougères nouvelles. J. Bot. Agric. 1: 266-273. (http://gallica.bnf.fr/ark:/12148/bpt6k96848g) 18 FERN GAZ. 20(1):15-18. 2015

FRASER-JENKINS, C.R. & BENNIAMIN, A. 2010 [“2009”]. Fifty rarities and additions to the pteridophytic flora of Arunachal Pradesh, N.E. India. Panjab University Research Journal (Science) 59: 1-38. FRASER-JENKINS, C.R. 2008. Taxonomic revision of three hundred Indian subcontinental pteridophytes with a revised census-list. Bishen Singh Mahendra Pal Singh, Dehra Dun. 685 pp. HOOKER, W.J. & GREVILLE, R.K. 1831. Icones Filicum. Vol. 1, Prostant Venales Apud Treuttel et Würtz, Treuttel Fil. et Richter etc., London. (http://dx.doi.org/10.5962/bhl.title.3867) MOORE, T 1857. Index Filicum. W. Pamplin, London. 396 pp. (http://dx.doi.org/10.5962/bhl.title.19640) VRIESE, W.H. DE & HARTING, P. 1853. Monographie des Marattiacées. Noothoven van Goor, Leiden & Arnz, Düsseldorf. 60 pp. FERN GAZ. 20(1):19-22. 2015 19



Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad, Iran *Author for correspondence: e-mail: [email protected]

Keywords: fern, , tetraploid.

ABSTRACT The chromosome number and ploidy level of Cystopteris fragilis (L.) Bernh. are documented for the first time from Iran. The count was tetraploid, with 2n= 4x= 168. This agrees with previous reports of tetraploid C. fragilis from , Asia and the United States. INTRODUCTION Cystopteris Bernh. (Cystopteridaceae) comprises about 27 species distributed throughout the temperate regions and tropical mountains (Rothfels et al., 2013). Three species occur in Iran: C. alpina (Lamark) Desvaux, C. dickieana Sim and C. fragilis (L.) Bernh. (Roux 2001; Khoshravesh et al., 2009). The two latter species have also been reported from high elevations in the Hindu Kush Mountains of neighboring Afghanistan (Breckle, 1987). In general, these species of Cystopteris are found in shady, rocky, wet habits (Bhellum and Razdan, 2012). The taxonomy and distribution of its species in Iran have been reported by Khoshravesh et al. (2009). Cystopteris fragilis differs from C. dickieana in having spiny spores (vs. verrucate in C. dickieana). The chromosome numbers of n=84 and n=126 (tetra- and hexaploid, respectively) of C. fragilis were first reported from Europe (Manton, 1950). Vida (1976) later reported octaploids in Central Europe. In North America, tetraploids and hexaploids of C. fragilis have been found (Wagner, 1955; Haufler and Windham, 1991; Haufler et al.; 1995, Paler and Barrington, 1995). In Asia, tetraploids and hexaploids have been reported from India and Japan (Bhellum and Razdan, 2012; Mitui, 1970). In 2010, a pentaploid with 2n=210 was also recorded from Mongolia Altai by Kawakami et al. (2010). For Iran, however, chromosome numbers of C. fragilis have not been reported previously. This paper represents the first such report for the country.

MATERIAL AND METHODS Fixed cytological material and voucher specimens were collected by the first author from three populations in natural habitats in Akhlamad waterfall valley, ca. 65 km NW of Mashhad, NE Iran at an elevation 1500 m in May and June 2013. The populations occurred on stony or rocky slopes exposed to waterfall, within shaded habitats, and occasionally in the cement of the roadside. Vouchers of examined specimens are deposited in IAUM and NY. Our initial identification was checked by comparison with descriptions in the floristic literature (Khoshravesh et al., 2009) and confirmed by Robbin C. Moran at NY. The chromosome squash method used followed that of Windham and Yatskievych (2003). Pinnae with whitish sporangia were collected every half hour from 9 am to 1 pm. The pinnae were fixed in Farmer’s Solution (1:3 glacial acetic acid-ethyl alcohol) 20 FERN GAZ. 20(1):19-22. 2015 and Carnoy’s Solution (ethanol, glacial acetic acid and chloroform in the proportion of 6: 1: 3). Large numbers of sporangia were squashed in acetocarmine. Photography were taken with a digital camera Dino-Lite connected to Labomed LX 400 microscope.

RESULTS AND DISCUSSION We found n=84 pairs (bivalents) at meiotic metaphase I (Figure 1). This is the first chromosome count of C. fragilis from Iran. This number agrees with previous counts from Jammu and Kashmir ((Bhellum and Razdan, 2012), Mongolian Altai (Kawakami et al., 2010) in Asia; Finland, (Sorsa, 1961), and Iceland (Löve & Löve, 1943) in Europe; Ontario, British Columbia, and Greenland in Canada (Britton, 1953; Taylor and Mulligan, 1968; Dalgaard, 1989), and Michigan, USA (Wagner, 1955). Each contained 6–19 sporangia. The most important factor in obtaining meiotic figures at the proper stage was the time of fixation. Descriptions and the figures in the present paper refer to fixation at 11am. Tetrad spores were produced by the second meiotic division and many well filled, spiny spores were observed (Figure 2). The size of 25 spores from one leaf was measured in distilled water as followed: 38.48–48.60 × 23.40–28.65 mm. The size straddles that reported for tetraploids and hexaploids within Cystopteris by Blasdell (1963). He found that tetraploids had lengths from 32–42 mm and hexaploids from 38–48mm. It is not known what mounting media Blasdell used to measure his spores—whether distilled water, Hoyer’s solution, or Canada balsam. Use of different mounting media might explain why our results of spore length measurements do not fall exactly within the tetraploid range reported by Blasdell (1963).

ACKNOWLEDGMENTS We thank Dr. R. C. Moran (NY) for protocol advice, comments on the manuscript, and confirmation of the species identification. We also thank Mr. M. Joharchi for the original identification the species and M. Basiri for his assistance in the field.

REFERENCES BHELLUM, B.L. & RAZDAN, B. 2012. Cystopteris fragilis f. kashmiriensis Bhellum and Razdan- A new forma from India. International Journal of Scientific and Research Publication 2(4): 1– 4. BLASDELL, R.F. 1963. A monographic study of the fern genus Cystopteris. Memoirs of the Torrey Botanical Club 21(4): 1–102. BRAITHWAITE, A.F. 1964. A new type of apogamy in ferns. New Phytol. 63 (3): 293– 305. BRECKLE, S.-W. 1987. Distribution and ecology of Cystopteris () Within the Flora Iranica region. Plant Syst Evol. 155: 59–69. BRITTON, D.M. 1953. Chromosome studies on ferns. Am J Bot. 40: 575–583. DALGAARD, V. 1989. Additional chromosome numbers in vascular plants from the Disko Bugt area (west Greenland). Willdenowia 19: 199–213. HAUFLER, C.H., MORAN, R.C., & WINDHAM, M.D. 1995. Cystopteris. Pages 263– 270. In: Flora of North America Editorial Committee, editors. Flora of North America, volume 2. Pteridophytes and . Oxford University Press, New York. HAUFLER, C.H. & WINDHAM, M.D. 1991. New species of North American Cystopteris and Polypodium, with comments on their reticulate relationship. Am Fern J. 81: 7–23. BABAEE & HAJI MONIRI: CYSTOPTERIS FRAGILIS IN IRAN 21 fragilis . Scale bar= 20 μm. B B Explanatory diagram for a, showing 84 bivalents. Scale bars= 20 μm. at meiosis, n = 168; B. Chromosomes of C. fragilis Surface ( A ) and internal B features of spiny spores tetraploid C. A A Figure 2. Figure Figure 1. A. 1. Figure 22 FERN GAZ. 20(1):19-22. 2015

KAWAKAMI, S.M., KAWAKAMI, S., KATO, J., KONDO, K., SMIRNOV, S.V. & DAMDINSUREN, O. 2010. Cytological study of a fern Cystopteris fragilis in Mongolian Altai. Chromosome Botany 5: 1–3. KHOSHRAVESH, R., AKHANI, H. & GREUTER, W. 2009. Ferns and fern allies of Iran. Rostaniha (Botanical Journal of Iran) 10 (1): 1–129. LÖVE, A., LÖVE, D. 1943. The significance of differences in the distribution of diploids and polyploids. Hereditas 29: 145-163. MANTON, I. 1950. Problems of cytology and in the Pteridophyta. Cambridge: University Press. London. 316 Pp. MITUI, K. 1970. Chromosome studies on Japanese ferns. Japanese Journal of Botany 45: 84–90. PALER, M.H. & BARRINGTON, D.S. 1995. The Cystopteris fragilis × C. tenuis (Dryopteridaceae) and the relationship between its tetraploid progenitors. Syst Bot. 20: 528–545. ROTHFELS, C., WINDHAM, M.D. & PRYER, K.M. 2013. A phylogeny of the cosmopolitan fern family Cystopteridaceae (Polypodiopsida). SystBot. 38: 259–306. ROUX, J. P. 2001. Conspectus of southern African Pteridophyta. ABC Press, Epping Ind., Cape Town. 223 Pp. SORSA, V. 1961. Chromosome studies on Finish Pteridophyta II. Hereditas 47: 480– 488. TAYLOR, J.A., & MULLIGAN, G.C. 1968. Flora of the Queen Charlotte islands. Part 2: Cytological aspects of the vascular plants. Canada Department of Agriculture Press, first edition. 659 Pp. WAGNER, W.H. 1955. Cytotaxonomic observations on North American ferns. Rhodora 57: 219–240. WINDHAM, M.D., & YATSKIEVYCH, G. 2003. Chromosome studies of cheilanthoid ferns (Pteridaceae: ) from the western United States and Mexico. Am J of Bot. 90: 1788–1800. FERN GAZ. 20(1):23-45. 2015 23



Alterra, P.O. box 47, 6700AA Wageningen, Netherlands Email: [email protected]

Key words: , economic botany, ethnopteridology, trade history

ABSTRACT In England in the early 19th century at least two products went by the commercial name Dutch Rush, viz. the Rough Horsetail Equisetum hyemale L. used in cabinet making and similar crafts, and the Common Club-rush/Bulrush Schoenoplectus lacustris (L.) Palla used in matting and chair manufacturing. Some authors did not heed the scientific names and confused the properties and geo-cultural backgrounds of both products. Thus the myth took hold that E. hyemale was in culture in the Netherlands and that is was deliberately planted and cared for to protect that country from the sea. Scarce but widespread evidence of trade reveals that this species was economically insignificant. The idea that it owes its common name to imports from Holland could be correct; however, other parts of North and Central Europe, especially the upper Rhine Valley, are more likely to be the original sources from where the Dutch obtained the plants. North America can be reasoned to be an alternative origin, but evidence for this hypothesis is still lacking.

INTRODUCTION Ever since the sixteenth century, many authors in a number of Western European countries have reported the use of Equisetum hyemale L. by various trades (e.g. Fuchs, 1543; Gerarde, 1597; Bock & Sebisch, 1630; Pexenfelder, 1670; Ruppe & Haller, 1745; Anon., 1749; Pernety, 1771; Krünitz, 1785; J. E. Smith, 1802; Headrick, 1813; Stewart, 1815; Phillips, 1818; Hooker, 1821; Gill, 1822). Material evidence of its use is found in inventories of workshops (e.g. Giskes, 1979) and characteristic scratches from its siliceous skin on antique woodcraft (Esterly, 1998). In addition, several authors during the past two centuries showed that the vernacular name “Dutch Rush” used in England relates to large-scale imports from Holland1 (Pratt, 1846; Francis, 1851; Johnson & Sowerby, 1856; Moore, 1861; Pratt, 1866). Up to the present day the statement is repeated frequently (Weeda et al., 1985; Øllgaard & Tind, 1993; Page, 1997). The apparent source to which this can be traced back is Edward Newman (Newman, 1842, 1844), who also noted that the species was cultured in Holland and played an important role in the defence of the coast against the eroding action of the sea: “… for this purpose it is imported, under the name of “Dutch Rush” in large quantities, from Holland, where it is grown on the banks of canals and on the sea ramparts, which are often bound together and consolidated by its strong and matted roots.”

1Wherever the toponym Holland is used, literal citations excepted, it is to be understood in the strict sense, i.e. the western part of the Netherlands, or the present provinces Noord-Holland and Zuid-Holland. 24 FERN GAZ. 20(1):23-45. 2015

Table 1. Vernacular names used in British English

Name Source

Shave-grass (discussion in this paper) Dutch Rush (discussion in this paper) Rough horsetail From the French/Latin asprella (de Winter, 2012), introduction unknown Dutch shave-grass species list of Scarborough (Hinderwell, 1811) Holland rushes In advertisements (Glover, 1843; Measom, 1861) Dutch reed rarely (Bailey, 1756; Martin, 1813; Henslow, 1839; Trimmer, 1842) polishing rushes customs’ duties lists (Burn et al., 1831; Parnell, 1831; Ellis, 1837) shave weed (Aubrey, 1848) shauynge gyrs (Turner, 1538) dyßhewaßhynges (Turner, 1538) Pewterwort e.g. (Withering, 1776; Targioni Tozzetti, 1813; Wilkinson, 1858) scouring[-]rush American (Law Olmstedt et al., 1924)

This is at odds with the ecological preferences of the species, as well as with both its historical and present day distribution (de Winter, 2007). These problems can be summarised under three main points: 1. the alleged abundance of E. hyemale in Holland finds no evidence in its current distribution in that country, nor is it supported in historical Dutch reports; 2. the reported occurrence and even cultivation of E. hyemale along canals and the practice of using it for the solidification of dykes, dunes, or any kind of coastal protection constructions finds no reference in Dutch literature, nor in the present distribution and habitat preferences of European E. hyemale; 3. the plant detailed by Newman by its size and number of ridges suggests American E. hyemale subsp. affine (Engelm.) Calder & Roy L.Taylor rather than European E. hyemale subsp. hyemale, which casts doubt on the continental origin of the merchandise. If these doubts are well-founded, then the question must be addressed as to whether E. hyemale really was imported from Holland to England, and where the origin should be sought of the plants sold on London markets.

HISTORY OF THE NAME “DUTCH RUSH” A number of vernacular names have been used for E. hyemale in English, but most of them never became popular and have disappeared into oblivion (Table 1). The oldest historic one is “shave-grass”, which was first recorded in the 14th century: “Cauda equina, cauda Caballina idem est. angl, schauegres” (Anon., 1350-1400; Lancaster, 1887; Murray, 1971). To shave is derived from the Anglo-Saxon scafan through the Old English shaven, and Middle English schaven/schafen (Flexner & Hauck, 1993), whence there is a direct relation with the Old High German scafthawi (Graff, 1838) and the Dutch stem schaaf - as in schaafstro (de Winter, 2012). Although the verb “to shave” at present is predominantly used to refer to the process of removing the beard, the general meaning is to make a surface smooth (Webster, 1913). The name is continuous through Turner (1538) and Gerard (1597). “Shave-grass” has been largely replaced now by the more official “rough horse-tail” (Bolton, 1790; J. E. Smith, 1802), and “Dutch Rush”. DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 25

The latter name has been in use since around 1700 and first appears in manuals of practical trades (C.K., 1701; Artlove, 1730; Barrow, 1735; Anon., 1754; Dossie, 1758). Eighteenth century dictionaries show a more conservative attitude towards this emerging name and strictly print “shave-grass”. The name “Dutch Rush” therefore remains dormant in written language until the second edition of the cryptogamic volume of William Withering’s Botanical Arrangement was published (Withering, 1792), in which the name “Dutch Rush” is used equivalent to “Shave- grass”. The Botanical Arrangement was an immediate and sustained success that would see many reprints (Lee, 2001). Inclusion in this work apparently made the name “official” and acceptable for use in subsequent publications (Figure 1). This pattern strongly suggests that since the onset of the eighteenth century something has changed that made people name the product by its origin rather than its purpose. Just assuming that the increasing trade with the low countries flooded the market with Dutch horsetails seems unsatisfactory as an explanation. A size and/or quality difference as suggested by Newman might have changed the perception and initiated the desire to distinguish the new product by name.

IMPORTS OF HORSETAILS INTO BRITAIN Even though E. hyemale is not especially rare in Britain (Wardlaw & Leonard, 2005), it was certainly felt so (Camden, 1722; James, 1745). Since urbanisation and industrialization in England started long before the invention and acceptance of sand paper in the mid 19th century, it is conceivable that here, more than elsewhere in Europe, the discrepancy was felt between urban demand and rural supply. Especially the reconstruction of London after the fire (1666) may have elevated the demand. The earliest account of exports from the Netherlands are found in a manuscript of the late 17th century by John Aubrey (Aubrey, 1847): “Watchmakers and fine workers in brasse use it after smooth filing. They have it from Holland”. This can be dated more exactly, for in a letter to John Ray of August 5, 1691 he writes: “Shave-weed used by artists (which they have from Holland)” (Aubrey, 1848).

Figure 1. Relative usage of the names “Dutch Rush” (darker bars) and “Shave-grass” (lighter bars) in English literature from 1720-1860. Counts made per 20 years periods, based on 178 printed publications, not including advertising. 26 FERN GAZ. 20(1):23-45. 2015

The demand declined when glass paper came into use, but it never completely disappeared. In the USA it was still advertised in 1938 (Kaliban’s Grocery and Market, 1938). Nowadays some still prefer it to scrape their clarinet reeds (Intravaia & Resnick, 1965; Taillard & Dalmont, 2012). Testimony of imports into England, which lasted more than one and a half centuries, exists in lists of import duties of the English customs (Burn et al., 1831; Parnell, 1831; Ellis, 1837), but is absent in earlier legislation (e.g. Steel, 1796). Often the horsetails could be imported, transported, and exported tax-free (de Martens & Murhard, 1836; Anon., 1837; MacGregor, 1843; Anon., 1849a, 1853, 1858) or they were not considered important enough to justify their own category (Koninkrijk der Nederlanden, 1816, 1822). As a consequence, no accounts of their trade were kept and they do not show in yearly statistical surveys (Departement van Financiën, 1848). England and Holland were the two countries with the worst kept statistics on agriculture and trade (MacGregor, 1843). Yet, although far from complete, a few statistics of the early period remain, covering the imports into the port of London and allowing sampling of the existing data of imports of E. hyemale. However, in all of them the horsetail trade is conspicuous by its absence, viz. in January 1683 (Houghton, 1728), from May to October 1735 (Anon., 1735), and from January to June, 1776 (Whitworth, 1777b, 1777a). In the nineteenth century Dutch newspapers meticulously reported freight loaded and unloaded per port, frequently even per ship. Any product of trade of any importance would be expected to be listed. Nonetheless horsetails are not mentioned by any of their known names. With one exception, however: in the second half of 1855, 142 inland navigation barges with in total 6050 bundles of shave-grass arrived in Brussels (Anon., 1856). Estimating the total volume of Equisetum transported requires acquaintance with the size of such a bundle, which we do not know. A rough estimate for a bundle of 80 cm perimeter (as is used at present for bulrushes: de Vries, 2008) would be 500 – 1000 stems. Given that dense stands have 200 – 500 stems per m² (Rutz & Farrar, 1984), the annual production for Brussels only would have required the depletion of c. 0.6 – 3 ha. Such small amounts could easily have been found on Belgian territory, but apparently for reasons of quality or cost, long-distance transport per ship was more attractive. In the mid-nineteenth century, exports of reeds and rushes to Britain exceeded those to Belgium by about 50% (Departement van Financiën, 1848) and if the export of horsetail may be estimated proportionally to this, the economic value must have been insignificant. John Yeats, an English commercial-geographer, who lived in Holland in 1845/46, compares E. hyemale, which “is occasionally imported from Holland” with Scirpus lacustris L. (now Schoenoplectus lacustris (L.) Palla), saying that “Many vessels laden with this rush arrive annually in England from Holland and Belgium, bringing thirty or forty tons of rushes each voyage. This is a very large quantity considering the lightness of the material. More than 1,000 tons of bulrushes are annually imported into the United Kingdom” (Yeats, 1870). It should be noted, though, that these figures stem from the second half of the nineteenth century, when the use of E. hyemale was already on the decline in favour of sandpaper, of which mass production in London had started by 1833. But also much earlier, in 1827, international horsetail trade was but marginal. From an example record of the net produce of customs duties it can be deduced that the total value of polishing rushes legally imported into Britain must have been ₤13.25 for that year DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 27

(Parnell, 1830). CULTIVATION AND CARE IN HOLLAND: COAST PROTECTION Newman’s statement, that the Dutch grew E. hyemale “on the sea ramparts, which are often bound together and consolidated by its strong and matted roots” (Newman, 1844), is repeated with slight variations in later literature. The authors obviously have copied each other in sequence, rephrasing the allegation without adding new observations: “it is grown on the banks of canals and on the sea ramparts, which are often bound together and consolidated by its strong and matted roots” (Newman, 1844) “The Dutch are well acquainted with the value of its long and matted roots in restraining the wasting effects of the ocean, which would soon undermine their dykes were it not for the Equisetum hyemale which is planted upon them” (Francis, 1851) “the plant is of immense value in its native country from the extraordinary length and interlaced growth of its root-fibres, which mat together and consolidate the loose and swampy soil in which they grow, and thus form one of the most effectual water-dams of so level a land.” (Wilkinson, 1858) “this species is planted to support embankments, which it does by means of its branching underground stems” (Moore, 1861) Along long stretches of the coast of Belgium and Holland, and the outward coasts of the islands in the North, belts of natural and semi-natural sand dunes protect the country from the sea. The hybrid E. × moorei Newman (E. hyemale × E. ramosissimum Desf.) is found in great numbers at several locations in the dunes of Zuid-Holland and southern Noord-Holland. It was first reported by Du Mortier in 1825 (as E. trachyodon A.Br.) near Beverwijk (Du Mortier, 1869), where it still can be found today. It is locally abundant between Scheveningen and Hook of Holland, where it has been known since the late 18th century (Anon., 1796; van Hall et al., 1832). It is surprising that J.E. Smith (1793) did not note it when visiting the place. Leiden (L) has a number of specimens collected in the West of Holland during the 19th century; however, until 1870, when the first collection was made from the fore-dunes south of Scheveningen, all the collections originate from the interior dunes around Haarlem. In 1797, Jan Kops set out to report on the state of the dunes of Holland. He was an eager botanist, paid special attention to any plants that could be used to control shifting sand and, as an agronomist working for the ministry, was also keen to find plants that could be made profitable in any way (Baert, 1943). If Newman with “the consolidation of sea ramparts” referred to the fixation of shifting sands in the dunes, it is hard to imagine that this would have escaped Kops’s attention. Yet Kops explicitly states that no other means are known to him to fix the dunes than marram (Ammophila arenaria (L.) Link), straw, and reed mats (Kops, 1798; van Eys et al., 1799). Neither earlier (Montin, 1771; Le Francq van Berkheij, 1780; van Geuns, 1789) nor later authors (Spengler, 1891; Vuyck, 1898) of technical treatments about sand fixation mention Equisetum for this purpose, even though Le Francq van Berkheij lists “Paardestaart, Equisetum” among the plants of the fore-dunes. Despite the present abundance of the hybrid in the dunes of southern Holland never, from the 18th century to the present, has its distribution been said to be deliberately promoted by man, and the remarks of Francis and Newman above cannot be attributed to it. Where dunes are weak or absent, dykes have been built to protect the land, as in the topographically complex estuarine area of Zeeland and Zuid-Holland, along the rivers, along the entire coast of the Wadden Sea, and, before 1932, along the Zuiderzee and its inlets. Sea-dykes must be protected by a hard surface since no can form mats firm enough to protect the dyke’s surface from the action of the waves. At the onset of the 28 FERN GAZ. 20(1):23-45. 2015

19th century such hard-shells were not yet customary. Reed and rushes were recognised as effective agents to break the action of the waves and to prevent erosion of the banks of lakes (Meese, 1768), but they grow neither in seawater, nor at the high water limits of rivers. Sea-dykes were protected by stapling bundles of straw, eelgrass and rushes to the dyke-surface to form a soft shell that had to be renewed every autumn (Bréval, 1726; Schraver, 1807). Inland river dykes consist of a core of sand or clay, covered with a water resistant layer of clay and finished with a grass-covered top layer (Fliervoet, 1992). Mats of superficially rooting herbs protect the surface against erosion, but deep-rooting plants weaken the construction (de Haan et al., 2001; 2003) and can perforate the water resistant clay mantle (Technische Adviescommissie voor de Waterkeringen, 1985), without adding much to the stability (Sykora & Liebrand, 1987). High growing dense stands of E. hyemale could outcompete lower herbs that offer better protection. Finally, frequently grazed or mown low vegetation is less than optimal for this woodland species and at present it is not found on dykes, nor are there collections in the National Herbarium from such places. The hybrid E. × moorei has a number of stations along the river Rhine, but it is still rare on dykes. Altogether both technical and historical evidence for Newman’s statement appear to be entirely lacking.

OVER-REPORTING OF DUTCH RUSH DUE TO DUTCH RUSHES The likely source of the delusion is the 18th century bestseller, The Gardeners Dictionary (Miller, 1754, Ed. 4) that depicts how species of rush “grow on the Sea-shores, where they are frequently watered by the Salt-water. These two Sorts2 are planted with great Care on the Banks of the Sea in Holland, in order to prevent the Water from washing away the Earth; which, being very loose, would be in Danger of removing every Tide, if it were not for the Roots of these Rushes; which fasten themselves very deep in the Ground, and mat themselves near the Surface, so as to hold the Earth closely together. Therefore, whenever the Roots of these Rushes are destroyed, the Inhabitants immediately repair them to prevent farther Damage.” This paragraph is quoted from the section on as “Juncus acutus”. Miller must have meant the glaucous bulrush Schoenoplectus tabernaemontani (C.C. Gmelin) Palla that was cultured in great numbers in the brackish water of the Maas estuary, and possibly to some extent S. maritimus (L.) Lye, which was not cultured, but common in the more seaward parts of the estuary and known for its even greater capacity of promoting the sedimentation rate of silt than the former (Clevering & van Gulik, 1990; Weeda et al., 1994). Miller’s handbook on wild and cultivated plants went into eight editions3, and was copied (e.g. MacFarquhar & Gleig, 1797; Knight, 1833), and translated into French, German and Dutch. As Miller’s text was copied over and over during more than a century, gradually elements changed, and new ones slipped in (Table 2). Noteworthy is Loudon’s addition that combines the name Dutch rush, and its usability for scouring metals (Loudon, 1829). Rushes (for making chairs) were imported from the

2i.e. Juncus acutus, capitulis sorghi C.B.P. & J. acutus maritimus Anglicus Park.

3Cited here is the fourth edition of 1754, but essentially the same text is found in the Dutch translation of 1745 (Miller, 1745) and therefore in earlier English editions not seen by the author. DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 29

Table 2. The evolution of Miller’s account on bulrush-culture to Newman’s account of E. hyemale Period Interpretation 1724 The first and second Sorts grow on the Sea Shores, where they are frequently to washed by the Salt Water. These two Sorts are planted with great Care on the 1754 Banks of the Sea in Holland, in order to prevent the Water from washing away the Earth; which, being very loose, would be in Danger of removing every Tide, if it were not for the Roots of these Rushes; which fasten themselves very deep in the Ground, and mat themselves near the Surface, so as to hold the Earth closely together. Therefore, whenever the Roots of these Rushes are destroyed, the Inhabitants immediately repair them to prevent farther Damage. In the Summer-time, when the Rushes are fully grown, the Inhabitants cut them, and tie them up into Bundles, which are dried, and afterward carried into the larger Towns and Cities, where they are wrought into Baskets, and several other useful Things, which are frequently sent into England. These Sorts do not grow so strong in England, as they do on the Maese, and some other Places in Holland, where I have seen them upward of four Feet high (Miller, 1754). 1797 The conglomeratus, and acutus or marine rush, are planted with great care on the banks of the sea in Holland (MacFarquhar & Gleig, 1797) 1829 J. acutus and maritimus are planted on the sea-embankments of Holland, and also in some parts of our own coasts, and in America. The roots run deep into the sand, and form a matted body which holds it together. In Holland, when the plants are fully grown and in flower, they are cut down, dried, and bound up like corn. The J. acutus, being very rough, is used for scouring copper and other vessels, and is one of the plants imported into this country for that purpose, under the name of the Dutch rush. The other species, and often both, are plaited into mats, baskets, chair-bottoms, ropes, etc. (Loudon, 1829) 1833 (…) are made of bulrushes; these grow in this country, naturally but not very commonly, in deep slow streams. The demand for them is greater than the home supply, and a considerable quantity is imported from Holland. (Society for the Diffusion of Useful Knowledge, 1833) 1842 Equisetum hyemale: for this purpose it is imported, under the name of “ Dutch Rush,” in large quantities, from Holland, where it is grown on the banks of canals and on the sea ramparts, which are often bound together and consolidated by its strong and matted roots. Bundles of this imported Dutch Rush are exposed for sale by many London shopkeepers. (Newman, 1842)

Netherlands into England in large quantities (Yeats, 1870). Inevitably, they were sometimes designated as “Dutch rushes”, an adjective combination that never made it to a taxonym. Traders used the name indiscriminately for both E. hyemale and Schoenoplectus. In advertising in the first half of the century, it is rarely made explicit which kind of rush is offered (e.g. “Dutch polishing rushes” or “Dutch bull rushes”). In less than half of such advertisements the context, such as chairmakers’ or cabinet-makers’, revealed the nature 30 FERN GAZ. 20(1):23-45. 2015

Table 3. Advertisements with “Dutch Rushes” or “Holland Rushes” from before 1850.

Advertisements where product defined as: Number

Equisetum 4 Schoenoplectus 8 Unknown 13 Total 25

Sources: Bury and Norwich Post: 29 Nov 1815; Carlisle Journal: 09 Mar 1833, 24 Aug 1833; Carlisle Patriot: 28 Oct 1820; Chelmsford Chronicle: 03 Oct 1783; Chester Chronicle: 23 Mar 1849; Hampshire Advertiser: 15 Sep 1832; Hampshire Chronicle: 26 Jul 1819 2x; Hampshire Telegraph and Sussex Chronicle: 23 Jul 1832; Hampshire Telegraph: 02 Mar 1829, 25 Apr 1831; Hull Packet: 07 Jan 1801, 03 Mar 1801, 27 Feb 1835; Kentish Gazette: 07 Jan 1845; Liverpool Mercury: 13 Jun 1823, 17 May 1844; Norfolk Chronicle: 09 Dec 1815; Salisbury and Winchester Journal: 19 Sep 1803, 26 Feb 1821; Stamford Mercury: 21 Apr 1848; The Ipswich Journal: 22 Jun 1805, 14 Sep 1805; The Times: 08 Sep 1831 of the product offered (Table 3). It must have been this ambiguous use of the name that led Newman to his misunderstanding. Evidently, when writing about E. hyemale, he did not make the distinction and copied unreliable sources without checking.

CULTIVATION AND CARE IN HOLLAND: HARVESTING FOR EXPORT Whereas in English literature it is often stated that Dutch Rushes were cultivated and exported by the Dutch (Newman, 1842, 1844; Francis, 1851; Moore, 1861; Pratt, 1866), the continental literature remains almost taciturn about any culturing activities. The earliest mention of such a trade in Dutch literature is found in the Flora Batava (Kops & Van der Trappen, 1846), published shortly after Newman’s publication in The Phytologist (Newman, 1842). In the Netherlands, E. hyemale is not an abundant species. In the east of the country several healthy populations exist, but it is hard to imagine that these could have supported prolonged harvesting for both domestic and export markets. In Holland in the strict sense, i.e. the western part, E. hyemale is virtually absent, but the hybrid E. × moorei is quite common in the dunes and along railroads (de Winter & Lubienski, 2012). It would be conceivable that present day populations are not representative for the abundance in the 18th and 19th century. Both habitat destruction and harvesting might have caused such a decline. De Gorter (1781) had found the species in forests in the county Zutphen and Kops & Van der Trappen (loc. cit.) say it is abundantly found near Zutphen, at a number of locations in the West, the latter almost certainly to be attributed to the hybrid, and at some riverine locations for many of which the same applies. In the 17th century, Comelin (1683) saw it in stagnant-water ditches near the river Vecht near “den Bergh”4, at the border of the firm, sandy soil of Utrecht and the fen areas of Holland. E. hyemale is a species of moist woodlands. The area of woodland in the Netherlands has not declined since 1800, but has actually increased (Bijlsma, 2003), as before that time much had been cleared. Older sources often associate the species with ditches rather

4presumably Nederhorst den Berg, about 25km SE. of Amsterdam DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 31 than woodland, which may be explained by its habit of persisting long after the trees have been felled. Altogether it seems unlikely that the magnitude of the population would have permitted large scale harvesting to support an export trade over a long time and it must be questioned whether this country has ever been the source of such trade. Comelin (loc. cit.) noted that at den Bergh it was collected for turners to polish with it5. But later, from 1806, a number of annual reports have been compiled about the Dutch agriculture, including products gathered from uncultivated lands, such as reed (Phragmites), rushes (Schoenoplectus) and herbs (Kops, 1807, 1808, 1809, 1816, 1819, 1821, 1822c, 1822b, 1822a, 1829). No mention is made of any production or harvesting of Equisetums. Nozeman (1783) expresses his amazement that E. hyemale is imported into the Netherlands from Spain (see below), rather than collected from local stocks. Subsequent botanists agree: “This plant does grow in some places of our home country, in woods and sandy lands, but it is, as far as I have been able to find out, not used” (van Hall, 1854). “The shave-grass that the cabinet-makers use does not originate from our land, but is imported from elsewhere. From [the Netherlands] it is further transported to England, where it, completely undeserved, is designated by the name Dutch Rushes”(Oudemans, 1862; likewise: de Vries et al., 1870). An interesting phenomenon is that, analogous to the English “Dutch Rush”, in the Netherlands it was sometimes called “Spanish rush” (Noel Chomel & De Chalmot, 1778; Van Meerten-Schilperoort, 1830; 1843; van Hall, 1843). The epithet “Spanish” intermittently used in the Dutch names of the species does not indicate a Spanish origin. Though it is found in Spain (Prada, 1986; Salvo Tierra, 1990), it occurs too infrequently to allow any significant yield (C. Fraser-Jenkins, pers. com.). Also, historically and regionally the predicate has been applied to at least thirty other species (Heukels, 1907), which seem to have little in common, though for quite a few of them it might be interpreted as “conspicuous” or “foreign”. A few authors hold the view that that the Netherlands does or could produce commercial horsetails: “This wood [Zalkerbos] also furnishes very good shave-grasses to polish wood” (van der Aa, 1851), without revealing his source nor the scale of the exploitation. In almanacs beggar boys were urged to collect shave-grass (among other products of nature) to earn their own living (Heldring et al., 1837; Heldring, 1845). This may denote more about the low value and little enthusiasm to collect the stems in the Netherlands. Altogether, the conclusion seems justified that if Dutch-produced horsetails were ever imported into England, this must have taken place longer ago than the memory of the 19th century writers cited above, therefore no later than the first half of the 18th century.

CONTINENTAL TRADE AND ORIGIN OF THE SPECIES Even if the plants have never been cultivated in the Netherlands, nor commercially harvested from the wild, it is still possible that Dutch traders imported them into London from other continental or overseas sources. The question then is where they would have originated.

5Comelin’s habitat description is more typical for E. fluviatile L. with which E. hyemale was often confused, and so was his description: Equisetum foliis nudum, non ramosum sive junceum. B. pin. Hippuris foliis & nuda Tab. Equisetum majus aquaticum prim. icon J. Bauh. Belg. Groot Paarde-staart. However, his observation on collecting the plant shows that he was at least partially correct. 32 FERN GAZ. 20(1):23-45. 2015

There is evidence of horsetail trade elsewhere in Europe. Tariffs of the Zollverein (customs union, 1818-1871) name Schachtelhalm in near complete lists of items transported through the German lands (Anon., 1824; de Martens & Murhard, 1836; Anon., 1849a, 1849b, 1853, 1858). Likewise, levied a small duty on horsetails (MacGregor, 1850), as did , (Arntzen, 1830), France (Dujardin-Sailly, 1813) and Austria (Klenner, 1822; Anon., 1824, 1838), whereas Illyria (Anon., 1836) let them pass for free. A number of more incidental sources can be found too. According to an early 19th century encyclopedia, E. hyemale was a lucrative product and exports from the southern French department Bouches-du-Rhone amounted to 10,000 francs annually (Leman, 1826). The amount in francs suggests the figure applies to statistics postdating 1795, when this currency had been introduced. The 1802 overview of this department, however, makes no mention at all of such an export (Michel, 1802). Moreover, it is stated that the department is very poor in woodland, a condition that has not changed to the present, and this explains the near absence of E. hyemale in those parts (J. le Paslier & C. Déliry, pers. com.). Duval-Jouve also expressed his doubt about the figures he had from de Villeneuve (de Villeneuve, 1821), but after investigating the matter he found that they referred to E. ramosissimum and E. palustre L., of which at least 30,000 bundles were sold each year for kitchen usage (Duval-Jouve, 1864). Local trade in Provence was provided by vendors crying “Leis fretadous de coussooudo”6 (Achard, 1785), who were not likely to be selling the hard skinned E. hyemale, since their merchandise would have ruined the kettles. Abundant sources of E. hyemale were found in the forests along the Rhine, where specialised collectors took it to sell it to carpenters and cabinetmakers, or to plait rings of them (the so-called Schaftheukränze, Figure 2) to clean kitchen utensils and floors (Kirschleger, 1857). These rings, also called Schlutte (Acker, 1982), are identical to the one depicted by Bock (1630) and described by Bauhin: apud nos ancillae circulos (qualis figurae adpositus,) ad suppellectilem expoliendam conficiunt7 (Bauhin, 1658). Long distance commercial trade of E. hyemale for use by craftsmen to polish wood and metal has been reported from south-west Germany, where they were imported into Württemberg in great masses from the Rhine valley in Baden (von Martens & Kemmler, 1882). French collectors apparently drew a heavy toll on the population, since Hausser (1894) complained that “a few decades ago” at Neubreisach the traders took cartloads of E. hyemale, but that at the time of his writing they would have come in vain. Plants collected near Strasbourg (dept. Bas-Rhin) were sold in Nancy, 120 km to the west (Braconnot, 1828; Kirschleger, 1857) and to Paris (Duval-Jouve, 1864). Halfway between Strasbourg and Paris, in the Orient Forest near Troyes, it was available for collecting, but, to the amazement of the Troyans, traders did not know this, or preferred to pull it in other places (des Étangs, 1841). Plumier tells us, presumably from Lyon8, that it is “a plant brought to us from the mountains”. In southern France commercial trade existed where Villeneuve functioned as the hub selling horsetails collected in the Lot-valley to the market in Bordeaux (de Saint-Amans, 1821).

6“scouring pads of horsetails”

7at ours the maids make wreaths (illustrated adjacent) to polish the furniture

8The printer as well as the religious authorities approving the book resided in Lyon. DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 33

Yet one other possible origin should be considered, viz. North America. For this route I have found no historical data, unless it would account for the surprisingly large export volume of “reeds” from the USA (Wolcott, 1795). However, the species is abundant in the USA to the extent of becoming a nuisance (Millhollon, 1987), and the larger American subspecies would explain observations of Newman and contemporaries on the size of the marketed horsetails. Newman expressed uncertainty about the Dutch Rushes of the London markets being conspecific with English E. hyemale, and was supported in that opinion by other, not explicitly named, botanists. His doubts arose from the “much larger size than any British examples of E. hyemale” and “the much greater number of striæ, amounting in some instances to thirty-two” (Newman, 1844). Large size and high number of ridges (Figure 3) better conform to the American E. hyemale subsp. affine which has 14 – 50 ridges than the European subspecies with 14 – 26 ridges (Hauke, 1963). Johnson (Johnson & Sowerby, 1856), however, disagrees, stating that “British plant from Gamlingay Bog, Cambridgeshire, cultivated in my garden for thirty years, frequently rivals the imported “Dutch Rushes” in these respects, the number of ridges and smaller tubes varying from twenty to twenty-eight in the larger stems”. In the Zalkerbos near Zwolle, the only Dutch population which historically has been associated with usage of the plants as smoothing agents, currently has stems of 1.20m tall with 25 – 26 ribs. Although such thick specimens of E. hyemale subsp. hyemale are memorable in itself, there is still a gap between Newman’s 32 ridges, and the Dutch and Johnson’s 26-28 ridges. In the Netherlands, Van Hall (1854) wrote: “This plant does grow in some places of our home country, in woods and sandy lands, but it is, as far as I have been able to find out, not used, and it is by far not as heavy as the one that occurs in commerce and is brought in from elsewhere”. Herman van Hall, a student of Kops, was co-author of earlier volumes of the Flora Batava (Kops & Van Hall, 1828, 1832, 1836, 1844) and therefore likely to be very well aware of the note given there, that E. hyemale is exported to England. His 1854 remark reads as if he has double-checked the fact, while bearing in mind that a different, larger taxon might be involved. Oudemans (1862) agreed: “The reason why, both at ours and in England, the foreign shave-grass is preferred, seems to be the latter’s thicker stems”.

DISCUSSION At the onset of the eighteenth century, the name Dutch Rush for E. hyemale had become so established that it started to show up in print, at first with authors addressing the artificers and amateurs who would actually be using it and, almost a century later, also in the scientific botanical literature. Something must have happened with the product for people to dub it with that new name. Later authors ascribe the name to imports from Holland, but they all might have been deluded by the Schoenoplectusus/Equisetum- misconception, with the possible exception of John Smith (1802), who published in advance of the earliest date so far discovered that shows the entanglement. The best argument that Dutch import does explain the name is given by Aubrey’s 1691 letter. While he writes that the shave-grass comes from Holland, he does not use the name Dutch Rush, and therefore this is not likely to be an ad hoc explanation of that name. As for the scale of the imports during the 150 years between Aubrey and Newman, again we must sift through the mystery caused by the Schoenoplectusus/Equisetum- misconception. All the imports of Dutch Rush, in “great quantities” or similar wording, are likely to be ascribed to bulrushes. In the eighteenth century, the Netherlands counted 34 FERN GAZ. 20(1):23-45. 2015 over 4000 ha of land with culture of bulrushes (Maas, 2000) and this was an important export product. In 1827, 1600 loads of 63 bundles of bulrushes were declared at the British customs, accounting for over 360 times more duty revenues than received on polishing rushes (calculations based on Parnell, 1830). If, in spite of Yeats’s opinion, E. hyemale would have been of any observable economic significance, one would have expected it to have been seen in the exceedingly detailed port statistics, where it is not. Evidently, half a year could pass without any horsetails being legally imported. In accordance with these incidental observations, it is absent from English and Dutch trade statistics. The possibility that significant amounts of rushes have deliberately been kept from import registration is not very likely. Smugglers concentrated on low-volume contraband where the import duty exceeded the intrinsic value. It is not unreasonable to assume that anything that was taxed was also smuggled, but 20% is a low tariff compared with that levied on alcohol and tobacco, so the incentive for smugglers to bring in shave- grass must have been slight, given that at some points in British history, smuggling carried the death penalty (Richard Platt, pers. com.). The conclusion must be that even if Holland has been an important source in the horsetail trade, the trade as a whole served but a niche market. The emerging pattern of several disjunct historic observations is that of trade routes from scattered source regions into the most accessible larger centres of population. For unknown reasons source populations closer at hand were ignored. Like Oudemans, De St.-Amans (loc. cit.) thinks it a matter of quality: “Celle des bords du Lot est belle et plus estimée”. Sustained exploitation sometimes resulted in depletion of local stocks. In the upper Rhine Valley an infrastructure of collectors and transporters had developed who transported E. hyemale overland to France and downstream by river, as suggested by the toll-treaties of German rivers (Anon., 1837, 1853). The Dutch had virtually monopolised navigation on the river Rhine (MacGregor, 1843). When brought all the way down to the sea, goods would have ended up in the port of Rotterdam, ideally situated for further transport to Belgium and England. Since the species was “rather common in whole Europe, even in Russia, and especially in ” (de Chalmot & Chomel, 1792), it may have been that the collectors took the product from wherever it was available in north-west Europe, including the eastern part of the Netherlands and Russia (which then included Poland). Tariffs categorising horsetails are available from many north and central European countries. During the Napoleonic wars around 1800, reciprocal trade embargos caused interruptions in the supply of indispensable commodities. Since the French invasion of the Netherlands in 1794, bulrushes from Holland had become difficult to obtain (Anon., 1819) and consequently become expensive: “during the non-commercial intercourse between Holland and Great Britain, which lasted a number of years, a great scarcity existed in (…) Dutch Rushes, which were not to be had during the war, except in very precarious chance lots, occasionally smuggled over, at any price” (E. Smith, 1841). The same will have happened to E. hyemale from the and it may be hypothesised that in circumvention a new supply route from the United States was opened. The aberrant size of the plants reported by nineteenth century authors indicates the North American subspecies rather than the slenderer European form. If the shipping cost had allowed a profitable transport, the availability would have been virtually unlimited from the largely uncultivated American lands where the species is abundant. However, doubt exists whether the economic opportunities were recognised: “The rushes used for rubbing down are a valuable article, and grow abundantly in various parts of the United States, DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 35 they are found in our own neighbourhood in the State of New Jersey; though extensively used in Europe they are scarcely known to our workmen” (Gill, 1828). REFERENCES ACHARD, C. F. 1785. Dictionnaire de la Provence et du Comté-Venaissin. 2 vols. Vol. 2, Marseille; Jean Mossy. ACKER, A. 1982. Prêles, Encyclopédie de l’Alsace, 6146. Editions Publitotal, Strasbourg. ANONYMOUS. 1350-1400. MS. Arch. Selden. B. 35, Bodleian, Oxford. ANONYMOUS. 1735. Goods imported and exported to and from the port of London, from the 23rd Day of May, to the 16th Day of October inclusive, 1735. The Political State of Great Britain 50: 450-456. ANONYMOUS. 1749. Arts Companion, Or A New Assistant for the Ingenious. London, Dublin; I. Jackson. ANONYMOUS. 1754. The British legacy: or, fountain of knowledge. . London; Thomas Chandler. ANONYMOUS. 1796. Kannenkraut. In: HÖPFNER, L. J. F. [ed.], Deutsche Encyclopädie oder Allgemeines Real-Wörterbuch aller Künste und Wissenschaften von einer Gesellschaft Gelehrten, 97-102. Varrentrapp und Wenner, Frankfurt am Mayn. ANONYMOUS. 1819. Transactions of the Society, instituted at London for the Encouragement of Arts, Manufactures, and Commerce; with Ihe premiums offered in the year 1817. The Literary Panorama and National Register 8 (New Series): 754-764. ANONYMOUS. 1824. Sr. K.k. Majestät Franz des Ersten Politische Gesetze und Verordnungen für die Oesterreichischen, Böhmischen und Galizischen Erbländer. Vol. 50, Wien; K.K. Hof- und Staats-Druckerey. ANONYMOUS. 1836. Sammlung der politischen Gesetze und Verordnungen für das Laibacher Gouvernement-Gebiet im Königreiche Illyrien. Vol. 18, Laibach (Lubljana); Eger’schen Gubernial-Buchdruckerei. ANONYMOUS. 1837. Tarif de Douanes concerté entre la Prusse, I’Electorat de Hesse et le Grandduché de Hesse d’une part, et la Bavière et le Wurtemberg de l’autre part. En date du 31. Oct. 1833. In: DE MARTENS, G. F. & MURHARD, F. [eds.], Nouveau recueil de traités d’Alliance, de Paix, de Trève, de Neutralité, de Commerce, de Limites, d’Echange etc, et de plusieurs autres actes servant à la connoisancce des relations étrangères des Puissances et d’Etats de l’Europe., 736-777. Librairie de Dieterich, Göttingen. ANONYMOUS. 1838. Zoll-Tariff für die Ein- und Ausfuhr der Waaren in dem österreichischen Kaiserstaate. Wien; der kaiserliche und königliche Hof- und Staats-Arearial-Druckerey. ANONYMOUS. 1843. Verhandeling der Stuco, of zoogenaamd Kunstmarmer. Bouwkundig magazijn of schetsen voor handwerkslieden 1: 11-12. ANONYMOUS. 1849a. Traité entre l’Autriche, la Prusse, la Saxe, le Hannovre, le Danemarc, le Mecklenbourg-Schwerin , les duchés d’Anhalt et les villes libres anséatiques de Hambourg et de Lübeck pour regulariser les droits de douanes hanovriennes payables à Brunshaus (ci-devant à Stade) Sur l’Elbe. Conclu et signe ä Drèsde, le 13 Avril 1844. In: DE MARTENS, M. & MURCHARD, F. [eds.], Nouveau recueil général de traités: conventions et autres transactions remarquables, servant à la Connaissance des Relations étrangères des Puissances 36 FERN GAZ. 20(1):23-45. 2015

et États dans leurs Rapports mutuels, 473-525. Libraire de Dieterich, Göttingen. ANONYMOUS. 1849b. Acte additionel au traité du 23 Juin 1821 sur la navigation de l’Elbe, signé à Drèsde le 13 Avril 1844, entre l’Autriche, la Prusse, la Saxe-royale, le Hanovre, le Danemarc, le Grand-duché de Mecklenbourg-Schwerin, les Duchés d’Anhalt-Coethen, d’Anhalt-Dessau et d’Anhalt-Bernbourg et les villes libres anséatiques de Hambourg et de Lübeck. (Gesetzsamml. für die Königl. Preussischen Staaten. Jahrg. 1844. Nro 37 v. 1. Novemb.). In: DE MARTENS, G. F. & MURCHARD, F. [eds.], Nouveau recueil général de traités: conventions et autres transactions remarquables, servant à la Connaissance des Relations étrangères des Puissances et États dans leurs Rapports mutuels, 386-462. Libraire de Dieterich, Göttingen. ANONYMOUS. 1853. Traité de commerce et de douanes entre l’Autriche et la Prusse, signé à Berlin, le 9 Février 1853. In: DE MARTENS, C. & DE CUSSY, F. [eds.], Recueil manuel et pratique de traités, conventions et autres actes diplomatiques, 134-173. F.A. Brockhaus, Leipzig. ANONYMOUS. 1856. Binnenlandsche Berigten. Nederlandsche staatscourant, 28-03-1856. ANONYMOUS. 1858. Traité de commerce et de douanes entre la Prusse et l’Autriche) signé à Berlin, le 19 fébrier 1853. In: MARTENS, G. F. & SAMWER, C. [eds.], Nouveau recueil général de traités: conventions et autres transactions remarquables, servant à la Connaissance des Relations étrangères des Puissances et Etats dans leurs Rapports mutuels, 382-429. Libraire de Dieterich, Göttingen. ARNTZEN, K. A. 1830. Love, Anordninger, Kundgjørelser, aabne Breve, Resolutioner, m.m., der vedkomme Kongeriget Norges Lovgivning og offentlige Bestyrelse, for aaret 1829. Christiania; Chr. Grøndahl. ARTLOVE, M. 1730. The art of japanning, Varnishing, Pollishing, and Gilding. Being a collection of very plain directions and receipts. Written for the Use of those who have a mind to follow those diverting and useful Amusements, and Published at the Request of several Ladies of Distinction. London; T. Warner. AUBREY, J. 1847. The Natural History of Wiltshire. Wiltshire Topographical Society. AUBREY, J. 1848. Letter to Mr. Ray. In: LANKESTER, E. [ed.], The Correspondence of John Ray, 237-238. The Ray Society, London. BAERT, J. 1943. Jan Kops, pionier van Hollands landbouw. ‘s Gravenhage; MArtinus Nijhoff. BAILEY, N. 1756. Universal Etymological English Dictionary. 4th ed. Vol. 2, London; T. Waller. BARROW, J. 1735. Dictionarium polygraphicum: or, the Whole Body of Arts Regularly Digested. Vol. II, London; C. Hithch & C. Davis. BAUHIN, G. 1658. Caspari Bauhini theatri botanici. Vol. 1, Basel; Joannes König. BIJLSMA, R. J. 2003. Bosrelicten op de Veluwe. Een historisch-ecologische beschrijving. Alterra, Wageningen, report no. 647. BOCK, H. & SEBISCH, M. 1630. Kräutterbuch Weylandt des Weitberhüm[m]ten vndt Hocherfharnen Herren Hieronymi Tragi genant Bock. Auff Vielfaltiges begeren vnd freundtliches ersuchen Mit vleiss übersehen, vnd mit der Kräutter zunamen, der Namen ursachen, qualitete[n], deren gradibus, gebrauch in den Apotecken, vnd sehr vielen in[n]erlichen vnd eusserlichen experimentis gemheret und gebessert. Durch Melchiorem Sebizium, der Artzeney Doctorem, vnd Professorem, auch der Statt Strassburg bestellten Physicum. Sampt Angehenckten Speisskammer ermelten DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 37

herrn H. Bocken, darin[n] aber für dissmhal nichts geändert worde[n]. 4th ed. Strassburg; Wilhelm Christian Glasern. BOLTON, J. 1790. Filices Britannicae. 2 vols. Vol. 2, London. BRACONNOT, H. 1828. Recherches chimiques sur la nature des Prêles. Annales de chimie et de physique 39: 5-24. BRÉVAL, J. D. 1726. Remarks on several parts of Europe: relating chiefly to the History, Antiquities and Geography, of those countires through which the author has traveled : as France, the Low Countries, Lorrain, Alsacia, Germany, Savoy, Tyrol, Switzerland, and Spain. Vol. 1, London; Bernard Lintot. BURN, R., CHITTY, J. & CHITTY, T. 1831. The Justice of the Peace and Parish Officer. 6 vols. Vol. 2, London; S. Sweet & A. Maxwell. C.K., 1701. Art’s master-piece: or, A companion for the ingenious of either sex. In two parts. London; G. Gonyers. CAMDEN, W. 1722. Britannia: Or a Chorographical Description of Great Britain and Ireland (etc.) Written in Latin and Translated Into English. 2nd ed. Vol. 1, London; Awnsham Churchhill. CLEVERING, O. A. & VAN GULIK, W. J. M. 1990. De aanleg van biezenbegroeiingen: mogelijkheden voor her-introduktie van biezen in het Haringvliet - Hollandsch Diepgebied. Oostvoorne; Instituut voor Oecologisch Onderzoek. COMELIN, J. 1683. Catalogus plantarum indigenarum Hollandiae, cui praemissa Lamb. Bidloo dissertatio de re herbaria. Amsterdam; Henr. & Viduam Theod. Boom. DE CHALMOT, J. A. & CHOMEL, N. 1792. Vervolg op M. Noël Chomel Algemeen huishoudelyk-, natuur-, zedekundig- en konst-woordenboek. Vol. agtste deel, zijnde het XV. deel van het woordenboek, Campen; J.A. de Chalmot. DE GORTER, D. 1781. Flora VII provinciarum Belgii foederati indigena. Haarlem; Bohn & Filius. DE HAAN, W. A., VAN DE PAVERD, M., VAN RIJNSBERGEN, J., LEENDERS, P. H. J. A., WORMGOOR, J. A. & VAN GELDER, P. H. A. J. M. 2001. Dijken als onderdeel van een systeem van bescherming tegen overstromingen. Delft; Delft University. DE MARTENS, G. F. & MURHARD, F. 1836. Publication officielle du Tarif de Douanes concerté entre la Hesse électorale d’une part et entre la Prusse et la Hesse grandducale de l’autre part, en exécution du Traité de douanes et de commerce, conclu le 25 Août 1831 entre les dits Etats. En date du 30 Décembre 1831. (Kurhess. Gesetz-Sammlung 1831. Dezemb. Nr.XXII.), Supplément au Recueil de principaux traités d’Alliance, de Paix, de Trêve, de Neutralité, de Commerce, de Limites, d’Echange etc conclus par les puissance de l’Europe tant entre elles., 495-521. Librairie de Dieterich, Göttingen. DE SAINT-AMANS, J. F. B. 1821. Flore Agenaise ou Description Methodique Des Plantes observées dans le département de Lot-et-Garonne et dans quelques parties des départements voisins. Agen; Prosper Noubel. DE VILLENEUVE, C. 1821. Statistique du département des Bouches-du-Rhône: avec Atlas Vol. 1, Marseille; Antoine Ricard. DE VRIES, H. 2008. De laatste biezentelers. Reformatorisch Dagblad, 10-10-2008. DE VRIES, H., VAN DER SANDE LACOSTE, C. M. & SURINGAR, W. F. R. 1870. Flora, Algemeene statistiek van Nederland: beschrijving van den maatschappelijken 38 FERN GAZ. 20(1):23-45. 2015

toestand van het Nederlandsche volk in het midden der negentiende eeuw, 172-241. Vereeniging voor de Statistiek in Nederland / A.W. Sijthoff, Leiden. DE WINTER, W. P. 2007. What’s Dutch about the Dutch rush? The Pteridologist 4: 195-197. DE WINTER, W. P. 2012. In Nomine Equiseti. Varen-Varia 25: 4-7. DE WINTER, W. P. & LUBIENSKI, M. 2012. Equisetum ×ascendens Lubienski & Bennert: de eerste triploïde paardestaartbastaard in Nederland. Gorteria 36: 1-17. DEPARTEMENT VAN FINANCIËN. 1848. Statistiek vand den handel en de scheepvaart van het Koningrijk der Nederlanden over het jaar 1846. ‘s-Gravenhage; Departement van Financiën. DES ÉTANGS, S., ed. 1841. Notes sur quelques plantes observées dans le département de l’Aube.Mémoires de la Société no. 77-78. Troyes; Société académique d’agriculture, des sciences, arts et belles-lettres du département de l’Aube. DOSSIE, R. 1758. The handmaid to the arts. London; J. Nourse. DU MORTIER, B. 1869. Bouquet du littoral belge. Bulletin de la Societe de Botanique de Belgique 7: 318-371. DUJARDIN-SAILLY, E. 1813. Tarif chronologique des douanes de l’empire française: avec des explications. 8th ed. Paris; Dujardin-Sailly. DUVAL-JOUVE, J. 1864. Histoire Naturelle des Equisetum de France. Paris; J.B. Baillière et Fils, Libraires de ‘Académie Impériale de Médicine. ELLIS, R. 1837. The Laws and practical Regulations forming the water-side and general Practice of the Customs. The Laws and practical Regulations of the Customs. 4 vols. Vol. 3, London; O.H. Bailey & Co. ESTERLY, D. 1998. Grinling Gibbons and the Art of Carving. New York; Harry Abrahams Inc. FLEXNER, S. B. & HAUCK, L. C. 1993. Random House unabridged dictionary. 2nd ed. New York; Random House. FLIERVOET, L. M. 1992. Aanleg en beheer van grasland op rivierdijken. Den Haag; Unie van Waterschappen etc. FRANCIS, G. W. 1851. An Analysis of the British Ferns and their Allies. London; Simpkin, Marshall & Co. FUCHS, L. 1543. Den nieuwen Herbarius, dat is dat boeck van den cruyden, int welcke met groote neersticheyt beschreven is niet alleen die gantse historie, dat is, die namen, tfaetsoen, natuere, cracht ende operatie van meesten deel de cruyden, die hier ende in anderlanden wassende sijn, midtsgaders der plecken oft stede, ende den tijt waer ende wanneer sij wassen, maer oock allede wortelen, , bladeren, bloemen, saet, vruchten, ende insumma dat gantse wesen ende gelegenheyt van den selven, is hierinne so constelijcken ende natuerlicken na dleven gefigureert ende geconterfeyt, dat desghelijcks voormaels noyt ghesien en heeft gheweest, noch oyt inde printe ghecomen en is. Basel; Michel Insingrin. GERARDE, J. 1597. The Herball or Generall Historie of Plantes. London; John Norton. GILL, T. 1822. The Technical repository. Vol. II, London; T. Cadell. GILL, T. 1828. Gill’s technological repository. 6 vols. Vol. 2, London; Thomas Hurst, Edward Chance & Company. GISKES, J. H. 1979. Jaarboeken van het genootschap Amstelodanum 71: De vioolmakersfamilies Boumeester en Menslage te Amsterdam. Debussy Ellerman Harms bv., Amsterdam. DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 39

GLOVER, S. 1843. The history and directory of the borough of Derby, a guide. Derby; S. Clover & Son. GRAFF, E. G. 1838. Althochdeutscher Sprachschatz: oder Wörterbuch der althodeutschen Sprach. Vol. 4, Berlin; Beim Verfasser und in Commission der Nikolaischen Buchhandlung. HAUKE, R. A. 1963. A taxonomic review of the genus Equisetum subgenus Hippochaete. Beiheft Nova Hedwigia B: 1 - 123. HAUSSER, E. 1894. Auf welche Weise fördern wir die Kenntnis unserer einheimischer Flora? Mitteilungen der Philomathischen Gesellschaft in Elsaß-Lothringen 2: 19-25. HEADRICK, J. 1813. General View of the Agriculture of the County of Angus, or Forfarshire; With Observations on the Means of Its Improvement. Edinburgh; Board of Agriculture, Great Britain. HELDRING, O. G. 1845. Eene vergelijking tusschen Geldersche en Noord hollandsche zeden, Geldersche volksalmanak, 71-101. G. van Eldik Thieme, Arnhem. HELDRING, O. G., VAN ELDIK THIEME, G. & NIJHOFF , I. A. 1837. Gedachten van Volksvrienden, Geldersche volksalmanak, (s.n.). C.A. Thieme, Arnhem. HENSLOW, J. S. 1839. Description and Physiological Botany. new edition ed. London; Longman &c. HEUKELS, H. 1907. Woordenboek der Nederlandsche volksnamen van planten: uit de gegevens verzameld door de Commissie voor Nederlandsche plantennamen. ‘s-Gravenhage; Versluys. HINDERWELL, T. 1811. The history and antiquities of Scarborough and the vicinity. 2nd ed. (1st ed. 1798) ed. York; Longman et al. HOOKER, W. J. 1821. Flora scotica, or, A description of Scottish plants. London; Archibald Constable etc. HOUGHTON, J. 1728. A collection for the improvement of husbandry and trade: Consisting of many valuable materials relating to corn, cattle, coals, hops, wool, &c. With a compleat catalogue of the several sorts of earths, ... With many other useful particulars, communicated by several eminent members of the Royal Society, to the collector, John Houghton, F.R.S. Now revised, corrected, and published, with a preface and useful indexes, by Richard Bradley, ... In three volumes. London; Woodman and Lyon. HULTÉN, E. 1962. The circumpolar plants. 1, Vascular cryptogams, , . . Handlingar / Kungl. Svenska vetenskapsakademiens (Fjaerde series). Vol. 4:8:5, Stockholm; Almqvist & Wiksell. INTRAVAIA, L. J. & RESNICK, R. S. 1965. A Research Study of a Technique for Adjusting Clarinet Reeds. Journal of Research in Music Education 16: 45-58. JAMES, R. J. 1745. A Medicinal Dictionary, Including Physic, Surgery, Anatomy, Chymistry, and Botany, in All Their Branches Relative to Medicine; Together with a History of Drugs, an Account of Their Various Preparations, Combinations, and Uses; and an Introductory Preface, Tracing the Progress of Physic and Explaining the Theories Which Have Principally Prevail’d in All Ages of the World. 3 vols. Vol. 2, London; T. Osborne. JOHNSON, C. & SOWERBY, J. E. 1856. The Fern Allies: a supplement to the Ferns of Great Britain. London; J.E. Sowerby. KALIBAN’S GROCERY AND MARKET. 1938. Advertisement of Kaliban’s Grocery and Market. The mount Vernon Hawkeye-Record and the Lisbon Herald, 24 Nov. 1938. 40 FERN GAZ. 20(1):23-45. 2015

KIRSCHLEGER, F. 1857. Flore d’Alsace. 3 vols. Vol. 2, Strasbourg, Paris; Victor Masson etc. KLENNER, F. W. 1822. Allgemeiner Zoll-Tariff für den österreichischen Kaiserstaat. 147 vols Wien; der k.k. Hof- und Staats-Arearial-Druckerey. KNIGHT, C. 1833. Vegetable Substances: materials of manufacturers. The Library of Entertaining Knowledge. London; Society for the Diffusion of Useful Knowlledge. KONINKRIJK DER NEDERLANDEN. 1816. Wet van den derden October 1816 over de heffing der regten van in- en uitvoer: benevens het tarief derzelve regten voor het Koningrijk der Nederlanden. P. den Hengst & Zoon, ‘s-Gravenhage & Amsterdam. KONINKRIJK DER NEDERLANDEN. 1822. Wet van den 26. Augustus 1822, houdende het Tarief van regten op den In-, Uit- en Doorvoer van alle goederen, waren en koopmanschappen. Staats-Secretarij, ‘s-Gravenhage. KOPS, J. 1798. Algemeen rapport der Commissie van superintendentie over het onderzoek der duinen; vol.1: Tegenwoordige staat der duinen van het voormaalig gewest Holland. 2 vols. Vol. 1, Leyden; Herdingh en Du Mortier. KOPS, J., ed. 1807. Staat van den landbouw in het Koninkrijk van Holland gedurende den jare 1806. Haarlem; Loosjes. KOPS, J., ed. 1808. Staat van den landbouw in het Koninkrijk van Holland gedurende den jare 1807. Haarlem; Loosjes. KOPS, J., ed. 1809. Staat van den landbouw in het Koninkrijk van Holland gedurende den jare 1808. Haarlem; Loosjes. KOPS, J., ed. 1816. Staat van den landbouw in de Vereenigde Nederlanden gedurende den jare 1814. Den Haag; Algemeende ‘s Lands Drukkerij. KOPS, J., ed. 1819. Staat van den landbouw in het Koninkrijk der Nederlanden, (Noordelijke provincien) gedurende het jaar 1815. Brussel; Stapleaux. KOPS, J., ed. 1821. Staat van den landbouw in het Koninkrijk der Nederlanden, (Noordelijke provincien) gedurende het jaar 1818. Brussel; Stapleaux. KOPS, J., ed. 1822a. Staat van den landbouw in het Koninkrijk der Nederlanden, (Noordelijke provincien) gedurende het jaar 1821. Brussel; Stapleaux. KOPS, J., ed. 1822b. Staat van den landbouw in het Koninkrijk der Nederlanden, (Noordelijke provincien) gedurende het jaar 1820. Brussel; Stapleaux. KOPS, J., ed. 1822c. Staat van den landbouw in het Koninkrijk der Nederlanden, (Noordelijke provincien) gedurende het jaar 1819. Brussel; Stapleaux. KOPS, J., ed. 1829. Staat van den landbouw in de Vereenigde Nederlanden gedurende het jaar 1828. ‘s Gravenhage; Algemeende Lands Drukkerij. KOPS, J. & VAN HALL, H. C. 1828. Flora Batava of Afbeelding en Beschrijving van Nederlandsche Gewassen. Vol. 5, Amsterdam; J.C. Sepp en Zoon. KOPS, J. & VAN HALL, H. C. 1832. Flora Batava of Afbeelding en Beschrijving van Nederlandsche Gewassen. Vol. 6, Amsterdam; J.C. Sepp en Zoon. KOPS, J. & VAN HALL, H. C. 1836. Flora Batava of Afbeelding en Beschrijving van Nederlandsche Gewassen. Vol. 7, Amsterdam; J.C. Sepp en Zoon. KOPS, J. & VAN HALL, H. C. 1844. Flora Batava of Afbeelding en Beschrijving van Nederlandsche Gewassen. Vol. 8, Amsterdam; J.C. Sepp en Zoon. KOPS, J. & VAN DER TRAPPEN, J. E. 1846. Flora Batava of Afbeelding en Beschrijving van Nederlandsche Gewassen. Vol. IX, Amsterdam; J.C. Sepp en Zoon. KRÜNITZ, J. G. 1785. Kannen-kraut, Kandelkraut, Kandelwisch. In: KRÜNITZ, J. G., FLOERKEN, F. J., FLÖRKE, H. G., KORTH, J. W. D., HOFFMANN, C. O. & DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 41

KOSSARSKI, L. [eds.], Oekonomisch-technologische Encyklopädie, oder allgemeines System der Stats- Stadt- Haus- und Land-Wirthschaft, und der Kunst-Geschichte, in alphabetischer Ordnung, 146-183. Joachim Pauli, Berlin. LANCASTER, J. 1887. Alphita, a medico-botanical glossary from the Bodleian manuscript, Selden B. 35. Oxford; Clarendon Press. LAW OLMSTEDT, F., COVILLE, F. V. & KELSEY, H. P. 1924. Standardized plant names : a catalogue of approved scientific and common names of plants in American commerce. Salem; American Joint Committee on Horticultural Nomenclature. LE FRANCQ VAN BERKHEIJ, J. 1780. Antwoord op de vraage, voorgesteld door de Hollandsche Maatschappye der Weetenschappen, te Haarlem: Welke Boomen, Heesters en Planten zyn ‘er, behalve den Helm ( Arundo Arenaria) en den Sleedoorn (Prunus Sylvestris), dewelke op de Zandduinen ter weeringe der Zandverstuivinge kunnen geplant worden ? Kan men ook eenige andere Planten aan onze Zee-stranden met voordeel gebruiken ? Zyn daar mede hier te Lande al eenige proeven gedaan ? En welke is de uitkomst daar van geweest ? Verhandelingen uitgegeeven door de Hollandse Maatschappye der Weetenschappen te Haarlem. XIX: 1-80. LEE, M. R. 2001. William Withering (1741-1799): A Birmingham Lunatic. Proceedings of the Royal College of Physicians of Edinburgh 31: 77-83. LEMAN, M. 1826. Prêle. In: CUVIER, F. [ed.], Dictionnaire des sciences naturelles. F.G. Levrault, Strasbourg, Paris. LOUDON, J. C. ed. 1829. An encyclopædia of plants: comprising the description, specific character, culture, history, application in the arts, and every other desirable particular respecting all the plants indigenous, cultivated in, or introduced to Britain, combining all the advantages of a Linnean and Jussieuean species plantarum, an historia plantarum, a grammar of botany, and a dictionary of botany and vegetable culture. The whole in English; with the synonymes of the commoner plants in the different European and other languages. London; Longman, Rees, Orme, Brown & Green. MAAS, G. J. 2000. Historische geomorfologie Maas en benedenrivieren : Oude Maas, Merwede-Hollandse Biesbosch, Afgedamde Maas en Maaskant. Alterra-rapport;075. Wageningen; Alterra, Research Instituut voor de Groene Ruimte. MACFARQUHAR, C. & GLEIG, G. 1797. Encyclopædia Britannica: Or, A Dictionary of Arts, Sciences, and Miscellaneous Literature. Edinburgh; A. Bell & C. Macfarquhar. MACGREGOR, J. 1843. Commercial statistics: A digest of the productive resources, commercial legislation, customs tariffs, of all nations. Including all British commercial treaties with foreign states. 1st. ed. 2 vols. Vol. 1, London; Whittaker & Co. MACGREGOR, J. 1850. Commercial statistics: A digest of the productive resources, commercial legislation, customs tariffs, of all nations. Including all British commercial treaties with foreign states. 5 vols. Vol. 2, London; Whittaker & Co. MARTIN, T. 1813. The circle of the mechanical arts; containing practical Treatises on the various manual Arts, Trades, and Manufactures. London; Richard Rees. MEASOM, G. S. 1861. Official Illustrated Guide to the Great Northern, Manchester, Sheffield, and Lincolnshire, and Midland Railways. London; Griffin Bohn. 42 FERN GAZ. 20(1):23-45. 2015

MEESE, D. 1768. Antwoord op de vraag, door de Hollandsche Maatschappy der Weetenschappen te Haarlem, in den jaare 1764. Voor de eerfte, en in het jaar 1766. Voor de tweedemaal opgegeeven , om voor het begin van het jaar 1767. Te beantwoorden: welke zyn de beste en minst kostbaare middelen, om het afnemen der oevers van het Haarlemmer Meer te beletten? Verhandelingen uitgegeeven door de Hollandse Maatschappye der Weetenschappen te Haarlem. 10: 1-46. MICHEL, J. E. 1802. Statistique du département des Bouches du Rhône. Paris; Hachette. MILLER, P. 1745. Groot en algemeen kruidkundig, hoveniers, en bloemisten woordenboek, behelzende de manier om moes-, bloem-, vrugt-, kruid-tuinen, wild-bossen, wijngaarden, Oranje-huizen, stook-kassen, enz. aanteleggen, enz. en allerlei gewassen te kweeken [...]. II vols. Vol. I, Leiden; Pieter vander Eyk, en de weduwe Jakob vander Kluis. MILLER, P. 1754. The Gardeners Dictionary: Containing the Methods of Cultivating and Improving All Sorts of Trees, Plants, and Flowers, for the Kitchen, Fruit, and Pleasure Gardens; as Also Those which are Used in Medicine. With Directions for the Culture of Vineyards, and Making of Wine in England. In which Likewise are Included the Practical Parts of Husbandry. 3 vols. Vol. II, London; John & James Rivington. MILLHOLLON, R. W. 1987. Control of Equisetum hyemale on drainage ditches in sugarcane. Journal of the American Society Sugar Cane Technologists 7: 57-64. MONTIN, L. 1771. Bemerkungen über den Anbau des Flugsandes. Der Königl. Schwedischen Akademie der Wissenschaften neue Abhandlungen aus der Naturlehre, Haushaltungskunst und Mechanik 30: 276-283. MOORE, T. 1861. British Ferns and their Allies. London; George Routledge and Sons. MURRAY, J. A. H., ed. 1971. Compact edition of the Oxford English dictionary, complete text reproduced, micrographically. 2 vols. Vol. 2; Oxford; Oxford University Press. NEWMAN, E. 1842. Art. LXXVI. - A History of the British Equiseta. The Phytologist 2: 273-278. NEWMAN, E. 1844. A History of British Ferns and Allied Plants. Second (revised) edition. London; John van Voorst. NOEL CHOMEL, M. & DE CHALMOT, J. A. 1778. Algemeen huishoudelijk-, natuur- , zedekundig-, en konst- woordenboek. 2nd. ed. Vol. 4, Joh. le Mair. NOZEMAN, C. 1783. Verhandeling over heermoes, unjer of kattestaart (Equisetum). Antwoord op de vraeg der Maatschappy ter Bevordering vav de Landbouw: Welke zyn de Eigenschappen van de verschillende soorten van het Equisetum, by ons bekend onder den naam van Heermoes, Unjer, of Kattestaart ? Waarin bestaat de aart en hoedanigheid van deszelfs Vruchtdeelen en Voortplanting? Welk nadeel wordt doorhetzelve aan het Wei en Bouw- land toegebragt ? En welk is het door de ondervinding beproefde beste middel om hetzelve op de minst kostbaare wyze uitteroeijen ? Verhandelingen uitgegeeven door de Maatschappy ter Bevordering van de Landbouw 2: 1-66. ØLLGAARD, B. & TIND, K. 1993. Scandinavian Ferns. Copenhagen; Rhodos, International Science & Art Publishers. OUDEMANS, C. A. J. A. 1862. De natuurlijke historie van Nederland : de flora van Nederland. 1st ed. ed. 3 vols. Vol. 3, Haarlem; A.C. Kruseman. PAGE, C. N. 1997. The Ferns of Britain and Ireland. Cambridge; Cambridge DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 43

University Press. PARNELL, H. 1830. On Financial Reform. London; J. Murray. PARNELL, H. 1831. On Financial Reform. 3rd ed. London; John Murray. PERNETY, D. 1771. The history of a voyage to the Malouine, or Falkland, islands, made in 1763. London; T. Jeffrys. PEXENFELDER, M. 1670. Apparatus eruditionis tam rerum quam verborum per omnes artes et scientias. Nürnberg; Michael & Joh. Friedrich Endter. PHILLIPS, R. 1818. Book of english trades and library of the useful arts. New Enlarged ed. London; J. Souter, at the Juvenile Library. PRADA, C. 1986. Equisetum. In: CASTROVIEJO, S., LAÍNZ, M., LÓPEZ-GONZÁLEZ, G., MONTSERRAT, P., MUÑOZ GARMENDIA, F., PIAVA, J. & VILLAR, L. [eds], Flora Iberica, 21-29. Real Jardín Botánoco, C.S.I.C., Madrid. PRATT, A. 1846. Wild flowers of the year. London; The Religious Tract Society. PRATT, A. 1866. The Flowering Plants, Grasses, Sedges, and Ferns of Great Britain. Vol. VI: British grasses, sedges and ferns. London; Frederick Warne & Co. RIJNLAND, H. V. 2003. Waterkeringsbeheerplan 2003-2007 - Katern 3: Beplantingsbeleid. Leiden, report no. RUPPE, H. B. & HALLER, A. 1745. Flora Jenensis Henrici Bernhardi Ruppii, ex posthumis auctoris schedis et propriis observationibus aucta et emendata. Jena; C.H. Cuno. RUTZ, L. M. & FARRAR, D. R. 1984. The habitat characteristics and abundance of Equisetum × ferrissii and its parent species, Equisetum hyemale and , in Iowa. American Fern Journal 74: 65-76. SALVO TIERRA, E. 1990. Guía de helechos de la Península Ibérica y Baleares. Madrid; Pirámide,. SCHRAVER, A. 1807. Antwoord op de vraag: Is ‘er eenig middel, om het Kram- en Mat-werk aan de zeedijken, het zij met daartoe geschikte kruiden, heesters, of andere gewassen, tusschen hetzelve te planten; het zij op eenige andere wijze, duurzamer, en tegen den slag van ‘t zeewater meer bestand ie maken ? Nieuwe Verhandelingen Van Het Zeeuwsch Genootschap der Wetenschappen 1: 67-131. SMITH, E. 1841. Proposed cultivation of the Dutch species of rushes in England &c. The Mechanics’ Magazine, Museum, Register Journal, and Gazette 35: 34-35. SMITH, J. E. 1793. A Sketch of a Tour on the Continent, in the Years 1786 and 1787. Vol. 1, London; James Edward Smith. SMITH, J. E. 1802. English Botany; or, coloured figures of British Plants, with their essenntial characters, synonyms, and places of growth, to which will be added occasional remarks. Vol. XIV, London; J. Sowerby. SOCIETY FOR THE DIFFUSION OF USEFUL KNOWLEDGE. ed. 1833. Vegetable substances: materials of manufactures.The Library of Entertaining Knowledge London; C. Knight. SPENGLER, G. C. 1891. Het vastleggen en opbosschen van duinen en zandverstuivingen. Ede; Menger. STEEL, D. 1796. New and complete tables of the net duties payable, and drawbacks allowed, on goods, wares, and merchandises, imported into, exported from, and carried coastwise in, Great-Britain, agreeable to the act of the 27th Geo. III. called the Consolidation-Act, and corrected to the year 1796. London; D. Steel. STEWART, J. A. 1815. The young woman’s companion; or, Female instructor. Oxford; 44 FERN GAZ. 20(1):23-45. 2015

Bartlett & Newman. SYKORA, K. V. & LIEBRAND, C. I. J. M. 1987. Natuurtechnische en civieltechnische aspecten van rivierdijkvegetaties. Wageningen; Landbouwuniversiteit. TAILLARD, P.-A. & DALMONT, J.-P. 2012. Scraping technique for clarinet reeds derived from a static bending simulation. Proceedings of the Acoustics 2012 Nantes Conference, Nantes, France: 2735-2740. TARGIONI TOZZETTI, O. 1813. Istituzioni botaniche del dottore Ottaviano Targioni Tozzetti. 3rd ed. Vol. III, Firenze; Guglielmo Piatti. TECHNISCHE ADVIESCOMMISSIE VOOR DE WATERKERINGEN. 1985. Leidraad voor het ontwerpen van rivierdijken. 2 vols. Vol. 1, Den Haag; Staatsuitgeverij. TRIMMER, J. 1842. Practical chemistry for farmers and landowners. London; John W. Parker. TURNER, W. 1538. Libellus de herbaria novus, in quo herbarum aliquot nomina greca, latina, & Anglica habes, una cum nominibus officinarum, in gratiam studiose inventutis nunc primum in lucem aeditus. William Turner : Libellus de re herbaria, 1538. The names of herbes, 1548. London; Ray Society, facsimile 1965. VAN DER AA, A. J., ed. 1851. Aardrijkskundig woordenboek der Nederlanden.13 vols. Vol. 13 (Z & app.). Gorinchem; J. Noorduyn. VAN EYS, J. N., REINHART VAN MARLE, C. G., TEMMINCK, M., VAN DER GOES, F. & VAN STYRUM, J. 1799. Ontwerp tot vruchtbaarmaking der duinen: zijnde het tweede deel van het Algemeen Rapport der Commissie van Superintendentie over het Onderzoek der Duinen. 2 vols. Vol. 2, Leyden; Herdingh en Du Mortier. VAN GEUNS, S. J. 1789. Antwoord op de vraag, voorgesteld door de Hollandsche Maatschappye der Weetenschappen, te Haarlem: Welken zyn de onderwerpen betreffende de Natuurlyke Historie onzes Vaderlands, waarvan men met gegronde reden te verwachten hebbe, dat eene verdere naspooring ten nutte van het Vaderland verstrekken zal? Verhandelingen uitgegeeven door de Hollandse Maatschappye der Weetenschappen te Haarlem. XXVI: 231-316. VAN HALL, H. C. 1843. De Heermoes of kwade aard en de middelen tot hare verdelging voorgeslagen. Tijdschrift ter bevordering van Nijverheid 8: 1. VAN HALL, H. C. 1854. Neerlands plantenschat, of landhuishoudkundige flora, behelzende eene beschrijving der onkruiden, vergiftige en nuttige inlandsche planten en der in onzen landbouw gekweekte gewassen. Leeuwarden; G.T.N. Suringar. VAN HALL, H. C., MIQUEL, F. A. W. & DASSEN, M. 1832. Flora belgii septentrionalis, sive index plantarum indigenarum, quae hucusque in 7 provinciis foederatis repertae sunt = Flora van Noord-Nederland, of korte aanwijzing der in het wild wassende planten, welke tot nu toe in de voormalige 7 vereenigde gewesten gevonden zijn. Pars 1: Equisetaceae, Filices, Marsiliaceae, , Musci et Hepaticae = Bedektbloeijende planten, (paardestaartigen, varens, wortelzadigen. 2 vols. Vol. 2, Amsterdam; J.C. Sepp. VAN MEERTEN-SCHILPEROORT, A. B. 1830. Een theekistje à la milionade of à la chinoise. Penélopé, of Maandwerk aan het vrouwelijk geslacht toegewijd 6: 85-87. VON MARTENS, G. M. & KEMMLER, C. A. 1882. Flora von Württemberg und Hohenzollern. 3rd ed. 2 vols. Vol. 2, Heilbronn; Gebr. Henniger. VUYCK, L. 1898. De plantengroei der duinen: overzicht van het voorkomen der DE WINTER: HISTORY AND MYTH OF THE EQUISETUM TRADE 45

planten in het duingebied en de veranderingen die in de flora zijn opgetreden in de laatste vijftig jaar in het bijzonder met het oog op den aanleg van duinwaterleidingen. PhD, Leiden University, Leiden. WARDLAW, A. C. & LEONARD, A., eds. 2005. New Atlas of Ferns & Allied Plants of Britain & Ireland. BPS Special Publication no. 8. London; The British Pteridological Society. WEBSTER, N. 1913. Webster’s Revised Unabridged Dictionary of the English Language. G. & C. Merriam Co. WEEDA, E. J., WESTRA, R., WESTRA, C. & WESTRA, T. 1985. Nederlandse Ecologische Flora. 5 vols. Vol. 1, Amsterdam; IVN. WEEDA, E. J., WESTRA, R., WESTRA, C. & WESTRA, T. 1994. Nederlandse Ecologische Flora. 5 vols. Vol. 5, Amsterdam; IVN. WHITWORTH, C. 1777a. A register of the trade of the port of London; specifying the articles imported and exported, arranged under the respective countries; with a list of the ships entered inwards and cleared outwards. Number I. January, February, and March, 1776. London; G. Kearsly, W. Flexney, G. Robinson, J. Robson, J. Walter, T. Cadell, J. Sewell. WHITWORTH, C. 1777b. A register of the trade of the port of London; specifying the articles imported and exported, arranged under the respective countries; with a list of the ships entered inwards and cleared outwards. Number II. April, May, and June, 1776. London; G. Kearsly, W. Flexney, G. Robinson, J. Robson, J. Walter, T. Cadell, J. Sewell. WILKINSON, C. C. 1858. Weeds and wild flowers: their uses, legends, and literature. London; John van Voorst. WITHERING, W. 1776. A botanical arrangement of all the vegetables naturally growing in Great Britain. 1st ed. 2 vols. Vol. 2, London; T. Cadell etc. WITHERING, W. 1792. A botanical arrangement of British Plants. 2nd ed. 3 vols. Vol. 3, Birmingham; G.G.J. & J. Robinson, etc. WOLCOTT, O. 1795. Abstract of the Goods, Wares, and Merchandize, exported from the United States, from the 1st October 1793, to the 30th September 1794. Philadelphia; Francis Childs. YEATS, J. 1870. The natural history of commerce. London; Cassell, Petter & Galpin. FERN GAZ. 20(1). 2015 46



Two fern collections from Gunung Gaharu, Serian District, 1st Division, Sarawak, on the border with Kalimantan in Borneo, have usually been filed with grammitid ferns in herbaria. They are Anderson SAR 15696 and Ilias & Azahari SAR 35673, both seen in K and SAR, and said to be in L also. Both collections grew on sandstone cliffs, at 610 m and 790 m respectively, and are conspecific. They differ from grammitids in numerous morphological characters, however, and actually belong to Plagiogyria. The Flora Malesiana treatment of Plagiogyria (Zhang & Nooteboom, 1998) provides the most up-to-date account of the genus in Borneo, but the specimens were difficult to key to species because although they had the crenate sterile pinna margins of P. egenolfioides (Baker) Copel., the sterile pinnae were slightly narrowed at base, not auriculate which is a character of this species. The key to varieties of P. egenolfioides indicates that the collections belong to var. egenolfioides, being dwarf and with obscure aerophores only at the stipe base, but the description of this variety also states that most of the sterile pinnae are auriculate. Plagiogyria minuta Copel. is listed in the synonymy of P. egenolfioides var. egenolfioides and examination of the image of the type, MICH 1190812, shows that not only is it the same taxon as the Gunung Gaharu plants, but also that it would not key to P. egenolfioides var egenolfioides either, because of the shape of the pinna bases. Copeland (1915) noted that P. minuta was “an evident and near relative of P. egenolfioides, but much smaller throughout, and the sterile pinnae usually narrowed at the base instead of cordate or auricled”. The Flora Malesiana account needs to be revised to take into account the differences between the two taxa.

REFERENCES COPELAND, E. B. 1915. Notes on Bornean Ferns. Philippines Journal of Science C Botany 10(2): 145-149. ZHANG, X. C. & NOOTEBOOM, H. P. 1998. Plagiogyriaceae, in Flora Malesiana Series II, Volume 3: 295-316, Flora Malesiana Foundation.

B. S. Parris Fern Research Foundation Kerikeri New Zealand Email: [email protected] 47 FERN GAZ. 20(1). 2015



Acrosorus nudicarpus P.M.Zamora & Co. is a grammitid fern (Polypodiaceae) known only from ultramafic substrates on Palawan Island in the Philippines (Zamora & Co, 1981) and Mount Kinabalu in Sabah, Malaysia (Borneo) (Parris et. al., 1992 as Xiphopteris nudicarpa (P.M.Zamora & Co) Parris). Although it was described in Acrosorus, it differs from all other member of the genus in having hydathodes at the vein endings on the adaxial surface of the lamina, a character shared with several species of Xiphopterella. The following new combination is proposed:

Xiphopterella nudicarpa (P.M.Zamora & Co) Parris, comb. nov., based on Acrosorus nudicarpus P.M.Zamora & Co, Nat. Appl. Sci. Bull. Univ. Philipp. 32: 47 (1981). Type: Philippines, Palawan, Mt Mantalingahan, Iloilo Ridge, c. 1700 m, March-April 1977, Co 1776 (holotype PNH 199345!; isotypes A, K, L, LWG, MICH barcode 1115915!, PUH).

REFERENCES PARRIS, B. S., BEAMAN, R. S. & BEAMAN, J. H. 1992. The Plants of Mount Kinabalu. 1. Ferns and Fern Allies. Royal Botanic Gardens, Kew. 165 pp. ZAMORA, P. M. & CO, L. L. 1981. New species of ferns (Filicopsida) from Palawan, Philippines. Nat. Appl. Sci. Bull. Univ. Philipp. 32: 43-51.

B. S. Parris Fern Research Foundation Kerikeri New Zealand Email: [email protected] 48 FERN GAZ. 20(1). 2015


PAPERS should not usually exceed 20 printed pages and are generally expected to be considerably shorter. As well as reports of original research, review articles and short notes, e.g. new records, are encouraged. The senior author should supply an email address to facilitate correspondence. REVIEWS should cover recent developments in any aspect of research on ferns and lycopods. This can include unpublished research by the author, and/or the possible direction of future research. They should be aimed at the general reader who may not be an expert in the chosen topic. Proposals for a review should be discussed first with the review editors. MANUSCRIPTS should be submitted in English (British) in electronic format in 10-point Times New Roman font and double spaced. Electronic versions of text and tables should be compatible with WORD, with figures as pdf or jpg files, and sent as email attachments or CDroms. All manuscripts will be refereed. THE TITLE should reflect the content of the paper and be in BOLD CAPITALS (11-point) and centrally aligned. Generic and specific names should be in italics and any title containing a generic or specific name should be followed by the family name in brackets e.g. SPECIOSUM () IN SOUTHERN SPAIN AUTHOR ABBREVIATIONS should follow Pichi Sermolli’s (1996) Authors of scientific names in Pteridophyta, Royal Botanic Gardens, Kew. MAIN HEADINGS: should be in BOLD CAPITALS (10-point) and centrally aligned. SUBSIDIARY HEADINGS: should be in bold, the first letter of each word in capitals, the rest in lower case and left-aligned. AUTHORS’ NAMES AND FULL ADDRESSES (including email address): follow the title and are centrally aligned. KEY WORDS: up to ten. ABSTRACT: should reflect the content of the paper. FIGURES: there is no distinction between photographs and line drawings in numbering. All should be presented in a form ready for reproduction, colour, TIF format and 300 dpi (please contact editor with queries), with a scale bar where appropriate. Lettering or numbers (Arabic) should be in the bottom left using uppercase Times Roman. Figure captions should be on a separate sheet. TABLES: can be printed in either portrait or landscape format. Authors should consider this when preparing tables. Authors should ensure that tables fit the printed page size in a legible form. PAGESIZE: The Gazette has a useable page size of 123 × 204 mm. This can be used in portrait or landscape mode but any supplied artwork should not be larger than this. MEASUREMENTS: follow the metric system. CHECKLISTS: follow the format of Baksh-Comeau, Fern Gaz. 16(1, 2): 11- 122. REFERENCES: follow the style of a recent issue of The Fern Gazette, e.g.:- HOOKER, W.J. 1864. Species Filicum, 5. Dulau & Co., London. MORTON, C.V. 1947. The American species of Hymenophyllum, section Sphaeroconium. Contr. U.S. Natl. Herb. 29(3): 139-201. STEVENSON, D.W. & LOCONTE, H. 1996. Ordinal and familial relationships of pteridophyte genera. In: CAMUS, J.M., GIBBY, M. & JOHNS, R.J. (Eds) Pteridology in perspective, pp. 435-467. Royal Botanic Gardens, Kew. JOURNAL ABBREVIATIONS: should follow Botanico Periodicum Huntianum & Supplements. Alterations from the original text at proof stage will be charged for unless they are minor points of detail. A pdf will be provided free to the senior author. FERN GAZ. 20(2):49-54. 2015 49


Naturalis Biodiversity Center, PO box 9517, NL-2300 RA Leiden, The Netherlands Email: [email protected]

Keywords: Diplazium, taxonomy, rhizome scales, Sulawesi, Seram,

ABSTRACT Two new species of Diplazium are described from Indonesia: D. walkeri Hovenk. from Sulawesi, and D. murkele Hovenk. from Seram.

INTRODUCTION During identification of ferns collected by A.C. Jermy and T. G. Walker in 1969 in the Latimojong Mountains, Sulawesi Selatan (Indonesia), a number of specimens of Diplazium (Woodsiaceae) could not be identified with any known species. Comparison with the collections in BM, K and L revealed similarities with Diplazium mutabile Hovenk. (Hovenkamp & De Joncheere, 1988), described from a different mountain area on Sulawesi, with D. furculicolum Alderw. (Van Alderwerelt van Rosenburgh, 1918), described from Seram, and with a number of collections from Seram identified by Kato et al. (Kato, 1994) as D. vestitum C.Presl. With D. mutabile the unidentified specimens share a characteristic combination of evanescent basal scales and pinnule rachis ornamentation. The basal scales characteristically disintegrate starting with the outer cell walls (Figure 1A), and the abaxial surfaces of the pinna and pinnule rachis are ornamented with a combination of somewhat fleshy hair-like protrusions and appressed flat scales (Figure 1B). The same rachis indument is found in D. furculicolum. and in the Seram collections identified as D. vestitum, but accompanied by different types of scales. The scales of D. furculicolum have the same thin texture as those of D. mutabile, but retain their general outline more strongly; the scales of the Seram “D. vestitum” specimens are thick, brown and fleshy, but also appear disintegrated somewhat especially at the margin and the surface of the scale, leaving the exact shape and outline of the scales uncertain. Diplazium vestitum, as represented in L by collections from the Philippines and Borneo, differs significantly in having basal scales that are translucent with a dark dentate margin and smooth abaxial rachis surfaces and thereby is clearly different from the Seram specimens. Notwithstanding the general state of ignorance we are in about the exact species delimitation in Malesian Diplazium, the four groups of specimens appear to be sufficiently distinct to warrant the description of two new species. Table 1 provides a synopsis of the major morphological similarities and differences between the four species discussed here.

METHODS Photographs were taken with a Zeiss Discovery V20 microscope equipped with a Zeiss Axiocam MRC5 camera, and processed with Axiovision software (Axiovision Vs40x64 V. 4.9.10). For scanning electron microscopy (SEM) spores were sputter-coated with gold and examined in a JEOL JSM 5300 microscope (Figure 2A) or coated with c. 5 nm Pt/Pd and observed with a Jeol JSM 7600F FEG-SEM (Figure. 2B, C, D). 50 FERN GAZ. 20(2):49-54. 2015

Figure 1. Diplazium walkeri. A: Rhizome scale, showing the fibrous transverse cell walls remaining after the disappearance of the superficial walls (rehydrated). Scale bar 2mm. B: Abaxial surface of penultimate midrib, showing combination of fleshy hairs and appressed flat scales (in sicco). Scale bar 1mm (Walker 12367, BM).

Figure 2. Spores: A: Diplazium mutabile B: Diplazium walkeri C: Diplazium murkele, inset: detail of scaly outer layer D: Diplazium furculicolum Scale bars 10µm except inset in C 1µm. (Hennipman 5571 (L): A; Kato et al. C 5043 (L): C; C 1124 (L): D, Walker T12373 (BM): B). HOVENKAMP: TWO NEW DIPLAZIUM (WOODSIACEAE) SPECIES 51 Diplazium furculicolum 1-3.5cm 6-15mm 1-3 7-13cm 1.5-5mm Crenate or rarely more deeply incised 0-2 pairs Often proliferous erect Thin, translucent, more or less persistent Ornamented narrow wings 5 - 10.5cm 1.5 - 3mm Diplazium murkele Ascending to erect 1 Up to 36cm 9 -17cm Deeply incised, to 1-3cm from midrib 7-9 pairs Not proliferous regulate, surface Weakly eroding Thick, spongy, evanescent? Thick, spongy, Up to 10cm Thin, translucent, evanescent Diplazium walkeri Ascending or erect 3-6 Up to 27cm c. 5cm c. 1.5cm Deeply incised, to 2-3mm from midrib 7-10 pairs Not proliferous rugulate Weakly Ascending or erect Thin, translucent, evanescent 1-2 (bipinnate specimens) 3-5cm 1.5-1.7cm Serrate-dentate 2-4 pairs rugulate Weakly Not proliferous Diplazium mutabile 22-26cm 6-10cm Table 1 . Diagnostic characters Table for the four taxa. Reduced pinnae Scales on rhizome and base of stipe Pinnae length Pinnae width Pinnules width Basal pinnules free ornamentation Spore Rhizome Pinnules length Pinnules margin Lamina apex 52 FERN GAZ. 20(2):49-54. 2015

DESCRIPTION OF NEW TAXA Diplazium walkeri Hovenk., spec. nov. Type: Trevor G. Walker 12373 (holo BM; iso L), Indonesia, Sulawesi Selatan, Enrekang, above the village of Rantelimo, 25km SE of Kalosi, on the south-western slopes of Mt. Rantemario, 1950-2100m, on ridge above northern branch of R. Pasui, 24 November 1969. Terrestrial fern, rhizome ascending or erect, incl. stipe-base remnants 2-3cm thick; scaly but glabrous in older parts; rhizome scales basifix, periclinal cell walls very thin and quickly disappearing, often leaving the scale disintegrating into a fibrous network, shape, margin, apex of the scales accordingly not observed. Fronds to c. 130cm long; stipes to c. 46cm, sparsely scaly at base with scales similar to those on the rhizome, upwards with papillose multicellular protrusions; lamina bipinnate, to c. 90 by 50cm, widest at c. 1/3-1/5, gradually narrowing from c. halfway up; free pinnae 12-15 pairs, lowest 1-6 somewhat reduced, alternate to subopposite, largest pinnae to 2 cm stalked, to 37 by 18cm; pinnules alternate to opposite, basal 7-10 pairs free, to 5 by 1.5cm, symmetric at base, incised to 1.5-3mm from midrib, apex obtuse to acute, lobes truncate; pinnae and pinnule rachis on the adaxial surface with two flanges interrupted at the vein junctions, on the abaxial surface with flat translucent scales and papillose multicellular protrusions, veins free. Sori diplazioid, on all veins of a vein-group, running from costule to c. 1mm from the margin, spores c. 50 µm long, very lightly rugulate. Distribution – Indonesia: Sulawesi Selatan (Enrekang District, Latimojong Mountains). Ecology – From lowland forest to upper montane forest, in deep shade, in ravines or by streams, 1700-2150m. Etymology - The species is named after Trevor G. Walker (1927-2006), British pteridologist. Note - Diplazium walkeri differs from D. mutabile mainly in the more highly dissected lamina. Diplazium furculicolum differs from both in a much smaller size, basal scales that retain more of their shape and in much more strongly ornamented spores (Figure 2).

Diplazium murkele Hovenk., spec. nov. Type: M. Kato, K. Ueda & U.W. Mahjar C 1292 (holo L; iso TI, not seen, BO, not seen), Indonesia, Seram, Tehoru & Seram Utara: Manusela National Park: along a trail between Hatumete (seaside) and Maraina (810 m) in Manusela Valley via Hoale Pass (1770m) in Murkele Ridge, 03° 10-14' S, 129° 35-37' E, 18 Nov. 1983. Terrestrial fern, rhizome ascending or erect, incl. stipe-base remnants 2-3cm thick; scaly but glabrous in older parts; rhizome scales basifix, to c. 10 by 2mm wide, thick, fleshy, margin entire, in the basal part thin, flaccid; apex obtuse. Fronds to c. 120 or longer; stipes to 75cm, naked or sparsely scaly at base with thick brown scales to 10 by 6 mm, lamina bipinnate, to c. 70 by 50cm, widest at c. 1/4, free pinnae 8-12 pairs, lowest pair slightly reduced, alternate to opposite, largest pinnae to 1.5cm stalked, to 37 by 17cm; pinnules alternate to opposite, basal 7-9 pairs free, to 8.5 (10.5) by 1.5-2 (3)cm, symmetric at base, incised to 1—3 mm from midrib, apex obtuse to acute, lobes truncate; pinnae and pinnule rachis on the adaxial surface with two flanges interrupted at the vein junctions, on the abaxial surface with flat translucent scales and papillose multicellular protrusions, veins free. Sori diplazioid, on all veins of a vein-group, running from costule to c. 1mm from the margin, spores c. 40µm long when well- HOVENKAMP: TWO NEW DIPLAZIUM (WOODSIACEAE) SPECIES 53 formed, many collapsed or otherwise malformed, perispore with a thin almost smooth, eroded, scaly outer layer uncovering a finely irregularly porous inner layer. Distribution – Indonesia, Moluccas: Seram. Ecology – Terrestrial, in deep shade, near brooks, in ravines etc., in lowland to montane forest, disturbed forest, often noted for calcareous areas, lowland to 1350m, possibly higher. Etymology - The epithet is to be treated as a word in apposition and derives from Murkele Ridge, the location of the type specimen and a number of other specimens. Note – Diplazium murkele differs from D. walkeri mainly in the basal scales that are not thin and translucent but thick and fleshy. However, they often also appear to show signs of disintegration, which may explain the scarcity of intact scales in the collected specimens. The lamina is somewhat smaller, and has distinctly fewer reduced pinnae near the basis, the pinnules on the other hand are a bit larger. The outer layer of the spores appears to erode away, leaving only a partial, scaly cover (Figure 2 C).

Additional Specimens Seen:

Diplazium mutabile Sulawesi. Palolo: Hennipman, E. 5003 (L) 21 April 1979; Track to Roroka Timbu, Sopu-valley: Hennipman, E. 5151 (L) 30 April 1979; Sopu-valley: Hennipman, E. 5571 (L) 23 May 1979; 5587 (L) 1979, 5625 (L) 26 May 1979; 5640 (L); Gumbassa: Hennipman, E. 5711 (L) 30 May 1979; Rantemario: Walker, T.G. 11985 (BM) 1 November 1969; 12369 (BM) 24 November 1969.

Diplazium murkele. Moluccas. Manusela National Park: Iwatsuki et al. C 1027 (L) 12 November 1983; Kato et al. C 801 (L) 11 November 1983, C 1292 (L) 18 November 1983; C 1475 (L) 22 November 1983; C 1773 (L) 29 November 1983, C 2924 (L) 30 December 1984; C 3223 (L) 2 January 1985; C 3553 (L) 3 January 1985, C 5043 (L) 22 January 1985, C 12022 (L) 24 July 1986; C 13799 (L) 26 August 1986; C 14089 (L) 27 August 1986.

Diplazium walkeri. Sulawesi. Rantemario: Walker, T.G. 11958 (BM, L) 1 November 1969; 11959 (BM,L) 11961 (BM,L) 12365 (BM,L) 24 November 1969; 12368 (BM,L) 12373 (BM,L)

Diplazium furculicolum. New Guinea. Sattelberg: Clemens 7059 (BM) 10 September 1937; Moluccas. Manusela National Park: Kato, M et al. C 949 (L) 16 November 1983, C 1602 (L) 25 November 1983; C 3216 (L) 2 January 1985, C 6887 (L) 17 February 1985; C 7437 (L) 20 February 1985, C 12191 (L) 27 July 1986; C 12431 (L) 28 July 1986; C 12582 (L) 31 July 1986; C 14325 (L) 7 September 1986; C 14457 (L) 1 September 1986; C 1124 (L) 17 November 1983; C 1207 (L) 16 November 1983; Parris, B.S. 11089 (L) 27 August 1987.

ACKNOWLEDGMENTS A visit to the Natural History Museum, London to identify collections from Sulawesi was supported by Synthesys grant GB-TAF-4095. I gratefully acknowledge the patience of the Curator of Pteridophytes at BM for allowing me the loan of a number of specimens for an extensive period. 54 FERN GAZ. 20(2):49-54. 2015

REFERENCES HOVENKAMP, P. & DE JONCHEERE, G.J. 1988. Additions to the fern flora of Sulawesi. Blumea; 33(2): 395-409. KATO, M. 1994. Taxonomic studies of Pteridophytes of Ambon and Seram. J. Fac. Sc. Tokyo, III. Botany 15: 135-152. VAN ALDERWERELT VAN ROSENBURGH, C. R. W. K. 1918. New or interesting Malayan Ferns 10. Bull. Jard. Bot. Buitenzorg sér. 2 28: 1-56. FERN GAZ. 20(2):55-64. 2015 55


Y.L. CHANG1,2, M.H. TSENG2,3, W.L. CHIOU4 & Y.M. HUANG4,5

1 Dr. Cecilia Koo Botanic Conservation Center, 31 Tongsing Rd., Gaoshu Township, Pingtung 90646, Taiwan. 2 These authors contributed equally to this work. 3 Department of Applied Physics and Chemistry, University of Taipei, 1 Ai-Guo West Rd., Taipei 10048, Taiwan. 4 Taiwan Forestry Research Institute, 53 Nanhai Rd., Taipei 10066, Taiwan. 5 Corresponding author, e-mail: [email protected]

ABSTRACT Short-lived chlorophyllous spores need special treatment to extend their longevity and vigour for ex situ conservation. Germination rates, mean longevity and germination times of chlorophyllous spores of the rare fern Osmundastrum cinnamomeum were determined to assess their viability, lifespan and vigour at 18–25°C (room temperature, RT), 4°C and -80°C during 96 weeks of storage. Results showed that at the low temperatures, the viability and vigour were significantly maintained, and the lifespan of O. cinnamomeum spores was extended. Spores stored at RT had completely lost all ability to germinate within 12 weeks. Some spores retained their viability for more than 96 weeks at 4°C and -80°C. Mean longevities were 6.1, 41.8, and >96 weeks with storage at RT, 4°C and -80°C respectively. The mean spore germination time (MGT), herein regarded as an indicator of vigour, was affected by both storage temperature and time. Spores stored at RT and 4°C began to lose their vigour after three weeks. However, it did not decline at -80°C until 24 weeks of storage. This study demonstrates that low temperature (4°C) and cryopreservation (-80°C) significantly extended the longevity and vigour of chlorophyllous spores of O. cinnamomeum.

Keywords: cryopreservation, mean germination time.

INTRODUCTION As plants are threatened by habitat loss, pollution, invasive species, and other factors, in situ conservation is considered as the primary focus for conserving threatened species. Ex situ germplasm storage, on the other hand, is an important complementary aspect of plant conservation (Pence, 2004). A germplasm bank, storing living propagules in a small space, can efficiently maintain genetic variability (Dickie et al., 1990; Smith et al., 2003; Pence, 2008). Conservation plans for a number of threatened fern species include propagation from spores (e.g., Estrelles et al., 2001; Lusby et al., 2002; Simabukuro et al., 1998). Spore banks have low financial costs and have been developed for more than 20 years to conserve ferns ex situ (Page et al., 1992; Ibars & Estrelles, 2012). Most fern spores can typically retain their viability for a long period but there is great disparity among species (Okada, 1929). One of the principal features causing such 56 FERN GAZ. 20(2):55-64. 2015 variation is the presence of chlorophyll pigments. Fern spores are primarily divided into chlorophyllous spores (also called green spores) and non-chlorophyllous spores (non- green spores), depending on whether or not chlorophyll pigments exist in them (Gabriel y Galán & Prada, 2011). Compared with non-chlorophyllous spores, which retain their viability for several months to years, chlorophyllous spores are relatively short-lived, surviving for only a few weeks or months at room temperature (Lloyd & Klekowski, 1970). Chlorophyllous spores quickly lose their viability because of a lack of desiccation tolerance, a high metabolic rate, and the absence of dormancy (Kato, 1976; Li & Shi, 2014). Therefore they require specialised treatment for long-term conservation in germplasm banks (Ballesteros et al., 2011). It is well documented that the viability of chlorophyllous spores can usually be extended under low temperatures (Ballesteros, 2011; Gabriel y Galán & Prada, 2011). The longevity of chlorophyllous and non- chlorophyllous spores has been studied for over 50 years, and some protocols have been established to optimise spore preservation in fern spore banks (Ibars & Estrelles, 2012). For example, low temperature (-70°C) storage substantially prolonged chlorophyllous spore viability of Equisetum hyemale L. for over 16 months (Whittier, 1996), and at -20°C for more than 28 months for Osmunda regalis L. (Magrini & Scoppola, 2012). The “viability” of spores has been variously defined, such as the length of time that spores retain their ability to germinate (Raghavan, 1989; Gabriel y Galán & Prada, 2011), or the capacity of a spore to germinate (Qunitanilla et al., 2002). The former one is mutually used with the term “longevity” (e.g., Aragón & Pangua, 2004, Pence, 2004). We herein define “spore longevity” (the lifespan of a spore) to be the period from spore harvest to loss of germination ability as per Windham et al. (1986) and Ballesteros et al. (2011). “Spore viability” is defined as the spore germination ability as indicated by the germination rate (%) (Gabriel y Galán & Prada, 2011). The quality of spores (spore vigour) is related to the germination rate and ageing during storage. Because spore germination time is prolonged with storage time (Smith & Robinson, 1975; Camloh, 1999), germination time was used to indicate spore vigour in this study. Osmundastrum cinnamomeum (L.) C.Presl is a temperate fern, usually growing in swamps and widely distributed from Asia to the Americas (Metzgar et al., 2008). However, the population number is low (≤4) and relatively small (<200 mature individuals in the largest population) in Taiwan (Chiu et al., 2013). It was evaluated to be a vulnerable species based on IUCN red list categories and criteria (Wang et al., 2012). The goal of this study was to determine the effect of various storage temperatures on the longevity and vigour of chlorophyllous spores of O. cinnamomeum. The results provide useful information for long-term ex situ conservation of this rare fern in spore banks.

MATERIALS AND METHODS Fresh spores of O. cinnamomeum were obtained from 12 fertile fronds of six plants growing in a marsh at Tsaopi (24°46'03"N, 121°36'26"E, elev. 809m), Yilan County, northeastern Taiwan, in March 2012. The fertile fronds released spores after being air-dried for three days. Spores were isolated from sporangia and transferred to microtubes (Labcon Screw-Cap Microcentrifuge Tubes, 2.0ml) for dry storage. Microtubes of spores were stored at 18−25°C (room temperature, RT), 4°C or -80°C for up to 96 weeks. CHANG et al.: EFFECT OF TEMPERATURE ON CHLOROPHYLLOUS SPORES 57

Spores stored at RT, 4°C and -80°C were sown separately onto four sheets of membrane filters (with a pore size of 0.45µm and diameter of 47mm; Gelman Laboratory) after 0 (fresh spores), 1, 2, 3, 4, 8, 12, 24, 48 and 96 weeks of storage. Each sheet was laid on a 4cm thick layer of moist sphagnum in a plastic box (114×86×102mm, PHYTATRAY IITM, Sigma). All cultures were incubated under white fluorescent illumination at approximately 25−35µmol m-2 s-1 for 12h d-1 at 20 ± 1°C. Germination refers to the breakage of the spore wall and the appearance of the first rhizoid or cell (Lloyd & Klekowski, 1970). To calculate the germination rate (%), 200 spores (50 spores/filter × 4 filters) were randomly observed under a microscope (Leica, Wild M8) every week after sowing until no more germination was detected. The mean germination time (MGT) was calculated for each replication per treatment according to the equation: MGT = Σ(tn)/(Σn), where t is the time in weeks starting from spore sowing and n is the number of spores that had germinated in week t (modified from De Brum & Randi, 2006). The spore lifespan was indicated by the spores’ mean longevity calculated by the equation: ML = Σti (pi − pi+1), where ti is the th th spore storage time (i week), pi is the germination rate in the i week, and pi+1 is the germination rate for the next storage time (i+1th week). Germination rates and mean germination times for each temperature were analysed by a one-way analysis of variance (ANOVA) and Fisher's least significant difference (LSD) test (p<0.05) to estimate significant differences of means among different storage times.

RESULTS All (100%) fresh spores of O. cinnamomeum germinated within one week. With RT storage, the spore germination rate remained at almost 100% for four weeks in storage. Then it decreased significantly to nearly 50% after eight weeks in storage, and spores had completely lost viability after 12 weeks in storage. When the storage temperature was lowered to 4°C, spores retained almost all of their viability for 24 weeks in storage. Then it decreased significantly to 69.5% and 3.0% after 48 and 96 weeks of storage, respectively. For spores stored at -80°C, the germination percentage was almost 100% during 48 weeks of storage and still remained at 91.5% even after 96 weeks of storage (Table 1). The MGT of fresh spores was one week. Under RT storage, the MGT increased significantly to 1.4 weeks after three weeks in storage, and up to 2.0 and 2.1 weeks after four and eight weeks in storage, respectively. The MGT of spores stored at 4°C did not increase within two weeks of storage, then slightly (but significantly) increased to 1.3−1.5 weeks during 3−12 weeks of storage, and again significantly increased to 2.4−2.5 weeks after 24−48 weeks of storage. However MGT was found to have decreased to 2.0 weeks after 96 weeks of storage. The MGT was ≤1.2 weeks when spores were stored at -80°C after 12 weeks in storage, but significantly increased to 2.2 weeks after 24 weeks of storage, then decreased to 1.9 and 1.7 weeks respectively when spores were stored for 48 and 96 weeks (Table 2). Spores had completely lost their viability after 12 weeks of storage at RT, whereas some viability was retained at 4°C and -80°C storage at the end of the observation period (96 weeks) (Table 2). Thus the lifespan of at least some proportion of spores at 4°C and -80°C was more than 96 weeks. The mean longevity of spores significantly 58 FERN GAZ. 20(2):55-64. 2015

Table 1. Germination rates of spores of Osmundastrum cinnamomeum at different storage times and temperatures

Germination rate (%)1,2)

Storage temperature Storage time

(weeks) RT3) 4°C - 80°C

0 100a -- --

1 100a 100a 100a

2 100a 100a 100a

3 100a 100a 100a

4 99.5±1.0a 100a 100a

8 51.5±4.4b 100a 100a

12 0c 100a 100a

24 0c 97.0±3.5a 96.5±4.7a

48 0c 69.5±10b 98.5±1.9a

96 0 c 3.0±1.2c 91.5±5.3b

1) Data are the mean±standard deviation (n=4). 2) Means with the same superscript letter do not significantly differ within a temperature (p<0.05; LSD test). 3) Room temperature of 18−25°C. CHANG et al.: EFFECT OF TEMPERATURE ON CHLOROPHYLLOUS SPORES 59

Table 2. Mean germination times of spores of Osmundastrum cinnamomeum at different storage times and temperatures

Mean germination time (weeks)1,2)

Storage temperature Storage time

(weeks) RT3) 4°C - 80°C

0 1.0a -4) -

1 1.1±0.0a 1.1±0.0a 1.1±0.0a

2 1.1±0.1a 1.1±0.0a 1.1±0.1a

3 1.4±0.2b 1.3±0.1b 1.1±0.0a

4 2.0±0.2c 1.3±0.0b 1.2±0.1a

8 2.1±0.2c 1.5±0.0c 1.1±0.0a

12 --5) 1.4±0.1bc 1.1±0.0a

24 -- 2.5±0.2e 2.2±0.3d

48 -- 2.4±0.2e 1.9±0.1c

96 -- 2.0± 0.0d 1.7±0.2b

1) Data are the mean±standard deviation (n=4). 2) Means with the same superscript letter do not significantly differ within a temperature (p<0.05; LSD test). 3) Room temperature of 18−25°C. 4) -, no data. 5) --, spores did not germinate. 60 FERN GAZ. 20(2):55-64. 2015 differed among the three temperature treatments, with 6.1, 41.8, and >96 weeks at RT, 4°C and -80°C storage, respectively.

DISCUSSION The viability of chlorophyllous spores under RT conditions is known to be lost rapidly but there are great disparities among species. For example, longevity of chlorophyllous spores of Equisetum hyemale, Osmunda regalis, and sp. are two weeks, ca. one month, and more than one year, respectively (Stokey, 1951; Lloyd & Klekowski, 1970; Lebkuecher, 1997). This study demonstrated that the lifespan of a proportion of spores of O. cinnamomeum at RT reached eight weeks, slightly longer than previous records from Japan (1.4–1.8 months) (Okada, 1929). This difference may have resulted from plant genetic variations and/or spore maturity. Fully mature spores usually have greater longevity than do immature ones (Huang et al., 2014). Some physiological mechanisms and compounds, including the respiration rate, water content, catalase content and granules of lipids and lipoproteins, were documented to affect chlorophyllous spore viability (Okada, 1929; Gullvåg, 1969; Olsen & Gullvåg, 1973). The decay of fern spore viability might also result from the loss of some housekeeping substances, such as sugars, amino acids and proteins (Beri & Bir, 1993; Ballesteros et al., 2004). Spore viability is also related to various kinds of sulphur metabolites, such as glutathione (GSH), glutathione disulphide (GSSG), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH), which are involved in modulating protein activities or other metabolites under stress (Giovanelli et al., 1985; Dixon et al., 2005; Michelet et al., 2005; Loenen, 2006; Roje, 2006). Chang et al. (2013) further documented that GSH/GSSG and SAM/SAH ratios of spores of O. cinnamomeum significantly declined as they lost viability. Apparently those physiochemical changes affect the germination rates of O. cinnamomea spores and probably are correlated with the loss of viability in other chlorophyllous fern spores. Chlorophyllous spores tend to germinate quickly because their photosynthetically active can rapidly transform light energy to metabolic activity (Raghavan, 1989). The MGT of chlorophyllous spores at RT is ca. 1.5 days for the Equisetaceae, Osmundaceae, Grammitidaceae, Hymenophyllaceae, Blechnum nudum (Labill.) Mett. ex Luerss., Lomariopsis sorbifolia (L.) Fée, and some species of Christiopteris, Marginariopsis, Matteuccia, and Onocleopsis (Lloyd & Klekowski, 1970). It is obvious that the germination rate of chlorophyllous spores should be measured at short intervals. Daily observations would provide more accurate data than the weekly observations that were used in this study. Nevertheless, correlations between spore vigour and different storage temperatures were distinguishable. The results of this study show that the MGT increased before the germination rate declined. However, the MGT of O. cinnamomeum spores increased significantly after three weeks of storage at 4°C, then decreased after 96 weeks of storage. A similar situation also occurred with storage at -80°C. The increase and decrease in the MGT may have resulted from the self-regulation of spores in response to stress during storage (Chang et al., 2013). More studies are needed to determine whether the MGT is a good indicator for evaluating the germination vigour of fern spores. The successful establishment of individuals and populations of a species in a new habitat after spore dispersal depends on how long the spores retain their viability and vigour (Sheffield, 1996; Shorina, 2001; Gabriel y Galán & Prada, 2011). When the spore vigour decreases, subsequent gametophytes may grow abnormally and even CHANG et al.: EFFECT OF TEMPERATURE ON CHLOROPHYLLOUS SPORES 61 affect subsequent production (Smith & Robinson, 1975; Beri & Bir, 1993) and population establishment (Gabriel y Galán & Prada, 2011). An increase in the storage time results in decline in spore viability and vigour as previously reported for O. cinnamomeum and a few other ferns (Smith & Robinson, 1975; Ballesteros et al., 2011, 2012). Evidence was presented that suggests spore age may be partly attributable to the declining ability of spores to synthesise metabolites essential for germination (Gabriel y Galán & Prada, 2011). It is well known that chlorophyllous spores have shorter lifespans compared with non-chlorophyllous spores (Lloyd & Klekowski, 1970). Using low temperature to prolong their lifespan and vigour was documented (Pence, 2000, 2008; Ballesteros et al., 2006, 2011, 2012; Ballesteros, 2011; Gabriel y Galán & Prada, 2011). It was reported that chlorophyllous spores of Osmunda regalis and O. japonica Thunb. stored at low temperatures (-25 and 4°C) also retained viability for up to 24 and 12 months respectively (Ballesteros et al., 2011; Li & Shi, 2014). This study also showed that spores of O. cinnamomeum rapidly lost viability at RT storage, and the lowest temperature of -80°C provided the best conditions to retain their spore viability and vigour. Cryopreservation is, therefore, recommended for the long-term storage of spores of this species, and this procedure may be applicable to other fern spores in terms of ex situ conservation.

CONCLUSIONS Chlorophyllous spores of ferns have shorter lifespans at room temperature storage compared with non-chlorophyllous spores. However, cryogenic temperatures might suppress metabolic activities and effectively extend their longevity and retain the vigour of chlorophyllous spores as shown in this study. It is suggested that cryopreservation is a feasible method for establishing long-term germplasm of the chlorophyllous spores of Osmundastrum cinnamomeum.

REFERENCES ARAGÓN, C.F. & PANGUA, E. 2004. Spore viability under different storage conditions in four rupicolous L. taxa. Amer. Fern J. 94: 28-38. BALLESTEROS, D. 2011. Conservation of fern spores. In: FERNÁNDEZ, H., KUMAR, A. & REVILLA, M.A. (Eds) Working with Ferns: Issues and Applications, pp. 165-172. Springer press, New York. BALLESTEROS, D., ESTRELLES, E. & IBARS A.M. 2006. Responses of pteridophyte spores to ultrafreezing temperatures for long-term conservation in germplasm banks. Fern Gaz. 17: 293-302. BALLESTEROS, D., ESTRELLES, E., WALTERS, C. & IBARS, A.M. 2011. Effect of storage temperature on green spore longevity for the ferns and Osmunda regalis. Cryoletters 32: 89-98. BALLESTEROS, D., ESTRELLES, E., WALTERS, C, & IBARS, A.M. 2012. Effect of temperature and desiccation on ex situ conservation of nongreen fern spores. Amer. J. Bot. 99: 721-729. BALLESTEROS, D., IBARS, A.M. & ESTRELLES, E. 2004. New data about pteridophytic spore conservation in germplasm banks. In: PLANTA EUROPA COMMITTEE (Eds) 4th European Conference on the Conservation of Wild Plants, Valencia. BERI, A. & BIR, S.S. 1993. Germination of stored spores of Pteris vittata L. Amer. Fern J. 83:73-78. 62 FERN GAZ. 20(2):55-64. 2015

CAMLOH, M. 1999. Spore age and sterilization affects germination and early gametophyte development of bifurcatum. Amer. Fern J. 89: 124-132. CHANG, Y.L., HSIEH, C.L., HUANG, Y.M., CHIOU, W.L., KUO, Y.H. & TSENG, M.H. 2013. Modified method for determination of sulfur metabolites in plant tissues by stable isotope dilution-based liquid chromatography-electrospray ionization- tandem mass spectrometry. Anal. Biochem. 442: 24-33. CHIU, T.Y., CHIOU, W.L. & HUANG, Y.M. 2013. Phenological differences between sterile and fertile fronds in a dimorphic fern, Osmundastrum cinnamomeum (L.) C. Presl (Osmundaceae). Taiwan J. Biodivers. 15: 311-322. DE BRUM, F.M.R. & RANDI, A.M. 2006. Germination of spores and growth of gametophytes and of Rumohra adiantiformis (Forst.) Ching (Dryopteridaceae) after spore cryogenic storage. Revista Brasil. Bot. 29: 489-495. DICKIE, J.B., ELLIS, R.H., KRAAK, H.L., RYDER, K. & TOMPSETT, P.B. 1990. Temperature and seed storage longevity. Ann. Bot. 65: 197-204. DIXON, D.P., SKIPSEY, M., GRUNDY, N.M. & EDWARDS, R. 2005. Stress-induced protein S-glutathionylation in Arabidopsis. Plant Physiol. 138: 2233-2244. ESTRELLES, E, IBARS, A.M., IRANZO, J. & MORALES, F. 2001. Recuperacio y reintroduccio de Marsilea quadrifolia L. en los arrozales del delta del Ebro (Tarragona, Españ). Bot. Complut. 25: 251-259. GABRIEL Y GALÁN, J.M. & PRADA, C. 2011. Pteridophyte spores viability. In: FERNÁNDEZ, H., KUMAR, A. & REVILLA, M.A. (Eds) Working with Ferns: Issues and Applications, pp. 193-205. Springer press, New York. GIOVANELLI, J., MUDD, S.H. & DATKO, A.H. 1985. Quantitative analysis of pathways of methionine metabolism and their regulation in Lemna. Plant Physiol. 78: 555-560. GULLVÅG, B.M. 1968. On the fine structure of the spores of var. verticillatum studied in the quiescent, germinated and non-viable state. Grana Palynol. 8: 23-69. HUANG, Y.M., LEE, P.H., FU, C.H. & CHANG, Y.H. 2014. The relation between spore maturity and viability of tree ferns. J. Nat. Taiwan Museum 67(2): 1-14. IBARS, A.M. & ESTRELLES, E. 2012. Recent developments in ex situ and in situ conservation of ferns. Fern Gaz. 19(3): 67-86. KATO, Y. 1976. The effect of freezing and organic solvents on viability of chlorophyllous fern spores. Cytologia 41: 387-393. LEBKUECHER, J.G. 1997. Desiccation-time limits of photosynthetic recovery in Equisetum hyemale (Equisetaceae) spores. Amer. J. Bot. 84: 792-797. LI, Y. & SHI, L. 2014. Effect of desiccation level and storage temperature on green spore viability of Osmunda japonica. Cryobiology 68: 446-450. LLOYD, R.M. & KLEKOWSKI, E.J. 1970. Spore germination and viability in pteridophyta: evolutionary significance of chlorophyllous spores. Biotropica 2: 129- 137. LOENEN, W.A.M. 2006. S-adenosylmethionine: jack of all trades and master of everything? Biochem. Soc. Transact. 34: 330-333. LUSBY, P., LINDSAY, S. & DYER, A.F. 2002. Principles, practice and problems of conserving the rare British fern ilvensis (L.) R. Br. Fern Gaz. 16: 350-355. MAGRINI, S. & SCOPPOLA, A. 2012. First results from conservation studies of chlorophyllous spores of the Royal fern (Osmunda regalis, Osmundaceae). Cryobiology 64: 65-69. CHANG et al.: EFFECT OF TEMPERATURE ON CHLOROPHYLLOUS SPORES 63

METZGAR, J.S., SKOG, J.E., ZIMMER, E.A. & PRYER, K.M. 2008. The of Osmunda is confirmed by phylogenetic analyses of seven plastid loci. System. Bot. 33: 31-36. MICHELET, L, ZAFFAGNINI, M., MARCHAND, C., COLLIN, V., DECOTTIGNIES, P., TSAN, P., LANCELIN, J.M., TROST, P., MIGINIAC- MASLOW, M., NOCTOR, G. & LEMAIRE, S.D. 2005. Glutathionylation of thioredoxin f is a redox signaling mechanism in plants. Proc. Natl. Acad. Sci. U.S.A 102: 16478-16483. OKADA, Y. 1929. Notes on the germination of the spores of some Pteridophytes with special regard to their viability. Sci. Rep. Tdhoku Imp. Univ. Biol. 4: 127-182. OLSEN, L.T. & GULLVÅG, B.M. 1973. A fine structural and cytochemical study of mature and germinating spores of . Grana 13: 113-118. PAGE, C.N., DYER, A.F., LINDSAY, S. & MANN, D.G. 1992. Conservation of Pteridophytes – the ex situ approach. In: IDE, J.M., JERMY, A.C. & PAUL, A.M. (Eds) Fern Horticulture; Past, Present and Future Perspectives, pp. 269-278. Intercept, Andover. PENCE, V.C. 2000. Survival of chlorophyllous and nonchlorophyllous fern spores through exposure to liquid nitrogen. Amer. Fern J. 90: 119-126. PENCE, V.C. 2004. Ex situ conservation methods for and pteridopytes. In: GUERRANT, E.O. Jr., HAVENS, K. & MAUNDER, M. (Eds) Ex Situ Plant Conservation Supporting Species Survival in The Wild, pp. 206-227. Island Press, Covelo. PENCE, V.C. 2008. Cryopreservation of bryophytes and ferns. In: REED, B.M. (Ed) Plant Cryopreservation: A Practical Guide, pp. 117-140. Springer, New York. QUINTANILLA, L.G., AMIGO, J. & PAJARÓN, S. 2002. Effect of storage method on spore viability in five globally threatened fern species. Ann. Bot. 90: 461-467. RAGHAVAN, V. 1989. Developmental Biology of Fern Gametophytes. Cambridge University Press, New York. ROJE, S. 2006. S-adenosyl-l-methionine: beyond the universal methyl group donor. Phytochemistry 67: 1686-1698. SHEFFIELD, E. 1996. From pteridopyte spore to sporophyte in the natural environment. In: CAMUS, M.G.J.M. & JOHNS, R. (Eds) Pteridology in Perspective, pp. 541-549. Royal Botanic Gardens, Kew. SHORINA, N.I. 2001. Population biology of gametophytes in homosporous polypodiophyta. Russ. J. Ecol. 32: 164-169. SIMABUKURO, E.A., DYER, A.F. & FELIPPE, G.M. 1998. The effect of sterilization and storage conditions on the viability of the spores of Cyathea delgadii. Amer. Fern J. 88: 72-80. SMITH, D.L. & ROBINSON, P.M. 1975. The effects of spore age on germination and gametophyte development in Polypodium vulgare L. New Phytol. 74: 101-108. SMITH, R.D., DICKIE, J.B., LININGTON, S.H., PRITCHARD, H.W. & PROBERT, R.J. 2003. Seed Conservation: Turning Science into Practice. Royal Botanic Gardens, Kew. STOKEY, A.G. 1951. Duration of viability of spores of the Osmundaceae. Amer. Fern J. 41: 111-115. WANG, J.C., CHIOU, W.L. & CHANG, H.M. 2012. A Preliminary Red List of Taiwanese Vascular Plants. Endemic Species Research Institute & Taiwan Society of Plant Systematics, Nantou. 64 FERN GAZ. 20(2):55-64. 2015

WHITTIER, D.P. 1996. Extending the viability of Equisetum hyemale spores. Amer. Fern J. 86: 114-118. WINDHAM, M.D., WOLF, P.G. & RANKER, T.A. 1986. Factors affecting prolonged spore viability in herbarium collections of three species of Pellaea. Amer. Fern J. 76:141-148. FERN GAZ. 20(2):65-78. 2015 65



15, rue Galilée, F-38000 Grenoble, France. E-Mail: 216, rue des Arènes, F-87000 Limoges, France. E-mail: 3 1, rue de la Faye, F-08160 Hannogne-Saint-Martin, France. E-mail: [email protected] 4 Universiteit Ghent, Vakgroep Biologie, Pteridologie, K.L. Ledeganckstraat 35, [email protected] Ghent, Belgium. E-mail: * [email protected] for correspondence

Keywords : Pleopeltis, hybrid, Polypodiaceae, Juan Fernández, Chile [email protected] ABSTRACT A fern hybrid of the genus Pleopeltis was discovered on Robinson Crusoe Island in the Juan Fernández Archipelago, off the coast of Chile, and is described as P. ×cerro-altoensis. Its putative parents are P. macrocarpa and P. masafuerae, two species present in the archipelago.

Mots-clés : Pleopeltis, hybride, Polypodiaceae, Juan Fernández, Chili

RÉSUMÉ Un hybride de fougère appartenant au genre Pleopeltis a été découvert sur l’île Robinson Crusoë, dans l’archipel Juan Fernández, au large du Chili, et est décrit sous le nom de P. ×cerro-altoensis. Ses parents probables sont P. macrocarpa et P. masafuerae, deux espèces présentes dans l’archipel.

Palabras clavas : Pleopeltis, híbrido, Polypodiaceae, Juan Fernández, Chile

RESUMEN Un híbrido de helecho que pertenece al género Pleopeltis ha sido descubierto en la isla Robinson Crusoe, en el archipiélago Juan Fernández, a la altura de Chile, y es descrito con el nombre de P. ×cerro-altoensis. Sus parientes probables son P. macrocarpa y P. masafuerae, dos especies presentes en el archipiélago.

INTRODUCTION Since 1997, the first author has undertaken, with the association ROBINSONIA , regular expeditions in the Juan Fernández Archipelago, off the coast of Chile, to study

1 th French association registered under Law 1901, on 9 October 2001, involved in the inventory1 and conservation of the Flora of Juan Fernández Archipelago, in relation with local communities and notably with the Corporación Nacional Forestal (CONAF) of Chile. 66 FERN GAZ. 20(2):65-78. 2015 both the indigenous and the introduced flora of the three islands that constitute this archipelago (Figure 1): the main island, Robinson Crusoe (or Masatierra) and the nearby Santa Clara Island (also called Île aux Chèvres), and the third one, Alejandro Selkirk Island (or Masafuera), located 180 km further west. During these expeditions, on 29th January 1999, a small isolated population of a polypodiaceous fern was discovered on a cliff of volcanic rocks, at Cerro Alto (Figures 2 & 3) on Robinson Crusoe Island (Masatierra). The presence of peltate scales on both surfaces of the lamina and its axes showed that it belongs to the genus Pleopeltis Humb. & Bonpl. ex Willd. (Figures 4 & 7). We compared this plant with the Pleopeltis species known from the area, but our review of botanical literature relating to Juan Fernández (Hemsley, 1884; Johow, 1892- 93, 1896; Christensen & Skottsberg, 1920; Skottsberg, 1953; Barrera Moscoso, 1997; Marticorena et al., 1998; Boudrie, 2003; Danton & Perrier, 2006) and to continental Chile (Gay, 1853; Looser, 1961, 1962, 1965-66, 1968; de la Sota, 1967; Marticorena & Rodriguez, 1995; Zuloaga et al., 2008) has shown that it was not recorded in any of the floras or floristic catalogues, nor any description applied to it. Therefore, whatever its identity or origin, we believe that it is a taxon new to the Juan Fernández Archipelago. Two other species of Pleopeltis (previously placed in the genus Polypodium) occur in the Juan Fernández Archipelago (Figure 5): - Pleopeltis macrocarpa (Bory ex Willd.) Kaulf., Berlin. Jahrb. Pharm. Verbundenen Wiss. 21: 41. 1820. Basionym: Polypodium macrocarpum Bory ex Willd., Sp. Pl. ed. 4, 5: 147. 1810. Syn. : Polypodium lanceolatum L., Sp. Pl. 2: 1802. 1753. Distribution: almost pantropical, America (from Central America to Chile and Argentina, Juan Fernández, Galapagos), tropical southern Africa, India. - Pleopeltis masafuerae (Phil.) de la Sota, Darwiniana 45(2): 239. 2007. Basionym : Polypodium masafuerae Phil., Linnaea 29: 107. 1857. Distribution: Juan Fernández Archipelago (only on Alexander Selkirk Island = Masafuera), Chile (province of Antofagasta); mentioned from southern Peru (Marticorena & Rodriguez, 1995), but not confirmed to our knowledge. Concerning the identity of P. masafuerae, its relationships with several South American Pleopeltis such as P. pinnatifida (Gillies) Hook. & Grev., P. pycnocarpa (C.Chr.) A.R.Sm. and Polypodium mollendense Maxon are unresolved (pers. comm. A.R. Smith, 2015). Although the circumscription of Pleopeltis has recently been enlarged, based both on molecular and on morphological studies (Otto et al., 2009; Smith & Tejero- Díez, 2014), Neotropical Pleopeltis taxa are a taxonomically difficult group, with polyploids and numerous hybrids, and a modern revision is needed to clarify the taxonomy. Tryon & Stolze (1993), treating the Peruvian species (in Polypodium), alluded to these complexities. Because neither of these two species matched the newly discovered plant, which thus might represent a new taxon (Danton et al. 1999), a more careful study of it was undertaken, as described below.

MATERIAL AND METHODS The material used for this study comprises: - Fronds collected in the field at Cerro Alto, now pressed and dried herbarium specimens: ● Danton B(652)619 (P, SGO, private herb. Ph. Danton) BOUDRIE et al.: PLEOPELTIS ×CERRO-ALTOENSIS 67

● Boudrie 3793 (P) - Fresh fronds collected at the Botanical Conservatory of Mulhouse, France, where a Pleopeltis plant collected by one of us (Ph. D.) at Cerro Alto on Feb. 27, 2002, has been in cultivation since 2002 under the number CBM 03-301, and now, extensively propagated. All the herbarium specimens mentioned below are issued from the CBM 03-301 plant. ● Ph. Danton – Mulhouse – 1 (private herb. R. Viane) ● Ph. Danton – Mulhouse – 2 (private herb. R. Viane) ● Ph. Danton – Mulhouse – 3 (private herb. R. Viane) ● Bizot 2905 (private herb. A. Bizot)

Figure 1: Location of the Juan Fernández Archipelago and Robinson Crusoe Island, west of Chile. Map base source : © Google maps, 2015

Figure 2: Alexander Selkirk Island (left) and Robinson Crusoe Island (right). © Ph. Danton & Ch. Perrier, 2004 68 FERN GAZ. 20(2):65-78. 2015

Figure 3: Pleopeltis ×cerro-altoensis in situ at Cerro Alto. © Photo Ph. Danton, 22/01/1999

Figure 4: Pleopeltis ×cerro-altoensis. © Ph. Danton, 25/01/1999. a - entire plant; b - portion of adaxial laminar surface; c - abaxial surface of fertile ; d – peltate-based lanceolate laminar scale; e – peltate-based lanceolate rhizome scale. BOUDRIE et al.: PLEOPELTIS ×CERRO-ALTOENSIS 69

Figure 5 – Left: Pleopeltis masafuerae (Phil.) de la Sota, rocks at the beginning of the Quebrada Las Casas, Alejandro Selkirk Island, 19/12/2003, Danton G(1319)1083. – Right : Pleopeltis macrocarpa (Bory ex Willd.) Kaulf., epiphytic in forest under the Mirador de Selkirk, Robinson Crusoe Island, 07/12/1998, Danton B(410)366. © Photo Ph. Danton, 26/05/2015. 70 FERN GAZ. 20(2):65-78. 2015

Figure 6: Pleopeltis ×cerro-altoensis Danton & Boudrie, Cerro Alto, Robinson Crusoe Island, Juan Fernández Archipelago, holotype (SGO). Danton B(658)619. © Photo Ph. Danton, 17/11/2014. BOUDRIE et al.: PLEOPELTIS ×CERRO-ALTOENSIS 71

Figure 7: Abaxial laminar surface of Pleopeltis ×cerro-altoensis, showing peltate- lanceolate scales and sori (from Bizot 2905). © Photo A. Bizot, 8/10/2014.

Figure 8: Peltate-based, lanceolate, fimbriate scale of abaxial laminar surface of Pleopeltis ×cerro-altoensis (from Bizot 2905). © Photo A. Bizot, 10/10/2014. 72 FERN GAZ. 20(2):65-78. 2015

Figure 9: and malformed spores of Pleopeltis ×cerro-altoensis (from Bizot 2905). © Photos A. Bizot, 8/10/2014 & 18/10/2014. BOUDRIE et al.: PLEOPELTIS ×CERRO-ALTOENSIS 73

- Dried herbarium specimens issued from the cultivated plant at Mulhouse under number CBM 03-301 : ● Boudrie 3966 (P) ● Boudrie 4500 (P) ● Danton Z(hs)1727 (private herb. Ph. Danton) - Other Pleopeltis taxa from Mexico, deposited in the personal herbarium of one of us (R. V.), but used, when freshly collected, for FCM analyses. Only one, P. guttata, is in cultivation in R.V.’s garden in Serskamp, near Ghent. ● Viane 11001: Pleopeltis guttata (Maxon) E.G. Andrews & Windham ● Viane 11005: Pleopeltis (Roem. ex Kunze) T.Moore var. erythrolepis (Weath.) Wendt ● Viane 11013: Pleopeltis polypodioides (L.) E.G.Andrews & Windham var. knoblochiana (Mickel) A.R.Sm. & Tejero Herbarium specimens of the respective private herbaria of A.B., Ph. D. and R. V., cited above, will be deposited in the future in official institutions. To establish its characters and identity, several methods have been applied to the material mentioned above.

Micromorphological observations Micromorphological observations were carried out by A.B. using a microscope Bresser triocular TRM-301 microscope at 400x to 1000x enlargements, equipped with a Bresser MicroCamLab-1 ocular camera. Fresh fronds from material in cultivation in the Botanical Conservatory of Mulhouse [Bizot 2905] were used. - Laminar scales were observed (Figures 7 & 8) on fresh fronds. Rhizome scales were not microscopically observed. However, despite their more lanceolate shape than the laminar scales, they show the same clathrate pattern of cells. - Stomata: Using tweezers, three epidermial slices were excised from the abaxial side of the lamina in the middle of a median pinna. The epidermis was observed in glycerin at 1000x enlargement. Eighty (80) stomata were measured. - Spores were observed (Figure 9) in glycerin liquid at 1000x enlargement. They were collected from several (3-4) sori of three different fresh fronds as well as from more than two sori of a frond collected in the field [Danton B(652)619]. Comparison of size of stomata of the Cerro Alto plant with stomata from plants of P. macrocarpa and P. masafuerae may reveal important information, but this investigation was impossible owing to the lack of adequate fresh material.

Measurements of rhizomes The width (rhizomes are slightly flattened) and the thickness of rhizomes of the 10 specimens of P. macrocarpa, four specimens of P. masafuerae, and four specimens of the Cerro Alto plant were measured from dried herbarium specimens. Sectioned pieces of rhizomes were observed under a binocular and measured using a plastic ruler with the smallest calibrated unit being 0.5mm.

DNA content using FCM A few fresh fronds (Botanical Conservatory of Mulhouse, France) of the Pleopeltis from Cerro Alto (in cultivation since 2002) were sent to the laboratory of Pteridology of Ghent University (Belgium) on 6th October 2014 for flow cytometric measurements (FCM). For this analysis, fresh samples were first chopped, using a sharp razor blade, 74 FERN GAZ. 20(2):65-78. 2015 in a glass Petri dish containing nucleus-isolation buffer (2.1% [w/v] containing citric acid monohydrate, 0.5% [w/v] Tween 20, and distilled water; this suspension was then filtered through a 50-µm nylon mesh. The nuclear suspension was supplemented with DAPI reagent [6.5% NaH2PO4.2H2O, 40µl DAPI stock solution (5mg DAPI/ml distilled water), distilled water], and each sample was measured three times. The samples were analysed using a flow cytometer (Ploidy Analyser, Partec GmbH, Münster, Germany), following the supplier’s instructions. To calculate the relative nuclear DNA content, Agave striata (2n = 2x = 60, 2C = 7.8pg (Zonneveld et al., 2005) was used as an internal standard. Fresh material of P. macrocarpa or P. masafuerae was unavailable, as the islands where they grow are difficult of access. To establish ploidy levels, our FCM results were compared with those of some Mexican Pleopeltis taxa collected in Chihuahua: diploid P. polylepis (Roem. ex Kunze) T.Moore var. erythrolepis (Weath.) Wendt (Viane 11005); P. guttata (Maxon) E.G.Andrews & Windham (Viane 11001); and triploid P. polypodioides (L.) E.G.Andrews & Windham var. knoblochiana (Mickel) A.R.Sm. & Tejero (Viane 11013). To confirm the FCM results, a chromosome count was made for P. guttata, revealing it to be triploid (Van den heede, pers. comm.). The three specimens from Cerro Alto submitted for FCM have been registered as “Ph. Danton - Mulhouse - 1" to "Ph. Danton -Mulhouse- 3", all sampled from the cultivated plant at Mulhouse under the number CBM 03-301.

RESULTS Micromorphology, spores and stomata Spores are either irregular in shape and in size or totally malformed (Figure 9), a condition typical for interspecific fern hybrids. Stomata range from 45 to 63µm long, with an average of standard deviation of 3.1µm.

Rhizome measurements 53µm and a Taxon Width (mm) Thickness (mm) min. mean max. min. mean max. Pleopeltis macrocarpa 1.2 2.1 2.5 0.7 1.4 2.0 Pleopletis masafuerae 3.0 3.3 4.0 1.8 2.0 2.4 Cerro Alto plant 2.3 2.5 2.8 1.5 1.7 2.1

Although the number of measurements is rather low, the values obtained for width and diameter of rhizomes from the Cerro Alto plant are intermediate between those of its putative parents, P. macrocarpa and P. masafuerae.

Flow cytometry Comparison of FCM values of the Cerro Alto plant with those of Mexican Pleopeltis taxa shows that it is most probably tetraploid (4x). Polyploidy is known within Pleopeltis, and, within P. macrocarpa, three cytotypes, diploid, tetraploid and hexaploid, have been reported (Evans, 1963; Jarret et al., 1968; Walker, 1973; Wagner BOUDRIE et al.: PLEOPELTIS ×CERRO-ALTOENSIS 75

& Wagner, 1975).

FCM results are presented in the table below:

* Taxon Voucher RCstriata Ploidy level Pleopeltis polylepis var. erythrolepis Viane 11005 1.56 2x Pleopeltis guttata Viane 11001 2.26 3x Pleopeltis polypodioides var. knoblochiana Viane 11013 2.54 3x Pleopeltis ×cerro-altoensis cult Mulhouse 3.06 4x

*RCstriata : average (based on 3 measurements) of relative DNA content compared with Agave striata. From the value of 3.06 for RCstriata for the new hybrid, we infer that it is tetraploid.

DISCUSSION Interspecific hybrids are well known among species of Pleopeltis, as redefined by Smith & Tejero (2014), and are discussed there, as well as in several floras, e.g., Mickel & Beitel, 1987; Mickel & Smith, 2004. Such hybrids, mostly in the Neotropics but a few in southern Africa, have sometimes been treated under the hybrid genus, ×Pleopodium, because historically they were thought to involve species of two different genera, Pleopeltis and Polypodium. Our study of the Cerro Alto plant shows that it is a hybrid that clearly belongs to Pleopeltis. Only two species of Pleopeltis occur in the Juan Fernández Archipelgo: P. macrocarpa, with entire, narrowly lanceolate laminae, and P. masafuerae, with deltate, pinnatisect laminae and undulate pinnae that are slightly decurrent onto the rachis. The latter species occurs on Alejandro Selkirk Island and on the coasts of Chile (Antofagasta province). Remarkably, P. masafuerae has never been found on Robinson Crusoe Island. However, due to the inaccessibility of large areas and infrequency of hybridization, it may have been overlooked. The overall lanceolate laminar shape of the Cerro Alto plant is similar to that of P. macrocarpa, whereas its division, though less incised (pinnatilobed to pinnatifid) and with distinctly decurrent but less undulate pinnae, is more similar to P. masafuerae. In addition, we have compared the laminar morphologies of the Cerro Alto plant with the Pleopeltis hybrids described by Wagner & Wagner (1975) and Mickel & Beitel (1987), in particular between species with entire, lanceolate laminae, such as P. macrocarpa or P. mexicana (Fée) Mickel & Beitel, and a species with deltate pinnatisect laminae, such as P. thyssanolepis (A.Braun ex Klotzsch) E.G.Andrews & Windham that has laminae similar to those of P. masafuerae. Two hybrids, P. ×leucospora (Klotzsch) T.Moore and P. ×tricholepis (Mickel & Beitel) T.Moore, show a laminar morphology very similar to that of the Cerro Alto plant. An interesting similarity is the irregularity of the shape of the laminae and of the length of the pinnae, pointed out by Wagner (1962), that we notice also in our plant. However, the Cerro Alto plant differs from P. ×leucospora by a shorter stipe and a shorter terminal segment, and from P. ×tricholepis also by a shorter stipe, broader pinnae and a lamina reduced at its base. These observations lead us to believe that P. macrocarpa and P. masafuerae are probably the parents of the Cerro Alto plant. As the hybrid is presumed tetraploid, based 76 FERN GAZ. 20(2):65-78. 2015 on FCM values, we assume that tetraploid P. macrocarpa is involved in the combination, and that P. masafuerae is also tetraploid. We conclude that the Cerro Alto plant is a hybrid new to the Juan Fernández Archipelago.

Pleopeltis ×cerro-altoensis Danton & Boudrie, hyb. nov. Abortivis sporis, tetraploideum, hybridum filix est, repente rhizomate, 5-7 cm in longitudine fronde, angustissime alato atrobrunneo petiolo, appressis, fimbriatis peltatisque paleis penitus pinnatifido lanceolato limbo atque infra atrobrunnea et super viridiluteola rhachidi. A P. macrocarpa pinno-pinnatifido limbo, et a P. masafuerae basi reducta lanceolato limbo praecipue differt.

Types: CHILE. Archipel Juan Fernández, île Robinson Crusoe, Cerro Alto, Quebrada Puerto Inglés, rochers en falaise sur le versant est-sud-est du cerro, 300 m, 22 janvier 1999, Danton B(652)619 [holotype: SGO (Figure 6); isotypes: P, private herb. P. Danton].

Description: Epilithic fern. Rhizomes creeping, 2.5-3 mm diam. (4-5 mm including the scales), branched, densely covered with oblanceolate, acute, clathrate, bicolorous scales, with dark centres and narrow, light brown, and finely dentate-fimbriate margins. 1 Fronds monomorphic, 0.5-1 cm apart, 6-15 × 0.6-4 cm. Stipes 2-5 cm, about /3 - ¼ as long as laminae, dark brown, with sparse, elongate scales. Laminae coriaceous, dark green, matt adaxially, light green to yellowish and slightly glaucous abaxially, narrowly elliptic to lanceolate, deeply pinnatifid, tapering proximally to reduced lobes, and below that long-decurrent (young fronds entire to sinuately lobed), distally ending in a long pointed to obtuse segment; laminar surfaces covered with numerous, scattered, peltate- based, appressed, bicolourous lanceolate scales with dentate or fimbriate margins, scales more abundant abaxially than adaxially. Dark brown rachis colour ending at the base of the apical segment abaxially, adaxially the colour ending at the first or second proximal pinnae and becoming green-yellowish distally. Pinnae 5-7 per side, triangular to oblong, 24-26 × 5.5-6 mm (for the longest ones), of variable length, oblique to rachis, alternate to more opposite in the distal half of the laminae, apices obtuse to rounded; laminar margins cartilaginous and slightly recurved. Stomata (45)- -(63) µm long. Sori round to slightly oblong, ca. 0.3 cm diam., exindusiate, mixed with peltate scales, median on both sides of the costae or (in less divided blades) of the rachis. Spores whitish to yellowish, malformed and irregular in shape and in size, (53)- -(90) µm long. Tetraploid (4x). 53.5

Etymology: The epithet of the taxon refers to the locality, Cerro Alto, where the plant was found. 73.5

Distribution and habitat: To our knowledge, endemic to Robinson Crusoe Island (Masatierra) in Juan Fernández Archipelago (Chile), on southern flanks of Cerro Alto. Plants grow in a limited population of four small, relatively close patches, on small rocky edges covered by thin soil, on subvertical unstable volcanic cliffs, exposed to the winds, at 300 m elevation (Figure 3). Due to its ability to revive, it can tolerate periods of drought. BOUDRIE et al.: PLEOPELTIS ×CERRO-ALTOENSIS 77

Specimens examined (paratypes): CHILE. Juan Fernández Archipelago, Robinson Crusoe Island, Quebrada Puerto Inglés, rebords de rochers en falaise et balmes sur le versant est-sud-est du Cerro Alto, 300 m, M. Boudrie et session SBF, 20 février 2002, Boudrie 3793 (P).

Specimens ex horto examined (paratypes): CHILE. Juan Fernández Archipelago, Robinson Crusoe Island, Puerto Inglés, rebords de rochers en falaise et balmes sur le versant est-sud-est du Cerro Alto, 300 m, ex horto Conservatoire Botanique de la ville de Mulhouse (Haut-Rhin, France), M. Boudrie et J.-P. Reduron, 22 août 2003, Boudrie 3966 (P); idem, Ph. Danton et M. Hildebrand, 13 juin 2006, Z(hs)1727 (private herb. Ph. Danton); idem, M. Boudrie et J.-P. Reduron, 5 octobre 2009, Boudrie 4500 (P); idem, M. Hildebrand, 29 septembre 2014, Bizot 2905 (private herb. A. Bizot), and P. Danton - Mulhouse - 1, 2 & 3 (private herb. R. Viane).

IUCN Status: “CR”

Conservation ex situ: A plant was previously cultivated, from 1999 to 2004, in the garden of the Administration of the National Park, on Robinson Crusoe Island, but is now dead. Another specimen has been maintained and propagated in the Botanical Conservatory of the town of Mulhouse (France) since 2002.

ACKNOWLEDGEMENTS We thank Mr. Christian Diaz (San Juan Bautista, Isla Robinson Crusoe, Chile), the Guards of the Parque Nacional Archipiélago Juan Fernández (San Juan Bautista, Isla Robinson Crusoe, Chile), MM. Hernan Gonzalez (San Juan Bautista, Isla Robinson Crusoe, Chile) and Ivan Leiva (San Juan Bautista, Isla Robinson Crusoe, Chile). We are also extremely grateful to Dr. Caroline Van den heede for the chromosome count of Pleopeltis guttata, Dr. Jacques Florence (IRD-MNHN, Paris) for the translation of the diagnosis into Latin, Dr. Alan R. Smith (University of Berkeley, USA) for useful recommendations, Mathieu Hildebrand (Mulhouse, France), Christophe Perrier (Saint- Crépin, France), Jean-Pierre Reduron (Mulhouse, France), as well as to the Société Botanique de France (Paris, France).

REFERENCES BARRERA MOSCOSO E., 1997. Helechos de Juan Fernández. Publ. Ocas. Mus. Nac. Hist. Nat., 51: 5-104. BOUDRIE M., 2003. Les ptéridophytes de l’île Robinson Crusoe (archipel Juan Fernández, Chili). J. Bot. Soc. Bot. France 24: 32-48. CHRISTENSEN C. & SKOTTSBERG C., 1920. The Pteridophyta of Juan Fernandez Islands. In Skottsberg C., Natural History of Juan Fernandez and Easter Island. Vol. II. Botany. Almqvist & Wiksells Eds., Uppsala, 46 p. DANTON P., BRETEAU E. & BAFFRAY M., 1999. Les îles de Robinson. Trésor vivant des mers du Sud – Entre légende et réalité. Yves Rocher, Nathan, Paris, 144 p. DANTON P. & PERRIER C., 2006. Nouveau catalogue de la flore vasculaire de l’archipel Juan Fernández (Chili). Acta Bot. Gallica 153 : 399-587. EVANS A.M., 1963. New chromosome observations on the Polypodiaceae and Grammitidaceae. Caryologia 16: 671-677. GAY C., 1853. Historia fisica y politica de Chile, Botanica. Tome VI. C. Gay (Ed.), 78 FERN GAZ. 20(2):65-78. 2015

Paris, Santiago, Chile, 531 p. HEMSLEY W.B., 1884. Report on the Botany of Juan Fernandez, the South-Eastern Moluccas and the Admiralty Islands [Voyage of H.M.S. Challenger]. Juan Fernandez and Masafuera. Botany, vol. 1, part 4, 100 pp. + Plates LIV-LWIII. JARRETT F.M., MANTON I. & ROY S.K., 1968. Cytological and taxonomic notes on a small collection of living ferns from Galapagos. Kew Bull. 22: 475-480. JOHOW F., 1892-93. Los Helechos de Juan Fernández. Anales Univ. Chile 82: 741-757, 977-1005. JOHOW F., 1896. Estudios sobre la Flora de las Islas de Juan Fernández. Imprenta Cervantes, Santiago de Chile, XI + 289 p. + XVIII láminas + 2 mapas. LOOSER G., 1961. Los Pteridofitos o helechos de Chile I. Revista Univ. (Santiago) 46: 213-262. LOOSER G., 1962. Los Pteridofitos o helechos de Chile (Excepto Isla de Pascua) II. Revista Univ. (Santiago) 47: 17-31. LOOSER G., 1965-66. Los Pteridofitos o helechos de Chile (Excepto Isla de Pascua) III. Revista Univ. (Santiago) 50-51: 75-93. LOOSER G., 1968. Los Pteridofitos o helechos de Chile (Excepto Isla de Pascua) IV. Revista Univ. (Santiago) 53: 27-39. MARTICORENA C. & RODRIGUEZ R. (Eds.), 1995. Flora de Chile. Vol. I, Pteridophyta – Gymnospermae. Universidad de Concepción, Concepción, Chile. MARTICORENA C., STUESSY T.F. & BAEZA C. M., 1998. Catalogue of the vascular flora of the Robinson Crusoe or Juan Fernandez Islands, Chile. Gayana, Bot. 55: 187-211. MICKEL J.T. & BEITEL J.M., 1987. Notes on ×Pleopodium and Pleopeltis in Tropical America. Amer. Fern J. 77: 16-27. OTTO E.M., JANSSEN T., KREIER H.-P. & SCHNEIDER H., 2009. New insights into the phylogeny of Pleopeltis and related Neotropical genera (Polypodiaceae, Polypodiopsida). Molec. Phylogen. Evol. 53: 190-201. SKOTTSBERG C., 1953. A supplement to the Pteridophytes and Phanerogams of the Juan Fernandez and Easter Island. The Natural History of Juan Fernandez and Easter Island. 2: 763-792. SMITH A.R. & TEJERO-DIéZ J.D., 2014. Pleopeltis (Polypodiaceae), a redefinition of the genus and nomenclatural novelties. Bot. Sci. 92: 43-58. SOTA E.R. DE LA, 1967. Sinopsis de las familias y géneros de Pteridófitas de Argentina, Uruguay y Chile (Incluyendo las Islas Juan Fernández y Pascua). Revista Mus.La Plata, Secc. Bot. 10: 187-221. TRYON R.M. & STOLZE R.G., 1993. Pteridophyta of Peru. Part V. 18. - . - 21. Polypodiaceae. Fieldiana, Bot. 32: 1-190. WAGNER W.H., 1962. Irregular morphological developments in hybrid ferns. Phytomorphology 12: 87-100. WAGNER JR. W.H. & WAGNER F.S., 1975. A hybrid polypody from the New World tropics. Fern Gaz. 11: 125-135. WALKER T.G., 1973. Additional cytotaxonomic notes on the pteridophytes of Jamaica. Trans. Roy. Soc. Edinburgh 69: 109-135. ZULOAGA F., MORRONE O. & BELGRANO M., 2008. Catálogo de las Plantas Vasculares del Cono Sur (Argentina, Sur de Brasil, Chile, Paraguay y Uruguay). Vol. 1: Pteridophyta, Gymnospermae, Monocotyledoneae. Missouri Botanical Garden Press, St. Louis, USA. FERN GAZ. 20(2):79-89. 2015 79



Associated contributor of the Biogeographic Institute of Trier University / Germany Neuwiese 13, D-54296 Trier, Germany Email: [email protected]

Keywords: Dryopteris remota, PCR-direct, PgiC 15/16 fragment, genotyping, phylogeny

ABSTRACT Since its discovery by A. Braun in 1834, there has been speculation on the origins of Dryopteris remota. The present contribution suggests that none of the previous assumptions are correct. Dryopteris remota is triploid and of hybrid derivation. Its parental species are the sexual allotetraploid D. carthusiana and an as yet unidentified sexual diploid species, most likely identical with the unknown parent of the apomictic diploid D. affinis. These assertions are based on comparison of traits, including habit, apomixis, colour of insertions of pinnae of the species under examination, and fragment length variation of the nuclear PgiC gene.

INTRODUCTION The phylogeny and taxonomic status of Dryopteris remota (A.Br.) Druce is probably one of the most controversially discussed of any Central European fern species. When Alexander Braun discovered the plant near Baden-Baden in 1834 (Baden-Württemberg/ S-Germany), he thought the new taxon to be a variation of D. villarii (Bellardii) Woynar ex Schinz & Tell. As such it was listed in 1843 by J. C. Döll in “Rheinische Flora”. In his book “Betrachtungen über die Erscheinung der Verjüngung in der Natur” of 1851, Braun revised his view in a footnote and described the taxon as a hybrid of D. filix-mas (L.) Schott and D. carthusiana (Vill.) H.P.Fuchs or D. dilatata (Hoffm.) A.Gray. He nonetheless attributed species rank to this taxon by calling it “Aspidium remotum”, i.e. in current nomenclature D. remota. Since then a substantial number of renowned fern experts have speculated on its origin and taxonomic classification. In 1973 Benl and Eschelmüller presented an exhaustive description of this continuing dispute among scientists. Further confusion had arisen following the discovery of a fern hybrid near Lake Windermere in north-west England in the middle of the 19th century. The morphology of this hybrid resembled very much that of D. remota, but it had abortive spores. In 1859 A. Braun found a single plant of D. remota among a large population of D. filix-mas near Aachen (Nordrhein-Westfalen/ Germany), which led him to the conclusion that D. remota must be a variation of D. filix-mas. He did not realize that this plant was not D. remota but actually the same hybrid as that from Lake Windermere. He transplanted this hybrid to the Botanical Garden of Berlin and for approximatively 15 years he sent incorrectly labelled exsiccata from there to herbaria and to individual scientists before this mistake was discovered (Benl & Eschelmüller, 80 FERN GAZ. 20(2):79-89. 2015

1973). Further examination of a frond sample in the herbarium at Bonn revealed that Braun’s finding was the second record of the tetraploid hybrid D. carthusiana × D. filix-mas, as discovered earlier at Lake Windermere (Krause et al., 2001), and which has now been typified as D. × brathaica Fraser- Jenk. & Reichst. (1977). It is now possible to distinguish the two taxa through cytology (Manton, 1950), morphology (Fraser-Jenkins & Reichstein, 1984; Krause et al., 2001; Freigang et al., 2013), chemotaxonomy (Widén et al., 1976) and molecular genetics (Freigang et al., 2013). Dryopteris remota is today recognised as a triploid species of hybrid derivation, but the dispute concerning its origin continues. From fundamental works by W. Döpp (1932; 1933; 1935; 1939; 1955) and I. Manton (1932; 1938; 1939; 1950), Döpp considered D. remota to be the hybrid of D. dilatata × D. affinis (Lowe) Fraser-Jenk. (Döpp, 1955). Reichstein, Fraser-Jenkins and others are of the opinion that the diploid apomictic D. affinis must be one of the parents due to the characteristics of D. remota (Fraser-Jenkins & Reichstein, 1984). In consequence the second parent would have to be a diploid sexual taxon that contributes the characteristics known from the D. carthusiana group to the genotype of D. remota. Dryopteris expansa (C.B.Presl) Fraser-Jenk. & Jermy was proposed (Widén et al., 1971, Fraser-Jenkins & Reichstein, 1984), even though chemo-taxonomic results did not match well (Widén et al., 1970; 1971; 1996). Furthermore, in an on-line discussion of the British Fern Group, Fraser-Jenkins (30 December 2008) reiterated doubts as to D. expansa being involved: “Although I agree that D. expansa is the most likely second ancestor of D. remota, it ought to have a ? along that line as it has never been proved in any way and the possibility of some other, even perhaps Asiatic species being involved still remains.” To shed light on the ongoing discussions, the nuclear PgiC-marker of many species of the genus Dryopteris, including D. remota, has been examined.

MATERIAL AND METHODS Material The samples used in the investigation are listed in Table 1. Herbarium vouchers, currently in the collectors’ private herbaria will be deposited in JE.

Methods Samples were analyzed at the nuclear PgiC gene (cytosolic 6-Phosphoglucose- isomerase), individual fragments were generated and their lengths compared. The intron fragment between exons 15 and 16 proved especially suitable for comparison as it shows a species specific length for a number of species of the genus Dryopteris, especially in the D. carthusiana group (Juslén et al., 2011, Freigang et al., 2013). For this fragment Ishikawa et al., 2002 constructed an ‘EPIC’- primer pair (Exon Primed Intron Crossing), which offers almost general applicability. Due to a high frequency of poor PCR yields or complete PCR failures, the method described in Freigang et al. (2013) was partly replaced by the PCR-direct method of ThermoScientific (Phire Plant Direct PCR Kit, F-130WH). This method worked without DNA isolation and PCR yields were often higher and, according to the producer instructions the workflow consisted of only two steps. 1. A disc of 0.3-0.5 mm diameter was punched out from fresh or silica gel-dried leaf material and put into PCR-solution. 2. PCR was carried out according to the producer instructions. The uncleaned amplicon was genotyped in a sequencer (ABI 3500, Applied Biosystems or MegaBace Instrument, Healthcare). Employment of different BUJNOCH: THE PHYLOGENY OF DRYOPTERIS REMOTA 81 sequencers made transformation of results necessary (marked with an asterisk). The analysis of the generated data was first made with Fragment Profiler software (Healthcare), then later with GeneMapper software (Applied Biosystems).Sometimes the sequencing device produced a double peak in consecutive basepairs (bp). This could be an artefact or may be the result of PCR-conditions. The relative height of signals within an electropherogram depended firstly on the number of sets of chromosomes causing the signal and, secondly, on the relative PCR yield of the 15/16-fragment of the existing alleles.

RESULTS The genotypes of samples 1 to 24 are summarised in Table 2. Signals of electropherograms which were not separated are noted in the same table-field. Electropherograms of a subset of samples are shown in Figures 1-3 (underlined in Tables 1 and 2).

DISCUSSION In the case of apomictic reproduction in ferns the sporophyte grows directly from a gametophyte with the unreduced number of chromosomes, and without gametes being involved. The sporophyte produces genetically uniform diplospores through a restitution nucleus; in the case of apomicts of Dryopteris, restitution occurs in the last mitotic cell division before the production of spore mother cells (Manton, 1950), resulting in the production of eight, rather than the usual 16 spore mother cells. Sporogenesis then results in the production of 32 (instead of the usual 64) spores, each with the same number of chromosomes as the sporophyte. Fischer (1909; 1919) detected apomixis in D. remota and Manton demonstrated that it is a triploid taxon, with three different (non-homologous) chromosome sets (i.e. a heterotriploid fern) as in the occasional production of a spore mother cell of the ‘16 cell type’, the chromosomes are unpaired (non-homologous), with 123 univalents (3x). The heterotriploid character is also shown by the electropherogram of PgiC 15/16 fragments of the species (Figure 1), where three distinct peaks are observed.The electropherogram shows the following: The genome of D. remota contains at least three chromosome sets possessing fragments of different length and therefore derived from different alleles. This supports the results of cytology that the species is heterotriploid. The two shorter fragments of 488 bp and 500 bp correspond to the results seen in D. carthusiana (Figure 2), an allotetraploid species; no other taxon of the European ferns of the genus Dryopteris examined up to now shows this pattern. Comparison with D. cristata (L.) A.Gray (sexual allotetraploid) and D. intermedia (Mühl. ex Willd.) A.Gray (sexual diploid) reveals that the peak at 488 bp differs from that of D. intermedia (500 bp), and is generated by the chromosome set derived from the common parental taxon of D. cristata and D. carthusiana, referred to as D. “semicristata” (Sc) by Walker (1955), a diploid species as yet unknown. The signal at 500 bp in D. remota is identical with that of D. intermedia (I). The chromosome sets of D. carthusiana can be symbolized by ScScII. The assumption that the parental species of D. carthusiana are D. intermedia and D. “semicristata” is supported (Table 2) and confirms the result of Sessa et al. (2012). The results are also in agreement with chloroplast sequence data by Juslén et al. (2011), which showed that D. carthusiana and D. remota share a maternally inherited set of chromosomes, referred to as “semicristata”. Table 2 shows that the 15/16-fragment length of D. expansa is the same 82 FERN GAZ. 20(2):79-89. 2015

Table 1: Genotyped samples (Nomenclature according Fraser-Jenkins & Reichstein, 1984 and Fraser-Jenkins, 2007). RF: numbers according to rasters (see Niklfeld, 1971). Abbreviations of the names of collectors: GZ (G. Zenner/ Kirn), HWB (H. W. Bennert/ Ennepetal), JF (J. Freigang/ Bergatreute), SJ (S. Jeßen/ Chemnitz), WB (W. Bujnoch, Trier). Collector/ Sample no. & Origin Determination/ Internal Data species Date Portugal/ Azores/ São Miguel/ N- MPb1458, 1. Dryopteris GZ 28.5.2013 side and ridge at Pico da Vara, DE807, aemula Dr 8 1100 m MSL RUN32/2H Portugal/ Madeira/ N Funchal/ c. JF/GZ MPb1724, 2. D. affinis 600 m S of restaurant Ribeiro Frio 25.8.2011 RUN45/7A at the roadside EN 103 JFV1086 Germany/ RP/ Palatinate/ NE Wilgartswiesen, rock at WB/GZ DE032, 3. D. affinis Wiligartsburg, ca. 230 m MSL, RF 6.8.1996 RUN21/11B 6713/32 Germany/ NRW/ Märkisches MPb1442, 4. D. HWB Land, SW Hagen/ near Hasper DE791, carthusiana 27.05.2013 dam, RF 4603 RUN32/1B Germany/ NRW/ niederrheinisches MPb1482, 5. D. N.Neikes Tiefland/ E Venlo/ Nettetal/ DE812, carthusiana 05.07.2009 Poelvenn–Kuhlen/ RF 4603/23 RUN32/3E Germany/ RP/ middle Mosel/ W 6. D. DE083, Trier/ Sirzenicher Bachtal, RF WB 17.7.2005 carthusiana RUN22/10B 6205/23 Germany/ BW/ Oberschwaben/ 7. D. JF 22.10.2006 DE385, NE Ravensburg/ SE Bergatreute/ carthusiana JF 2006 RUN20/12A near Kiliansweiher, RF 8124/32 Germany/ RP/ Eifel/ SSE Daun/ WB 28.8.1984 DE024 8. D. cristata Schalkenmehrener Maar, RF GWBT2 spore RUN20/G11 5807/13 cultured France/ Haut–Rhin/ Vosges/ S Col de la Schlucht/ Sentier des Roches/ GZ/P.Holveck MPb1665, 9. D. dilatata NE “les Trois Four“, 1050 m 27.8.2014 Dr 4 RUN40/11D MSL, RF 7908/31 Germany/ BY/ upper Allgäu/ MPb1229, West–Grünten/ N Burgberg/ N GZ 19.7.2004 DE695, 10. D. expansa „Weinhalde“, northern slope, 780– Dr 20 GWBT156 795 m MSL, RF 8427/41 RUN28/11E MPb1230 Switzerland/ Wallis/ S Leukerbad/ GZ 18.7.2000 DE696, 11. D. expansa Dala Valley/ lower Lochwald, Dr 1 GWBT182 1260 m MSL, RF 9611/23 RUN28/11F BUJNOCH: THE PHYLOGENY OF DRYOPTERIS REMOTA 83

HWB 12. D. USA/ / Rutland/ Leicester DE368 15.8.2001 intermedia Hollow Trail/ NNE Brandon RUN25/A11 SJ-3476 France/ Hérault/ Haut Languedoc,/W Bédarieux/ N GZ/WB DE067, GZ-58, 13. D. oreades Colombières-s-Orb/ near “La 23.7.1994 Dr 1 RUN18/11C Fage”, c. 760 m MSL Italy/ Toscana/ Appennino Tosco SJ/J.Müller Emiliano/ NW Pistoia/ W Abetone/ DE288, 14. D. oreades 12.8. 1992 W M Gomito/Val di Luce, c. 1480 RUN19/11B SJ-2226 m MSL MPb1562 15. D. pallida Italy/ Sicily/ Mount Etna above JF 1.1.2007 DE861 subsp. pallida Zafferana JFV542 RUN37/B12 Germany/BW/ Northern– FZ-90/7, GZ/WB Schwarzwald/ SSE Bad Herrenalb/ GWBT 32, 16. D. remota 1.8.1990 valley of Rothenbach, 440 m DE26, spore cultured MSL, RF 7216/21 RUN19/12G Germany/ BY/ W Berchtesgaden/ MPb1516, Ramsau/ SW of GZ/JF 5.8.2013 17. D. remota DE 824, Schwarzbachwacht, c. 900 m Dr 2, RUN35/11B MSL, RF 8343/31 Schwitzerland/ Berner Oberland/ MPb1235, DE SJ 21.7.1992 18. D. remota Innertkirchen/ Urbachtal in 637, RUN28/ SJ-2270 Vordertalwald, RF 9315/12 11C Austria/ Salzburg/ Pinzgau/ E GZ 6.8.2014 MPb1650, 19. D. remota Rauris/ valley of Geißbach, c. Dr 10 RUN44/11H 1060 m MSL, RF 8744/31 Romania/ S-Karpaten/ N- Fǎgǎraș/ MPb1236, NW des Rifugio “Negoiu”/ SSE SJ 5.8.1985 SJ- 20. D. remota DE638, Porumbacu de Sus/ Şerbota Tal c. 2560-2 RUN28/ 11D 1000 m MSL Germany/ BY/ West-Allgäu/ NW MPb1250, JF 4.6.2011 JF- 21. D. remota Scheidegg/ forest N “Lötz”, c. 700 DE710, V1008 m MSL, RF 8425/11 RUN28/12D North-Italy/ Prov. Varese /E Luino/ GZ/JF/SJ W Ponte Tresa/ Tresa-Valley near MPb1580, 22. D. remota 15.6.2014 Dr Biviglione, 250 m MSL, RF RUN40/9D 33 0018/21 SJ-3026, TR- Northeast-Turkey/ Rize/ E 4011, CRFJ MPb1738, 23. D. remota Trabzon, S Ardeşen, Cat - 4037, RUN47/A1 Ҫamlihemşin, c. 400m MSL 27.08.1973 Bulgaria / North-Pirin/ E des MPb1452 Kameniski vrah/ NW des Vihren/ SJ 24.7.1981 24. D. villarii DE801, W Bansko, Bela-reka-Valley, 2150 SJ-335 RUN32/2C m MSL 84 FERN GAZ. 20(2):79-89. 2015

Table 2: Genotyping results for the length of the 15/16 PgiC fragment of samples 1 to 24 in base pairs (bp). The sample numbers in Table 2 correspond with those in Table 1. The measurements in Table 2 indicate that the two shorter fragments of D. remota correspond with those of D. carthusiana and the third fragment of D. remota with that of D. affinis and D. oreades.

Fragment length Species in base pairs (bp)

1. Dryopteris aemula 525 2*. D. affinis subsp. affinis 530 3. D. affinis subsp. affinis 530 4. D. carthusiana 487 499 5. D. carthusiana 486 499 500 6. D. carthusiana 486 487 498 499 7. D. carthusiana 487 500 8. D. cristata 487 521 9. D. dilatata 499 517 10. D. expansa 499 500 11. D. expansa 499 500 12. D. intermedia 500 13. D. oreades 529 530 14. D. oreades 530 15. D. pallida subsp. pallida 516 16. D. remota 487 488 499 500 530 17. D. remota 487 500 531 18. D. remota 487 499 530 19*. D. remota 489 501 530 20. D. remota 487 500 530 21. D. remota 488 500 530 22. D. remota 489 501 531 23*. D. remota 489 501 531 24. D. villarii 532 533

* Employment of different sequencers made transformation of results necessary. BUJNOCH: THE PHYLOGENY OF DRYOPTERIS REMOTA 85 as that of D. intermedia. As mentioned in the introduction, the involvement of D. expansa in the parentage of D. remota has been the subject of much discussion. But this scenario would entail that the second parent of D. remota must be a diploid apomictic taxon of the D. affinis agg. with signals at 488 bp and 530 bp. Despite study of many taxa of the D. affinis agg., this combination has never been found. The same applies to the combination of signals at 500 and 530 bp. During the first half of the last century D. remota was thought to be a hybrid of D. carthusiana × D. filix-mas, and Döpp endeavoured to backcross D. remota with the supposed parental species. He was successful in hybridizing D. remota only with D. carthusiana (Döpp, 1935). This provides some support for the conclusion that D. carthusiana is one parent of D. remota. Already H. Fischer pointed to the fact that such a hybrid should also emerge in nature and he mentioned that the pharmacist H.K. Woynar (Graz) allegedly found intermediate forms in mixed stands of the two species at different sites in Tirol. Since 2011 putative hybrids of D. remota × D. carthusiana have been found at various locations in the wild (Freigang et al., in prep.). The remaining question relates to the unknown identity of the third signal in the electropherogram of D. remota, at 530 bp. The chromosome set causing this third signal at 530 bp would have to contribute two qualities to D. remota that are characteristic of it and cannot be derived from the genetic material of D. carthusiana - the obligatory apomixis and the blue-black colour at the insertion of the pinnae (Fraser-Jenkins & Reichstein, 1984). Both features also exist in the D. affinis agg., but not in that of D. filix-mas. Examination of the diploid taxon D. affinis (Figure 3) shows a single peak at 530 bp. According to current opinion D. affinis is a diploid apomict fern that shows non-homology of the two chromosome sets in 16-celled sporangia (Fraser-Jenkins, 2007) and it is presumed to have been derived from a cross between the sexual diploid D. oreades Fomin (OO) and a sexual diploid taxon, not yet identified but referred to as D. “semiaffinis” (SaSa) (Krause, 1998). The PgiC 15/16 fragments of both taxa correspond to the third peak in D. remota. As D. remota is triploid and its genome

Figure 1. Electropherogram of Dryopteris remota (Schwarzwald, Smp.No. 16)

Figure 2. Electropherogram of Dryopteris carthusiana (Bergatreute, Smp. No. 7)

Figure 3. Electropherogram of Dryopteris affinis (Wilgartswiesen, Smp. No. 3) 86 FERN GAZ. 20(2):79-89. 2015 contains two sets of chromosomes of allotetraploid D. carthusiana, D. affinis as the second parent is out of question, because as an apomictic taxon it would contribute both its chromosome sets to D. remota, resulting in a tetraploid taxon. This implies that the third peak (530 bp) in D. remota must be derived from a diploid sexual taxon with the 530 bp peak, and the only possible candidate from the results presented here are D. oreades or the missing diploid taxon D. “semiaffinis”. Unfortunately the length of PgiC 15/16 fragments of D. oreades and D. “semiaffinis” cannot be distinguished with the method used here. Of these two, D. “semiaffinis” is the more likely parent; D. oreades does not exhibit blue-black insertions, nor apomixis. We are left to infer that both diploid D. affinis and triploid D. remota have inherited these characteristics from D. “semiaffinis”, and hence the phylogeny of D. remota could be analogous to that of D. affinis, as shown in Table 3. All the evidence suggests that “semiaffinis” induces apomixis in its hybrids and species of hybrid derivation, in contrast to hybrids involving D. oreades. This is the case in all heterodiploid as well as heterotriploid taxa of the D. affinis complex, which are all fertile but to varying degrees. In hybrids of taxa of the D. affinis complex with D. filix-mas, apomixis is also expressed, despite that fact the “semiaffinis” chromosome set is only one of four or five sets in the nucleus. The hybrid of D. carthusiana and D. “semiaffinis” (Table 3) has three non-homologous sets of chromosomes and would be expected to be sterile, in contrast to D. remota which is fertile and can produce viable offspring in the wild. This case of D. remota, gives further support to the hypothesis that

Table 3. Diagram of the hypothetical genesis of Dryopteris remota and D. affinis Abbreviations of chromosome sets: O = oreades, Sa = “semiaffinis“, I = intermedia, Sc = “semicristata“

D. oreades D. “semiaffinis” D. carthusiana Parental Taxa homodiploid, sexual homodiploid, sexual allotetraploid, sexual

Chromosome sets of a somatic cell OO SaSa IScISc Chromosome set(s) O Sa ISc of a gamete


Primary hybrid OSa SaISc heterodiploid, sterile heterotriploid, sterile Development of apomixis

Progeny D. affinis D. remota

chromosome sets of a OSa SaISc somatic cell heterodiploid, heterotriploid, apomictic apomictic fertile fertile BUJNOCH: THE PHYLOGENY OF DRYOPTERIS REMOTA 87 the set of chromosomes from “semiaffinis” has the ability to transfer the qualities of dark insertion of pinnae, and apomixis coupled with fertility. The distribution of D. remota lies between the Pyrenees and the and thus falls within the distribution of D. carthusiana and D. affinis (Fraser-Jenkins & Reichstein, 1984). The genetic variability of D. remota was examined by the isozyme-method (Schneller & Holderegger, 1994) and also by RAPDs (Schneller et al., 1998). The first method did not reveal any genetic variability within or between populations, the second only little. The authors suggested that the species developed relatively recently and only once, presumably in Caucasus or the Mediterranean region, and spread over the now populated area by ‘long-distance spore dispersal’. Since spores of apomicts produce sporophytes without the need for sexual fertilization, then an individual spore has the potential to give rise to a new individual or eventually even a new population. During the past 180 years of discussion about the origin of D. remota other taxa have been proposed as possible parents, for example D.villarii (Braun in Döll, 1843), D. dilatata (Döpp, 1935; 1955), D. aemula (Aiton) O.Kuntze (Widén et al., 1971) or D.pallida subsp. pallida (Bory) Fomin and subsp. raddeana Fraser-Jenk. (Peroni et al., 1991). The results in Table 2 rule out almost all of these possibilities, with the exception of D. pallida subsp. raddeana, as samples of this taxon were not included in the study.

ACKNOWLEDGEMENTS I would like to thank Prof. Dr. M. Veith, head of the Institute of Biogeography at Trier University, for providing me with a working place and also PD. Dr. A. Hochkirch, head of laboratory and the technicians at the laboratory. In particular I would like thank my colleagues, listed in Table 1, who made samples available for me. I would like to thank S. Jeßen and G. Zenner for their discussion and their valuable suggestions, also E. Erfurth and J. Beninde who helped with the translation of this paper into English. Finally I would like to thank the two reviewers and the editor for help and advice.

REFERENCES BENL,G. & ESCHELMüLLER, A. 1973. über „Dryopteris remota“ und ihre Vorkommen in Bayern. Ber. Bayer. Bot. Ges. 44: 101-141. BRAUN, A. 1851. Betrachtungen über die Erscheinung der Verjüngung in der Natur, ins- besondere in der Lebens- und Bildungsgeschichte der Pflanzen. Engelmann, Leipzig. DöLL, J. C. 1843. Rheinische Flora. Brönner, Frankfurt a. M. DöPP, W. 1932. Die Apogamie bei Aspidium remotum A.Br. Planta 17: 86-152. DöPP, W. 1933. Weitere Untersuchungen an apogamen Farnen. Ber. Dtsch. Bot. Ges. 51: 341-347. DöPP, W. 1935. Versuche zur Herstellung von Artbastarden bei Farnen, I. Ber.Dtsch. Bot. Ges. 53: 630-636. DöPP, W. 1939. Cytologische und genetische Untersuchungen innerhalb der Gattung Dryopteris. Planta 29: 481-533. DöPP, W. 1955. Experimentell erzeugte Bastarde zwischen Dryopteris filix-mas (L.) und D. paleacea (Sw.) C.Chr. Planta 46: 70-91. FISCHER, H. 1909. über Aspidium remotum A.Br.: Kreuzung oder Mutation? – Ein neuer Fall von Apogamie. Ber. Dtsch. Bot. Ges. 27: 495-502. FISCHER, H. 1919. Apogamie bei Farnbastarden. Ber. Dtsch. Bot. Ges. 37: 286-292. 88 FERN GAZ. 20(2):79-89. 2015

FRASER-JENKINS, C. R. & REICHSTEIN, T. 1977. Dryopteris ×brathaica Fraser-Jenk. & Reichst. hybr. nov., the putative hybrid of D. carthusiana × D. filix-mas. Fern Gaz. 11: 337. FRASER-JENKINS, C. R. & REICHSTEIN, T. 1984. Dryopteris. In: Kramer, K. U. (ed.) Hegi, Illustrierte Flora von Mitteleuropa, 1(1), ed. 3, pp. 136-169. Paul Parey, Berlin & Hamburg. FRASER-JENKINS, C. R. 2007. The species and subspecies in the Dryopteris affinis group. Fern Gaz. 18: 1-26. FRASER-JENKINS,C. R. 2008. http://tech.groups.yahoo.com/group/ukferns/message/863. FREIGANG, J., BUJNOCH, W. & ZENNER, G. 2013. Erstfunde von Dryopteris ×brathaica Fraser-Jenk. & Reichst. (Dryopteridaceae, Pteridophyta) in Süd-Deutschland – morphologische und molekulargenetische Untersuchungen. Kochia 7: 67-86. ISHIKAWA, H., WATANO, Y., KANO, K., ITO, M. & KURITA, S. 2002. Development of primer sets for PCR amplification of the PgiC gene in ferns. J. Plant. Res. 115: 65-70. JUSLéN, A.,VäRE, H., WIKSTRöM, N. 2011. Relationships and evolutionary origins of polyploid Dryopteris (Dryopteridaceae) from Europe inferred using nuclear PgiC and plastid trnL-F sequence data. Taxon 60: 1284-1294. KRAUSE, S. 1998. Dryopteris Adans. (Dryopteridaceae). In: Wisskirch, R. & Haeupler, H. (Eds) Standardliste der Farn- und Blütenpflanzen Deutschlands, Vol. 1, pp. 182-183. Ulmer, Stuttgart. KRAUSE, S., HACHTEL, M. & BENNERT, H. W. 2001. Dryopteris ×brathaica Fraser-Jenk. & Reichst. (Englischer Wurmfarn; Drypoteridaceae, Pteridophyta) in Deutschland. Feddes Repert. 112: 247-260. MANTON, I. 1932. Contributions to the cytology of apospory in ferns in Britain. Nature 144: 291. MANTON, I. 1950. Problems of Cytology and Evolution in the Pteridophyta. University Press, Cambridge. MILDE, J. 1865. Die höheren Sporenpflanzen Deutschland’s und der Schweiz. Felix, Leipzig. NIKLFELD, H. 1971. Bericht über die Kartierung der Flora Mitteleuropas. Taxon 20: 545 – 571. PERONI, A., PERONI, G., RASBACH, H., RASBACH, K. & REICHSTEIN, T. 1991. Dryopteris remota (A.Braun) Druce in Italien. Farnblätter 23: 1-13. SCHNELLER, J. J. & HOLDEREGGER, R. 1994. Lack of isozyme variation in the agamosporous fern Dryopteris remota (A.Braun) Druce. Amer. Fern J. 84: 94-98. SCHNELLER, J. J., HOLDEREGGER, R., GUGERLI, F., EICHENBERGER, K. & LUTZ,E. 1998. Patterns of genetic variation detected by RAPDs suggest a single origin with subsequent mutations and long-distance dispersal in the apomictic fern Dryopteris remota (Dryopteridaceae). Amer. Fern J. 85: 1038-1042. SESSA, E. B., ZIMMER, E.A. & GIVNISH, T. J. 2012. Unraveling reticulate evolution in North American Dryopteris (Dryopteridaceae). BMC Evol. Biol. 12: 104-127. WALKER, S. 1955. Cytogenetic studies in the Dryopteris spinulosa complex I. Watsonia 3: 193-209. WIDéN, C.-J., EUW, J. VON, & REICHSTEIN, T. 1970. Trispara-aspidin, ein neues Phloroglucid aus dem Farn Dryopteris remota (A.Br.) Hayek. Helv. Chim. Acta 53: 2176-2188. BUJNOCH: THE PHYLOGENY OF DRYOPTERIS REMOTA 89

WIDéN, C.-J., VIDA, G., EUW, J. VON, REICHSTEIN, T. 1971. Die Phloroglucide von Dryopteris villarii (Bell.) Woynar und anderer Farne der Gattung Dryopteris sowie die mögliche Abstammung von D. filix-mas (L.) Schott. Helvetica Chimica Acta 54: 2824-2850. WIDéN, C.-J., LOUNASMAA, M., JERMY, A.C., EUW, V. J., REICHSTEIN, T. 1976. Die Phloroglucide von zwei Farnhybriden aus England und Schottland, von authentischem “Aspidium remotum” A. Braun und von Dryopteris aemula (Aiton) O. Kuntze aus Irland. Helv. Chim. Acta 59: 1725 -1744. WIDéN, C.-J., FRASER-JENKINS, C. R., REICHSTEIN, T., GIBBY, M. & SARVELA, J. 1996. Phloroglucinol derivatives in Dryopteris sect. Fibrillosae and related taxa (Pteridophyta, Dryopteridaceae). Ann. Bot. Fennici 33: 69-100. 90 FERN GAZ. 20(2). 2015 FERN GAZ. 20(2):91-94. 2015 91



J. MAZUMDAR Department of Biological Sciences, Burdwan Town School, Burdwan-713101, India Email: [email protected]

The fern name Hemionitis arifolia (Burm.f.) T.Moore (Pteridaceae) is based on Asplenium arifolium Burm.f., first proposed by Burmann in 1768. Morton (1974: 316) found a sheet in the Geneva Herbarium (G, barcode G00360112, Figure 1) carrying two specimens and labeled as “Typus”. Alston in August, 1952, had determined these specimens as juvenile aureum L., not Asplenium arifolium. As the type belonged to another taxon Morton (1974) suggested the next available synonym Hemionitis cordata Roxb. ex Hook. & Grev. as the correct name for this taxon. The sheet carries Burmann’s annotation “Arifolium. Asplenium Arifolio Petiv Tab 50 Fig 12” indicating that Burmann saw these specimens (Figure 1). But in diagnosis he mentioned “simple ovate auriculate sinuous frond” and these specimens showing non- auriculate, non-sinuous frond (Figure 1), and not the basis of description or diagnosis validating the name (McNeill et al. 2012, Art. 9.3). Thus these specimens are not Types and on that basis the name Asplenium arifolium cannot be reduced as synonym of Acrostichum aureum. Hemionitis arifolia is the correct name for Hemionitis cordata. In the protologue Burmann mentioned Petiver’s (1702, t. 50, f. 12) and van Rhede tot Drakestein’s (1692-1703, t. 10, Fig. 2zzz) figures showing auriculate sinuous fronds and his diagnosis was based on these figures. Although Morton (1974) used the term “Type” for the specimen in G (Figure 1), he did not accept it, as is clear from his comment “If this really is the type…,”; it is not acceptable as lectotype (McNeill et al. 2012, Art. 7.10). Here I select van Rhede tot Drakestein’s figure from Hortus Indicus Malabaricus (1703) as Lectotype. But, as no collection data is available for the specimen on which this drawing was based (Johnston 1970; Wagenitz 1978; McNeill et al. 2012, Rec. 8A.1 & 8A.2) an epitype is selected.

TYPIFICATION Asplenium arifolium Burm. f., Fl. Ind. 231. 1768 — Hemionitis arifolia (Burm. f.) T. Moore, Index Fil. 114. 1859. — Parahemionitis arifolia (Burm. f.) Panigrahi, Amer. Fern J. 83(3): 90. 1993. Lectotype (designated here): van Rhede tot Drakestein, Hortus Indicus Malabaricus 12: 21, t. 10, 1703 (Figure 2). Epitype (designated here): India. West Bengal: Burdwan, Kanchannagar, near bridge above canal, 30 m, 21 Nov 2007, J. Mazumdar 59 (CAL Figure 3).

ACKNOWLEDGEMENTS I sincerely thank Dr. I. Valette, Secrétaire Herbiers and Conservatoire et jardin botaniques de la Ville de Genève, Switzerland (G) for providing permission for publication of image of G specimens; and Dr. A.R. Smith, University of California, Berkeley, U.S.A. and Mr. N.J. Turland, Botanischer Garten und Botanisches Museum Berlin, Germany for useful discussions. 92 FERN GAZ. 20(2):91-94. 2015

REFERENCES BURMANN, N.L. 1768. Flora Indica etc. Apud Cornelium Haek, Amsterdam. (http://www.biodiversitylibrary.org/item/123275). JOHNSTON, M.C. 1970. Still No Herbarium Records for Hortus Malabaricus. Taxon 19(4):655.

Figure 1. Type sheet of Asplenium arifolium Burm.f. from G (barcode G00360112). Image © Conservatoire et jardin botaniques de la Ville de Genève, Switzerland, reproduced with permission. MAZUMDAR: NOMENCLATURAL NOTE ON HEMIONITIS ARIFOLIA 93

MCNEILL, J., BARRIE, F.R., BUCK, W.R., DEMOULIN, V., GREUTER, W., HAWKSWORTH, D.L., HERENDEEN, P.S., KNAPP, S., MARHOLD, K., PRADO, J., PRUD’HOMME VAN REINE, W.F., SMITH, G.F., WIERSEMA, J.H. & TURLAND, N.J. (eds) 2012. International Code of Nomenclature For algae, fungi, and plants (Melbourne Code) adopted by the Eighteenth International Botanical Congress Melbourne, , July 2011 . (http://www.iapt-taxon.org/nomen/main.php?page=title). MORTON, C.V. 1974. William Roxburgh's fern types. Contr. U. S. Natl. Herb. 38(7): 283-396. (http://www.biodiversitylibrary.org/item/13798). PETIVER, J. 1702. Catalogus Classicus & Topicus Omnium Rerum Figuratum in V. Decadibus, Seu primo [-secundo] volumine Gazophylacii naturae & artis; Singulis ad proprias Tabulas & Numeros relatis. Ex Officinâ Christ. Bateman, vico vulgò dicto Pater-noster-row, London. (http://bibdigital.rjb.csic.es/ing/Libro.php?Libro=4831). VAN RHEDE TOT DRAKESTEIN, H.1692-1703. Hortus Indicus Malabaricus etc. Vols. 11 & 12. Sumptibus Johannis van Someren, et Joannis van Dyck, Amsterdam. (http://www.biodiversitylibrary.org/item/14380). WAGENITZ, G. 1978. The “Plantae Malabaricae” of the Herbarium at Göttingen Collected near Tranquebar. Taxon 27(5/6): 493-494.

Figure 2. Lectotype of Asplenium arifolium Burm.f.: Tab. 10 from van Rhede tot Drakestein’s Hortus Indicus Malabaricus. 94 FERN GAZ. 20(2):91-94. 2015

Figure 3. Epitype of Asplenium arifolium Burm.f. (J. Mazumdar 59) (CAL). FERN GAZ. 20(2):95-97. 2015 95



J. MAZUMDAR Department of Biological Sciences, Burdwan Town School, Burdwan-713101, India Email: [email protected]

The small epiphytic fern Loxogramme lankokiensis (Rosenst.) C.Chr. was first reported to occur in India from the Tirap District, Arunachal Pradesh, by Singh & Panigrahi (1987). Later Singh & Panigrahi (2005) suspected that an earlier record (Bhargavan & Joseph 1978) of L. grammitoides (Baker) C.Chr., from Arunachal Pradesh, Lohit District, may also represent L. lankokiensis and not L. grammitoides (Baker) C.Chr. However, they were unable to locate the specimen at CAL and could not make a definite decision (Singh & Panigrahi 2005). During the taxonomic study of ferns of Eastern India, I found this specimen of Loxogramme lankokiensis: INDIA. Arunachal Pradesh: Lohit District (N.E.F.A.), forest around Hayuliang along Dalai river, J. Joseph 48934 (originally from CAL, now at BURD; Figures 1A–C). In recent revisions Fraser-Jenkins (2008, 2012) followed the opinion of Dr. M.G. Price (University of Michigan, Michigan) and claimed that the report of L. lankokiensis from India was based on the misidentification of small plants of L. grammitoides. Close examination of J. Joseph 48934 showed features that matched with characters of L. lankokiensisas described in literature (Singh & Panigrahi 2005, Zhang et al. 2013). Loxogramme lankokiensis has dimorphic fronds (Figure 1C) and rhizome scales with entire margins whereas L. grammitoides has mostly monomorphic fronds and rhizome scales with slightly dentate margins, according to Zhang et al. 2013. This specimen (J. Joseph 48934) confirms the earlier report of L. lankokiensis in India, Lohit District, Arunachal Pradesh by Singh & Panigrahi (2005), in addition to China, Thailand and Vietnam.

Selected digitized specimens examined— Loxogramme lankokiensis: VIETNAM. Lankok [“Roches moussues du Mont Bavi, prèt de la vallée de Lankok”], 12 Jun. 1888, B. Balansa 1934 (Isotypes B-20 0089150, BR-0000006979492, BM-001039924, BM- 001039925, P-01356591); Tonkin: Cao-Bang, D. Billet s.n., Herb. H. Christ (P- 01356590). THAILAND. Udawn: Phu (Mt.) Luang, about 900 m, 11 Jan. 1966, E. Hennipman 3631A (P-01353469).— Loxogramme grammitoides: CHINA. S. Wushan, West Hupeh, E.H. Wilson 620 (Isotypes B-20 0089149, US00067489, BM-001039922).

ACKNOWLEDGEMENTS I sincerely thank the Director, Botanical Survey of India and Prof. R. Mukhopadhyay, Pteridology Section, Department of Botany, The University of Burdwan for kindly providing opportunity to study the specimen and Dr M. Lehnert, University Bonn, Germany for constructive suggestions.

REFERENCES BHARGAVAN, P. & JOSEPH, J. 1978. Loxogramme grammitoides (Bak.) C.Chr.- a fern record for India. New Botanist 5: 43-44. 96 FERN GAZ. 20(2):95-97. 2015

FRASER-JENKINS, C.R. 2008. Taxonomic revision of three hundred Indian subcontinental Pteridophytes with a revised Census- List. Bishen Singh Mahendra Pal Singh, Dehra Dun, 685 pp. FRASER-JENKINS, C.R. 2012. Rare and Threatened Pteridophytes of Asia 2. Endangered Species of India—the Higher IUCN Categories. Bull. Natl. Mus. Nat. Sci., Tokyo, B. 38: 153-181. SINGH, S. & PANIGRAHI, G. 1987. Notes on two species of Loxogramme Presl (Pteridophyta) in Eastern . Bull. Bot. Surv. India. 27 (1-4): 252-254.

Figure 1. A.–C. Loxogramme lankokiensis (J. Joseph 48934 CAL): A. & B. Specimen from Arunachal Pradesh, India, C. Part of plant showing distinct fertile and sterile fronds. MAZUMDAR: LOXOGRAMME LANKOKIENSIS IN INDIA 97

SINGH, S. & PANIGRAHI, G. 2005. Ferns and Fern-allies of Arunachal Pradesh (Adiantaceae to Nephrolepidaceae). Vol. 1. Bishen Singh Mahendra Pal Singh, Dehra Dun, 426 pp. ZHANG, X.C., LU, S.G., LIN, Y.X., QI, X.P., MOORE, S., XING, F.W., WANG, F.G., HOVENKAMP, P.H., GILBERT, M.G., NOOTEBOOM, H.P., PARRIS, B.S., HAUFLER, C., KATO, M. & SMITH, A.R. 2013. Polypodiaceae. 758-850 pp., in WU, Z.Y., RAVEN, P.H. & HONG, D.Y. (eds), , Vol. 2-3 (Pteridophytes). (http://flora.huh.harvard.edu/china/mss/volume02/Flora_of_China_Volume_2_3_P olypodiaceae.pdf) 98 FERN GAZ. 20(2). 2015



Three new combinations are proposed for Ctenopterella (Polypodiaceae) in preparation for a monograph of the genus.

Ctenopterella lasiostipes (Mett.) Parris comb. nov. Based on Polypodium lasiostipes Mett., Ann. Sci. Nat. 4, 15: 76-77 (1861). Type: New Caledonia, Poila, Vieillard 1601 (lectotype P, chosen by Brownlie, Fl. Nouv.-Caléd. 3: 274 (1969); isolectotype NSW!). Synonym: Ctenopteris lasiostipes (Mett.) Brownlie, Fl. Nouv.-Caléd. 3, Pteridophytes: 273 (1969).

Ctenopterella thwaitesii (Beddome) Parris comb. nov. Based on Polypodium thwaitesii Beddome, Ferns Brit. India: t. 188 (1866). Type: Ceylon, Thwaites C. P. 3900 (lectotype K!, chosen by Parris, Fern Gaz. 16(5): 243 (2002); isolectotypes BM!, CAL 20454 images seen, CGE!, E 00195204!, GH!, K!, P 02141982!, PDA!). Synonym: Ctenopteris thwaitesii (Beddome) Sledge, Bull. B.M. (Nat. Hist.) Bot. 2(5): 155 (1960).

Ctenopterella undosa (Baker) Parris comb. nov. Based on Polypodium undosum Baker, J. Bot. 28: 108 (1890). Type: New Guinea, Mt Musgrave, 1889, MacGregor 61 (holotype K 000507064!; isotypes MEL!). Synonyms: Ctenopteris undosa (Baker) Copel., Philipp. J. Sci. 81(2): 103 (1953); Polypodium pycnophyllum C.Chr., Brittonia 2(4): 308 (1937). Type: Papua, Central Division, Mt Albert Edward, 3680 m alt., 5 July 1933, Brass 4421 (holotype BM; isotypes BRI!, MICH 1115954!, NY) (synon. nov.), Ctenopteris pycnophylla (C.Chr.) Copel., Philipp. J. Sci. 81(2): 102 (1953). B. S. Parris Fern Research Foundation, Kerikeri, New Zealand. Email: [email protected] FERN GAZ. 20(2). 2015 99


Farne als Lebensleidenschaft – Festschrift für H. Wilfried Bennert anlässlich seines 65.Geburtstages. Berichte des Instituts für Landschafts- und Pflanzenökologie der Universität Hohenheim. Beiheft 22. 2013. 238pp. 21.0 x 29.7cm (in German). Available from A. Kleinsteuber, Weiβdornweg 35, D-76149, Karlsruhe, Germany. email: [email protected]

This festschrift for Wilfried Bennert on the occasion of his 65th birthday is a collection of 11 papers gathered together by Karsten Horn and illustrated with a series of etchings of ferns by Reinhard Böcker. The subjects of the articles reflect Bennert’s wide interests in pteridology and ecology. The volume starts with a description of his life and career in science, the Bochum days, and how he fell under the spell of ferns, horsetails and clubmosses. He carried out fieldwork not only in Europe but also in California, Iceland, Southeast Asia and Russia, and Horn describes the work that culminated in one of his major fern projects, the publication of Die seltenen und gefährdeten Farnpflanzen Deutschlands in 1999. This is followed by a full list of his publications. The subjects and authors of the other papers are summarised below. Reinhard Bornkamm describes the ferns, horsetails, Ephedra and gymnosperms of Egypt and their role in plant communities. Herbert Sukopp looks at three ferns in C19th horticulture around Berlin. Andreas Sarazin et al. describe new records of neophyte fern taxa that have been discovered in Northwest Germany, including species of Adiantum, Cyrtomium and Pteris. Böcker describes the historical and present distributions of ferns, lycophytes and horsetails from the Stuttgart area, and assesses the impact of human activity. Marcus Lubienski provides photographs, detailed descriptions and distributional data for all known hybrids of Equisetum in Europe, and their morphological variability is discussed. Caroline Van den heede and Ronnie Viane report the discovery of Asplenium lolegnamense and A.×chasmophilum and discuss their origins. Wolfgang Jäger reports the occurrence of ascomycete fungi on ferns in the Bergisches Land, Northrhine-Westphalia. Karsten Horn has published an account of the life and scientific output of K.A.J. Milde, a great pteridologist of C19th, with illustrations from some of his significant monographs. Henning Haeupler reviews and clarifies the work on life forms to describe vegetation, first introduced by Humboldt in 1806 and extended by Raunkiaer in 1907. The volume concludes with plant colonisation in shallow waters - plants of the littoral zone, by Klaus Kaplan. Wilfried Bennert has been a member of BPS since 1975, and is active in the Group of European Pteridologists.

M. Gibby 100 FERN GAZ. 20(2). 2015


PAPERS should not usually exceed 20 printed pages and are generally expected to be considerably shorter. As well as reports of original research, review articles and short notes, e.g. new records, are encouraged. The senior author should supply an email address to facilitate correspondence. REVIEWS should cover recent developments in any aspect of research on ferns and lycopods. This can include unpublished research by the author, and/or the possible direction of future research. They should be aimed at the general reader who may not be an expert in the chosen topic. Proposals for a review should be discussed first with the review editors. MANUSCRIPTS should be submitted in English (British) in electronic format in 10-point Times New Roman font and double spaced. Electronic versions of text and tables should be compatible with WORD, with figures as pdf or jpg files, and sent as email attachments or CDroms. All manuscripts will be refereed. THE TITLE should reflect the content of the paper and be in BOLD CAPITALS (11-point) and centrally aligned. Generic and specific names should be in italics and any title containing a generic or specific name should be followed by the family name in brackets e.g. TRICHOMANES SPECIOSUM (HYMENOPHYLLACEAE) IN SOUTHERN SPAIN AUTHOR ABBREVIATIONS should follow Pichi Sermolli’s (1996) Authors of scientific names in Pteridophyta, Royal Botanic Gardens, Kew. MAIN HEADINGS: should be in BOLD CAPITALS (10-point) and centrally aligned. SUBSIDIARY HEADINGS: should be in bold, the first letter of each word in capitals, the rest in lower case and left-aligned. AUTHORS’ NAMES AND FULL ADDRESSES (including email address): follow the title and are centrally aligned. KEY WORDS: up to ten. ABSTRACT: should reflect the content of the paper. FIGURES: there is no distinction between photographs and line drawings in numbering. All should be presented in a form ready for reproduction, colour, TIF format and 300 dpi (please contact editor with queries), with a scale bar where appropriate. Lettering or numbers (Arabic) should be in the bottom left using uppercase Times Roman. Figure captions should be on a separate sheet. TABLES: can be printed in either portrait or landscape format. Authors should consider this when preparing tables. Authors should ensure that tables fit the printed page size in a legible form. PAGESIZE: The Gazette has a useable page size of 123 × 204 mm. This can be used in portrait or landscape mode but any supplied artwork should not be larger than this. MEASUREMENTS: follow the metric system. CHECKLISTS: follow the format of Baksh-Comeau, Fern Gaz. 16(1, 2): 11- 122. REFERENCES: follow the style of a recent issue of The Fern Gazette, e.g.:- HOOKER, W.J. 1864. Species Filicum, 5. Dulau & Co., London. MORTON, C.V. 1947. The American species of Hymenophyllum, section Sphaeroconium. Contr. U.S. Natl. Herb. 29(3): 139-201. STEVENSON, D.W. & LOCONTE, H. 1996. Ordinal and familial relationships of pteridophyte genera. In: CAMUS, J.M., GIBBY, M. & JOHNS, R.J. (Eds) Pteridology in perspective, pp. 435-467. Royal Botanic Gardens, Kew. JOURNAL ABBREVIATIONS: should follow Botanico Periodicum Huntianum & Supplements. Alterations from the original text at proof stage will be charged for unless they are minor points of detail. A pdf will be provided free to the senior author. FERN GAZ. 20(3):101-116. 2016 101



MARCUS LEHNERT1,2* & MICHAEL KESSLER3 1 Nees-Institut für Biodiversität der Pflanzen, Rheinische Friedrich Wilhelms-Universität Bonn, Meckenheimer Allee 170, D-53115 Bonn, Germany. 2 Staatliches Museum für Naturkunde, Am Löwentor, Rosenstein 1, D-70191 Stuttgart, Germany 3 Systematic and Evolutionary Botany, University of Zurich, Zollikerstrasse 107, CH-8008 Zurich, Switzerland.

* Author for correspondence, email: [email protected]

ABSTRACT Mycorrhizae, i.e., symbiotic associations between fungi and plant roots, occur in about 80% of land plant species and have been shown to benefit both the (carbon uptake) and plant (nutrient and water uptake, protection against pathogenic fungi). We here provide a brief overview of the state of knowledge of mycorrhization in ferns. Only about 62% of species studied to date have mycorrhizae, with arbuscular mycorrhizal fungi (mainly Glomeromycta) being the dominant partners, while other associations are made with ascomycetes and the so-called Dark Septate Endophytes. There is no clear phylogenetic signal in mycorrhization among ferns, with both basal (e.g., Anemiaceae, Gleicheniaceae, Cyatheaceae) and derived families (e.g., non-grammitid Polypodiaceae, Tectariaceae, Aspleniaceae) having <50% of species mycorrhizal. Ecologically, epiphytes have lower degrees of mycorrhization than terrestrial species, with aquatic taxa almost completely lacking mycorrhizae. This probably reflects the requirements of the fungi. There are no experimental studies on the benefits of mycorrhizae for ferns, but field experiments suggest that there is a fine balance between positive and negative effects, so that many fern species have disposed of the fungi either generally or facultatively. Much remains to be learnt about mycorrhization in ferns, especially in an evolutionary context in comparison with bryophytes and seed plants.

INTRODUCTION The term mycorrhiza (pl. mycorrhizae) was introduced by Frank (1885) for structures in angiosperm roots caused by fungi and translates as “fungus root”. Since then it has been shown that it is one of the most common and important symbioses in the land plants (Brundrett, 2002; 2004; 2009), even in plants like bryophytes and Psilotaceae that lack roots. It is estimated that ca. 80% of all land plants engage either in facultative or obligate mycorrhizal associations (Trappe, 1987; Wang & Qiu, 2006; Brundrett, 2009). It is likely that the colonization of land by early land plants was facilitated by mycorrhizae (Pyrozinski & Malloch, 1975; Remy et al., 1994; Blackwell, 2000; Selosse et al. 2015), even before roots first arose (Taylor et al., 1995; Brundrett, 2002; Kenrick & Strullu-Derrien, 2014). This assumption is based on the presence of structures very similar to extant symbiotic Glomeromycota in the earliest tracheophyte from the Rhynie Chert (Remy et al., 1994; Strullu-Derrien & Strullu, 2007; Selosse et al., 2015) 102 FERN GAZ. 20(3):101-116. 2016 and supported by the conserved nature of the genes regulating mycorrhizal associations across all land plant lineages (Wang et al., 2010). Mycorrhizal fungi are known to provide numerous benefits to their plant hosts (Selosse & Rousset, 2011). They enhance nutrient uptake, especially of phosphorous (Siddiqui & Pichtel, 2008), increase drought tolerance by facilitating water uptake (Faber et al., 1991; Warren et al., 2008), and compete with parasitic fungi thus protecting plants from parasitic fungal infections (Newsham et al., 1995; Brundrett, 2002). In return, the fungal partners receive carbohydrates from the host plant (Selosse & Rousset, 2011). The intensity of this symbiosis may vary with external ecological factors (Allen et al., 2003; Antunes et al., 2011) and apparently can be regulated more actively by the plant host than by the fungal symbiont (Barker et al., 1998; Jones et al., 2004). Most research on mycorrhizae has been conducted on seed plants because of their dominance, diversity and economic importance (Brundrett, 2004; 2009). In recent years, the scientific focus has shifted more to the origin of this symbiosis and hence to its presence in old land plant lineages like extant bryophytes and lycophytes (Read et al., 2000; Rimington et al., 2015). Mycorrhizae in ferns have been regularly screened for but rarely been in the focus of physiological or synthetic studies combining ecology and phylogeny (Wang & Qiu, 2006; Brundrett, 2009). In the present paper, we provide a brief overview of the still incomplete knowledge of mycorrhization in lycophytes and ferns (for simplicity called ferns in the following).

TYPES OF MYCORRHIZAE Four major types of mycorrhiza have been distinguished (Barker et al., 1998; Smith & Read, 2008; Siddiqui & Pichtel, 2008) with some characteristic deviations and aberrations in some major of host plants (Figure 1; Brundrett, 2002; 2004). Arbuscular mycorrhiza (AM) is an endosymbiosis, in which the fungi penetrate the cell walls of the root cortex and form characteristic organs in the living cells for nutrient exchange, the so-called arbuscules and vesicles (Figure 2a,b; Bonfante & Genre, 2008). It is the most common, and probably the ancestral form of mycorrhizae (Field et al., 2015a), occurring in ca. 80% of extant land plants (Wang & Qiu, 2006; Brundrett, 2009). The fungal partners are aseptate Eumycota, in the majority Glomeromycota (Schüßler et al., 2001), which are asexual and obligate symbionts (Brundrett, 2004; 2009). In some basal lineages of landplants (, some liverworts and lycophytes, a few ferns), Mucoromycotina, which are either basal to or sister to Glomeromycota, may also form a similar endomycorrhiza but without the presence of arbuscules (Desirò et al., 2013; Strullu-Derrién et al., 2014; Field et al., 2015a, b). Ectomycorrhizae (ECM) are mainly formed by the more derived septate and Basidiomycota (Taylor & Alexander, 2005; Taylor et al., 2010) but in a few cases also endogonalean fungi (Mucoromycotina, Hibbet et al., 2007). They build an internal network of hyphae that surround the intact root cells (Hartig net) and an external hyphal sheath around the root tips (Smith & Read, 2008; Brundrett, 2004). ECM occur in 2.0–4.7% of seed plants but dominate among tree species in temperate and boreal zones families (Smith & Read, 2008; Brundrett, 2009). Until now, ECM have not been confirmed for ferns. It has been suggested that ferns shift from AM to ECM in forests where ectomycorrhizal tree species are dominant (Cooper, 1976; Iqbal et al., 1981), but the claim that the investigated roots really belonged to ferns has been strongly contested (Smith & Read, 2008; Brundrett, 2002). Fungal symbioses interpreted as derivations of ECM are the Arbutoid mycorrhiza and the Monotropid mycorrhiza (Brundrett, 2004), LEHNERT&KESSLER: MYCORRHIZAL RELATIONSHIPS 103 . Diagram of the assumed relationships between the different main types of mycorrhizae (AM, ECM, ericoid, orchid) and their Figure 1 modifications (facultative, exploitative, mixed) based on studies AM of occur all AM in land are plants. confirmed; For exploitative ferns, only some gametophytes; ECMF and ericoid MF have been found in some lycophytes and ferns but without expressing the regular phenotype of the mycorrhiza (MF = mycorrhizal fungi). Numbers above arrows indicate instances of transitions (αAdapted from Brundrett (2002) = many). 104 FERN GAZ. 20(3):101-116. 2016

but these are also not reported for ferns. Ericoid mycorrhizae are formed with septate Ascomycota, which all are primarily decomposers and saprophytes (Hibbet et al., 2007). They are uncommon but typical of Ericaceae (Cullings, 1996; Brundrett, 2004) and some other plant groups occurring on nutrient-deficient substrates characterized by low primary production (Cairney & Meharg, 2003). Typical of this symbiosis is that the hyphae enter through root hairs and form hyphal coils in cortical cells (Read, 1996). ascomyceta infections of the Ericoid type have been reported for tropical ferns, mainly epiphytes, such as , grammitid ferns, Hymenophyllum and Trichomanes (Schmid et al., 1995; Lehnert et al., 2009). Records of the so-called Dark Septate Endophytes (DSE) in ferns (Dhillion, 1993; Jumpponen & Trappe, 1998; Fernández et al., 2008) may include still unrecognized Ericoid mycorrhizae since the taxonomic identity of most of these fungi is unknown (Jumpponen, 2001; Rains et al., 2003). Like the Ericoid mycorrhizal Ascomycota, DSE are more common in epiphytic fern species (Kessler et al., 2010). Orchid mycorrhizae involve a small group of Basidiomycota (Brundrett, 2002), and are characterized by the initial carbon dependency of the host plant seedling on the saprophytic fungal partner; several orchid lineages have independently developed an exploitative life-style from this condition (Brundrett, 2002; 2004). Such exploitative mycorrhizae are not known from the sporophytes of lycophytes and ferns, but gametophytes of Lycopodiaceae (Duckett & Ligrone, 1992; Winther & Friedmann, 2008), Ophioglossaceae (Schmid & Oberwinkler, 1994; Field et al., 2015c) and Psilotaceae (Duckett & Ligrone, 2005; Winther & Friedmann, 2009) are well known for being mycoheterotropic. These mostly involve AMF but recently Horn et al. (2013) reported the steady prevalence of basidiomycetes of the order Sebacinales (as found in orchid mycorrhiza and ECM) in gametophytes of Diphasiastrum alpinum (L.) Holub. This had been anticipated because basidiomycetes had already been documented as mycorrhizal endophytes for bryophytes (Pressel et al., 2010) Mycorrhizae are species-specific regarding their phenotype (i.e., one fungus and one plant always engage in a specific type of interaction) but not regarding the taxa involved: Ferns can host a mixture of up to five different mycorrhizal fungi at the same time but with one usually dominating (Jansa et al., 2008), and one fungus can colonize different

Figure 2a. Internal structures of arbuscular mycorrhiza (vesicles and arbuscules, lower ones partially released from cell due to preparation) in the root cortex of the tree fern Cyathea tortuosa R.C.Moran from Ecuador (Prov. Napo, 500 m) (20 x). Figure 2b. The roots of a typical non-mycorrhizal plant with pronounced root hair formation, represented by Elaphoglossum castaneum (Baker) Diels from Ecuador (Prov. Napo, 2000 m). (10 x) Photos Ramona Güdel. LEHNERT&KESSLER: MYCORRHIZAL RELATIONSHIPS 105 plant species (Gemma et al., 1992; van der Heijden et al., 2015). The true symbiotic nature of a fungal association can only be confirmed by physiological experiments aiming at the flow of nutrients (e.g., Jurkiewicz et al., 2010). As this has not been done for any fern yet, it is uncertain whether most observed associations are really symbiotic or functionally neutral (Brundrett, 2004; 2009). As Glomeromycota are fully dependent on the symbiosis and only known to occur independently from a host plant when they are dormant spores (Brundrett, 2009), it seems likely that a symbiotic relationship is involved in most cases but not necessarily with the roots in which they are found. Under high inoculum pressure in the substrate, typical non-mycorrhizal taxa may harbor AMF as neutral endophytes while the same mycelium probably forms a true mycorrhiza with a typical host plant nearby (Lekberg et al., 2015). Similarly, some typical non-mycorrhizal taxa have been found to be able to host AMF under nutrient deficiency (Brundrett, 2009). Furthermore, a low presence of fungi in the roots must not necessarily imply a low physiological activity between both partners. The categorization into obligate and facultative mycorrhizal plant species can only be evaluated in controlled experiments, e.g., by establishing sterile cultures of both partners and later re-inoculation (Pearson & Read, 1973), and not by visual estimation alone.

TAXONOMIC DISTRIBUTION OF MYCORRHIZAE The mycorrhizal status is regarded to be ancestral among ferns (Zhao, 2000; Wang & Qiu, 2006), as is likely for all land plants (Pirozynski & Malloch, 1975; Strullu-Derrién et al., 2014; Field et al., 2015a). The scant reviews of fern mycorrhizae have focused on the sporophyte and have come up with highly diverging results regarding the general presence of fungal symbionts as well as the distribution among the different groups. Newman and Reddell (1987) found 87% to be always mycorrhizal, 7% facultative, and 6% not mycorrhizal (based on 180 fern species), which is within the range of the values found for angiosperms (Brundrett, 2009). Others reported 75% of the studied fern species to have AM (Gemma et al., 1992). Zhao (2000) found only 33% of 256 species from China to be mycorrhizal (17% obligate + 16% facultative), and similarly Moteetee et al. (1996) reported 36% of the species to be mycorrhizal. Cooper (1976) found 100% of the investigated ferns to harbour mycorrhiza, but the sampling included only terrestrials and low epiphytes from humid subtropical to southern temperate forest, i.e., conditions that foster fungal growth. On the other extreme, Lesica & Antibus (1990) focused on epiphytes and found no mycorrhiza in the 12 investigated species. Wang & Qiu (2006) found 52% of all species and 92% of all families to harbour AMF. Brundrett (2009) summarized only more recent reports on mycorrhizae that he could trust, but contrary to his detailed summary on , he could not give an exact evaluation for ferns. Lehnert (2007) used all available data on fern mycorrhiza worldwide starting from Boullard (1958, 1979), and evaluated simple presence/absence of mycorrhizal colonisations in three classes (aseptate fungi = AMF; septate fungi = DSE/ascomycetes; unspecified mycorrhiza) in 675 taxa (ca. 6% of the global diversity; Smith et al., 2006). The observed average of 62% for fungal colonisation lies in the middle of the range of previous reports for ferns but is lower than the values for seed plants (Brundrett, 2009). AMF are dominant among the fungal symbionts with 66%, whereas the remaining 34% are made up of ascomycetes, DSE and unspecified mycorrhizations. Across the phylogeny at the family level ( Smith et al., 2006), below average mycorrhization (≤62%) was found among several families of derived Polypods (non-grammitid 106 FERN GAZ. 20(3):101-116. 2016

Polypodiaceae 23%, Tectariaceae 25%, Aspleniaceae 45%) but other families of the Polypods such as Davalliaceae and had 100% mycorrhization. Vice versa, among the other leptosporangiate ferns, several families had below average mycorrhization rates (Anemiaceae 50%, Gleicheniaceae 48%, Cyatheaceae 53%). Among the eusporangiate lineages, only the Equisetaceae showed low mycorrhization (22%) whereas the remainder had average to high values (Psilotacae 67%, Marattiaceae 91%). Families largely restricted to aquatic habitats showed low (; one of two samples) or no (, Salviniaceae) fungal colonization. Mycorrhizal epiphytes were found mainly among grammitid ferns (84% of epiphytes and 74% of all species mycorrhizal), Hymenophyllaceae (76% of epiphytes and 76% of all species mycorrhizal) and Dryopteridaceae (63% of epiphytes [mainly Elaphoglossum] and 62% of all species mycorrhizal). Interestingly, only 42% of the investigated sporophytes of Lycopodiaceae had mycorrhizae despite their predominantly mycoheterotrophic gametophytes (Winther & Friedmann, 2008). Later substantial additions to the survey (Kessler et al., 2009; 2010b; 2014a; Zubek et al., 2010; Ogura Tsujita et al., 2013; Muthukumar & Prabha, 2012; 2013; Lara-Pérez et al., 2015) have increased the species count but have not significantly changed the originally found total average and ratios between the substrates (Lehnert et al., unpublished data). A caveat to bear in mind when comparing many different reports is the often uneven approaches regarding evaluation and quantification of the presence of fungi, as has been pointed out for AMF by Brundrett (2009). However, considering that it is more likely that fungi present have been over-interpreted as symbionts rather than that true, functioning mycorrhizas have been overlooked, these numbers are more likely to drop than to rise after a thorough re-evaluation. Thus, the observed pattern that lycophytes and ferns have a below-average colonization rate compared with seed plants or land plants in general is expected to hold true. A similar but even stronger uncertainty lies in the data supporting the mutualistic nature of these colonizations, which would allow them to be addressed undisputedly as mycorrhizae (Brundrett, 2002; 2009). Most reports used only light microscopical preparation techniques to investigate the presence of fungi, which render the sample unsuitable for futher ultrastructural and molecular studies (pers. obs. for ferns, but see Ishii & Loynachan, 2004). Even if obligately mycorrrhizal fungi abound in the roots, this does not necessarily mean that the partners interact physiologically (Lekberg et al., 2015). Electron microscopy may allow the larger classification of the fungus and could reveal whether the host cell responded more in a symbiotic way or if it just tried to reject and contain the penetrating fungus (e.g. Schmid & Oberwinkler, 1994; Schmid et al., 1995). So far such ultrastructural and/or genetic investigations of mycorrhizae in the sporophyte generation covered only 12 species of lycophytes (Duckett & Ligrone, 1992; Winther & Friedmann, 2007a; Rimington et al., 2015) and 35 species of ferns (Schmid et al., 1995; Schmid & Oberwinkler, 1996; Pazmiño, 2006; Winther & Friedmann, 2007b; 2009; Lehnert et al., 2009; Field et al., 2015c; Rimington et al., 2015), which represents only a small fraction (3.7 %) of the 1,287 species of lycophytes and ferns for which reports are available (ca. 11 % of the global diversity; Lehnert et al., unpubl. data)

LIFE-STAGE DISTRIBUTION OF MYCORRHIZAE Mycorrhizal interactions with fern gametophytes are not as uniform as often stated (Mehltreter, 2010). With a few exceptions (Duckett & Ligrone, 1992), gametophytes of LEHNERT&KESSLER: MYCORRHIZAL RELATIONSHIPS 107

Lycopodiaceae, Psilotaceae (Duckett & Ligrone, 2005) and Ophioglossaceae are achlorophyllous and mycoheterotrophic, meaning not only do they receive water and minerals but also carbohydrates from the fungi (Schmid & Oberwinkler, 1994). By this means, the gametophytes may tap into the carbon source of another, photoautotrophic host of the fungus (Harley & Harley, 1987; Horn et al., 2013), possibly even the parental sporophyte (Winther & Friedmann, 2008). The anatomy of these mycoheterotrophic gametophytes, which are tuberous and relatively thick textured (Winther & Friedmann, 2007; 2008; 2009), allows an easy colonisation by fungi, whereas across fern lineages with photoautotrophic gametophytes, we see a gradual reduction of size (especially thickness) and longevity along the phyletic sequence (Smith et al., 2006). Mycorrhization rates seem to follow this pattern. The eusporangiate Marrattiaceae (Bower, 1923; Ogura-Tsuijita et al., 2013) and the basal leptosporangiate Osmundaceae, Plagiogyriaceae, Gleicheniaceae and Cyatheaceae (Ogura-Tsuijita et al., 2015) show relatively constant and high presence of AM fungi (58–97%) in the thick centre of the gametophytes. However, most leptosporangiate families have the common heart-shaped Polypodium-prothallium and offer only restricted space for fungal symbionts; accordingly fungal colonization is observed infrequently (e.g., Reyes-Jaramillo et al., 2005; Turnau et al., 2005; Muthukumar & Prabha, 2012) and most taxa are considered to be only facultatively mycorrhizal (Read et al., 2000). In leptosporangiate lineages with strongly dissected to filiform prothallia (e.g., some Hymenophyllaceae, grammitid Polypodiaceae), AM are not observed (Boullard, 1979). Even if both generations in the life cycle are mycorrhizal, the sporophyte does not directly inherit the fungal partner from the gametophyte but has to acquire it anew from the substrate (Schmid & Oberwinkler, 1995), so that each generation can have a different assortment of fungal partners, although usually with a strong overlap (Winther & Friedmann, 2008).

HABITAT DISTRIBUTION OF MYCORRHIZAE Among ferns, most studies have only considered selected species; whole fern communities have been studied only in Canada (Berch & Kendrick, 1982), China (Zhao, 2000), La Réunion (Kessler et al., 2010), and Ecuador (Kessler et al., 2014a). In general, the mycorrhization rate of soil-growing fern species is typically higher than that of epiphytic species (Gemma et al., 1992; Kessler et al., 2010). Thus, Lehnert (2007) reported putative mycorrhizal fungi mainly in terrestrial and saxicolous species (67% and 59%, repectively) but only in 53% of epiphytic species and in 9% of aquatic species. The percentage in epiphytes included a high presence of ascomycetes + DSE (67% of colonisations), whereas the remaining 33% of epiphytic mycorrhiza represented only 8% of the total of AMF colonisations in all lycophytes and ferns. The low rate of mycorrhizal infections among epiphytes may be caused by either the fungi or the ferns. Glomeromycetes, in particular, are expected to have a lower colonization potential than fungi with air-borne spores (e.g., ascomycetes, basidiomycetes) because they produce spores subterraneously and require stable substrate conditions for growth and development (Zubek et al., 2010), thus being hardly suited for the dynamic canopy habitat (Janos, 1993). It has also been suggested that epiphytic ferns may be less dependent on mycorrhizae because they have special adaptations (e.g., drought resistance, leaf litter traps, thick storage rhizomes) that limit requirements for mycorrhizae (Mehltreter, 2010). Among the ferns growing on the ground, there are also slight differences between 108 FERN GAZ. 20(3):101-116. 2016 substrates, with those on well-developed soil and rocks having high levels of mycorrhization than those on sand – again a habitat that is unsuitable for fungal growth (Brundrett, 2009) – having fewer mycorrhizal fern species (Gemma et al., 1992). A special case is provided by raw volcanic soils, especially on islands such as Hawai’i. It has been suggested that such soils are more easily colonized by nonmycotrophic or facultatively mycorrhizal fern species due to the paucity of fungal innoculum, perhaps facilitating later colonization of obligate mycotrophic ferns and angiosperms (Gemma & Koske, 1990; Gemma et al., 1992). This contrasts, however, with the regular presence of mycoheterotrophic gametophytes of Psilotum in volcanic soils (Holloway, 1938; Duckett & Ligrone, 2005) and observations on La Réunion, where the percentage of terrestrial fern individuals with mycorrhizae was higher on shallow, raw soils with high pH values, high base-saturation and low nutrient availability (Kessler et al., 2010). These contrasting results may be due partly to the fact that the Hawai’i studies were based on the species level, whereas the results from La Réunion are based on numbers of individuals. Mycorrhization is absent to very rare in aquatic environments (Boullard, 1958; 1979; Lehnert et al., 2007; Brundrett, 2009), presumably reflecting the paucity of fungi in such environments. Furthermore, the benefits mycorrhiza could bring are no longer advantageous in a medium in which water and most nutrients are freely available and often in surplus, but – at least in the case of fully submerged plants – assimilation is hampered by lower CO2 partial pressure and decreased light intensity. In some cases, amphibious species that grow both inundated and on dried-out soils are facultatively mycorrhizal, as found in some (Beck-Nielsen & Madsen, 2001; Radhinka & Rodrigues, 2007). Likewise, species of Equisetum are non-mycorrhizal in , but commonly mycorrhizal in dry environments (Harley & Harley, 1987; Marsh et al., 2000; Read et al., 2000; Brundrett, 2002). Focussing on climatic gradients, the degree of mycorrhization has been documented to decrease with elevation in Ecuador (Kessler et al., 2010). In addition to the commonly proposed fungus-oriented explanations based on reduced fungal growth at low temperatures (Read & Haselwandter, 1981; Olsson et al., 2004; Schmidt et al., 2008), these authors also proposed a fern-oriented one in that mycorrhization might decrease because the relative costs of allocating carbon to the fungal partner increase with elevation (where decreases: Kessler et al., 2014b) and possibly exceed the benefits of the fungal nutrient supply.

ECOLOGICAL EFFECTS OF MYCORRHIZATION IN FERNS Based on work on seed plants, it is known that fungi provide plants with soil nutrients (especially PO4), water, and other benefits (Blackwell, 2000; Wilkinson, 2001). In exchange, the fungi obtain organic C from the photosynthetic host plants (Smith & Read, 2008; Olsson et al., 2010), which allocate up to 20% of their net photosynthetic C to their fungal partners (Allen et al., 2003). The relative costs of this fungal C use have to be assessed in relation to the benefits derived from increased nutrient uptake, and presumably can be offset partly by higher rates of photosynthesis, if the plants are not light limited (Smith & Smith, 2011). In greenhouse experiments with potted plants, mycorrhizal fungi have been shown to increase leaf tissue nutrient concentrations, most importantly those of P (Stribley et al., 1980; Smith & Smith, 2011). For ferns, very few data are available, and contrary to seed plants there are no greenhouse experiments. In an early study, Cooper (1976) showed that mycorrhizal plants LEHNERT&KESSLER: MYCORRHIZAL RELATIONSHIPS 109 of the facultatively mycorrhizal Pteridium aquilinum (L.) Kuhn in New Zealand had stronger growth than non-mycorrhizal individuals. More recently, it has been shown both on La Réunion and in Ecuador that terrestrial mycorrhizal fern species are locally more abundant than those lacking mycorrhizae (Kessler et al., 2010; 2014a). For example, on La Réunion, the 78% mycorrhizal species made up 98% of all fern individuals. This suggests a clear ecological advantage provided by mycorrhizae to ferns. On the other hand, a study in Ecuador found that fern species with mycorrhizae had lower relative biomass increment per year than those without mycorrhizae (Kessler et al., 2010), suggesting a high C cost of mycorrhization. Even more surprisingly, non- mycorrhizal fern species had significantly higher concentrations of N, P, Mg, and Ca in their leaves. Negative growth effects of mycorrhization have previously been documented in a number of cases among seed plants (e.g., Johnson et al., 1992; Smith & Read, 2008; Veiga et al., 2011). These often reflect the parasitic nature of mycorrhizal fungi in situations in which the nutritional benefits for the plant do not outweigh the C loss to the fungi (Johnson et al., 1992; Karst et al., 2008). The very sparse data available until now thus provide evidence for benefits of mycorrhization in ferns as well as for negative effects. While this may initially be surprising, in fact it makes sense if we consider that not all plant species have mycorrhizae (Brundrett, 2009), even if they do not grow under special habitat conditions that limit fungal growth such as aquatic or epiphytic environments. If we further bear in mind that the ancestral state is most likely the presence of mycorrhization, then the 33% of terrestrial and 47% of epiphytic fern species lacking mycorrhizae (Lehnert, 2007) must have lost them over evolutionary times, suggesting that there must be a benefit of losing mycorrhiza. It thus appears that there is a fine balance between positive and negative effects of mycorrhization among land plants in general and ferns in particular. It is much too early to speculate under which conditions this balance switches either way, especially considering that the balance is likely to be not simply determined by a nutrient- carbon trade-off but also by water relations and the influence of symbiotic fungi on parasitic or pathogenic fungi and other biotic interactions.

OPEN QUESTIONS We conclude this overview of what is known about mycorrhizae in ferns with a series of questions that readily develop from the above text. This list is certainly incomplete, but it may offer some guidelines as to what we would like to know to better understand the role of mycorrhization in ferns and lycophytes: • Are all visually detectable fungal associations of ferns truly mutualistic? Most of the records are based on light microscopical screenings only and await further microstructural and molecular conformation. Until now, true mycorrhizae with mutual benefits have been confirmed only for lycophytes and basal fern lineages and involve only Glomeromycota and Mucoromycotina. • Why is the overall degree of mycorrhization lower among lycophytes and ferns (and liverworts) than among seed plants? Does this reflect some basic physiological difference between spore-bearing and seed-bearing vascular plants, e.g., in water use efficiency (Brodribb et al., 2009; Watkins et al., 2010) that tips the balance more in the direction of non-mycorrhization in ferns? • What is the role of mycorrhization in ferns that are very difficult to cultivate or transplant, such as Gleicheniaceae, Lycopodiaceae and grammitids (Page, 2002)? • For ferns with facultative or variable mycorrhization, does the degree of 110 FERN GAZ. 20(3):101-116. 2016

mycorrhization vary with ecological conditions? • Are there implications of mycorrhizal associations in ferns for herbivory or other ecological interactions?

ACKNOWLEDGEMENTS We thank Bridget Laue and Adrian Dyer for the invitation to do this review, and Mary Gibby and the reviewers for helping us improve the manuscript.

REFERENCES ALLEN, M. F., SWENSON, W., QUEREJETA, J. I., EGERTON-WARBURTON, L. M. & TRESEDER, K. K. 2003. Ecology of mycorrhizae: a conceptual framework for complex interactions among plants and fungi. Ann. Rev. Phytopath. 47: 271–303. ANTUNES, P. M., KOCH, A. M., MORTON, J. B., RILLIG, M. C. & KLIRONOMOS, J. N. 2011. Evidence for functional divergence in arbuscular mycorrhizal fungi from contrasting climatic origins. New Phytol. 189(2): 507–514. BARKER, S. J., TAGU, D. & DELP, G. 1998. Regulation of root and fungal morphogenesis in mycorrhizal symbioses. Plant Physiol. 116(4): 1201–1207. BECK-NIELSEN, D. & MADSEN, T.V. 2001. Occurrence of vesicular-arbuscular mycorrhiza in aquatic macrophytes from lakes and rivers. Aquatic Botany 71: 141– 148. BERCH, S. M. & KENDRICK, B. 1982. Vesicular-arbuscular mycorrhizae of Southern Ontario ferns and fern-allies. Mycologia 74: 769–776. BLACKWELL, M. 2000. Terrestrial life - Fungal from the start? Science 289: 1884– 1885. BOULLARD, B. 1958. La mycotrophie chez les ptéridophytes. Sa fréquence, ses caractéres, sa signification. Doctor thesis, Université de Caen. E. Droulliard, Bordeaux. BOULLARD, B. 1979. Consideration sur la symbiose fongique chez les Pteridophytes. Syllogeous 19: 1–59. BONFANTE, P. & GENRE, A. 2008. Plants and arbuscular mycorrhizal fungi: an evolutionary-developmental perspective. Trends Plant Sci. 13(9): 492–498. BOWER, F. O. 1923. The Ferns. Vol. I. University Press, Cambridge, 376 pp. BRODRIBB, T. J., MCADAM, S. A., JORDAN, G. J. & FEILD, T. S. 2009. Evolution of stomatal responsiveness to CO2 and optimization of water‐use efficiency among land plants. New Phytol. 183(3): 839–847. BRUNDRETT, M. C. 2002. Coevolution of roots and mycorrhizas of land plants. New Phytol. 154: 275–304. BRUNDRETT, M. C. 2004. Diversity and classification of mycorrhizal associations. Bot. Rev. 79: 473–495. BRUNDRETT, M. C. 2009. Mycorrhizal associations and other means of nutrition of vascular plants: understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant Soil 320: 37–77. CAIRNEY, J. W. G. & MEHARG, A. A. 2003. Ericoid mycorrhiza: a partnership that exploits harsh edaphic conditions. European Journal of Soil Science 54(4): 735–740. CAVAGNARO, T. R., GAO, L. L., SMITH, F. A. & SMITH, S. E. 2001. Morphology of arbuscular mycorrhizas is influenced by fungal identity. New Phytol. 151(2): 469– LEHNERT&KESSLER: MYCORRHIZAL RELATIONSHIPS 111

475. COOPER, K. M. 1976. A field survey of mycorrhizas in New Zealand ferns. N.Z. J. Bot. 14: 169–181. CULLINGS K. W. 1996. Single phylogenetic origin of ericoid mycorrhizae within the Ericaceae. Can. J. Bot. 74: 1896–1909. DESIRÒ, A., DUCKETT, J. G., PRESSEL, S., VILLARREAL, J. C. & BIDARTONDO, M. I. 2013. Fungal symbioses in hornworts: a chequered history. P. Roy. Soc. Lond. B Bio. 280(1759), p.20130207. DHILLION, S. S. 1993. Vesicular-arbuscular mycorrhizas of Equisetum species in Norway and the USA: occurrence and mycotrophy. Mycol. Res. 97: 656–660. DUCKETT, J. G. & LIGRONE, R. 1992. A light and electron microscope study of the fungal endophytes in the sporophyte and gametophyte of Lycopodium cernuum with observations on gametophyte-sporophyte junction. Can. J. Bot. 70: 58–72. DUCKETT, J. G. & LIGRONE, R. 2005. A comparative cytological analysis of fungal endophytes in the sporophyte rhizomes and vascularized gametophytes of Tmesipteris and Psilotum. Can. J. Bot. 83: 1443–1456. FABER, B. A., ZASOSKI, R. J., MUNNS, D. N. & SHACKEL, K. 1991. A method for measuring hyphal nutrient and water uptake in mycorrhizal plants. Can. J. Bot. 69(1), pp.87–94. FERNANDEZ, N., MESSUTI, M. I. & FONTENLA, S. 2008. Arbuscular mycorrhizas and dark-septate fungi in Lycopodium paniculatum (Lycopodiaceae) and (Equisetaceae) in a Valdivian temperate forest of Patagonia, Argentina. Amer. Fern J. 98: 117–127. FIELD, K. J., PRESSEL, S., DUCKETT, J. G., RIMINGTON, W. R. & BIDARTONDO, M. I. 2015a. Symbiotic options for the conquest of land. Trends Ecol. Evol. 30(8): 477–486. FIELD, K. J., RIMINGTON, W. R., BIDARTONDO, M. I., ALLINSON, K. E., BEERLING, D. J., CAMERON D. D., DUCKETT, J.G., LEAKE, J. R. & PRESSEL, S. 2015b. First evidence of mutualism between ancient plant lineages ( liverworts) and Mucoromycotina fungi and its response to simulated Palaeozoic changes in atmospheric CO2. New Phytol. 205(2): 743–56. FIELD, K. J., LEAKE, J. R., TILLE, S., ALLINSON, K. E., RIMINGTON, W. R., BIDARTONDO, M. I., BEERLING, D. J. & CAMERON, D. D. 2015c. From mycoheterotrophy to mutualism: mycorrhizal specificity and functioning in Ophioglossum vulgatum sporophytes. New Phytol. 205(4): 1492–1502. FRANK, B. 1885. Über die auf Wurzelsymbiose beruhende Ernährung gewisser Bäume durch unterirdische Pilze. Ber. dt. Bot. Ges. (3): 128–145. GEMMA, J. N. & KOSKE, R. E. 1990. Mycorrhizae in recent volcanic substrates in Hawaii. Am. J. Bot. 77 (9): 1193–1200. GEMMA J. N., KOSKE, R. E., & FLYNN, T. 1992. Mycorrhizae in Hawaiian pteridophytes: Occurrence and evolutionary significance. Am. J. Bot. 79: 843–852. HARLEY, J. L. & HARLEY, E. L. 1987. A check‐list of mycorrhiza in the British flora. New Phytol. 105(s1): 1–102. HIBBETT, D. S., BINDER, M., BISCHOFF, J. F., BLACKWELL, M., CANNON, P.F., ERIKSSON, O.E., HUHNDORF, S., JAMES, T., KIRK, P. M., LÜCKING, R. & LUMBSCH, H. T. 2007. A higher-level phylogenetic classification of the Fungi. Mycol. Res. 111(5): 509–547. HOLLOWAY, J.E. 1938. The embryo and gametophyte of Psilotum triquetrum. A 112 FERN GAZ. 20(3):101-116. 2016

preliminary note. Ann. Bot. (Lond.) 2: 807–809. HORN, K., FRANKE, T., UNTERSEHER, M., SCHNITTLER, M. & BEENKEN, L. 2013. Morphological and molecular analyses of fungal endophytes of achlorophyllous gametophytes of Diphasiastrum alpinum (Lycopodiaceae). Am. J. Bot. 100(11): 2158–2174. IQBAL, S.H., YOUSAF, M. & YOUNUS, M. 1981. A field survey of mycorrhizal associations in ferns of Pakistan. New Phytol. 87: 69–79. ISHII, S. & LOYNACHAN, T. E. 2004. Rapid and reliable DNA extraction techniques from trypan-blue-stained mycorrhizal roots: comparison of two methods. Mycorrhiza 14(4): 271–275. JANOS, D. P. 1993. Vesicular-arbuscular mycorrhizae of epiphytes. Mycorrhiza, 4(1): 1–4. JANSA, J., SMITH, F. A. & SMITH, S. E. 2008. Are there benefits of simultaneous root colonization by different arbuscular mycorrhizal fungi? New Phytol. 177(3): 779– 789. JOHNSON, N. C., COPELAND, P. J., CROOKSTON, R. K. & PFLEGER, F. L. 1992. Mycorrhizae: possible explanation for yield decline with continuous corn and . Agron. J. 84(3): 387–390. JONES, D. L., HODGE, A. & KUZYAKOV, Y. 2004. Plant and mycorrhizal regulation of rhizodeposition. New Phytol. 163(3): 459–480. JUMPPONEN, A. R. I. 2001. Dark septate endophytes–are they mycorrhizal? Mycorrhiza 11(4): 207–211. JUMPPONEN, A. R. I. & TRAPPE, J. M. 1998. Dark septate endophytes: a review of facultative biotrophic root‐colonizing fungi. New Phytol. 140(2): 295–310. JURKIEWICZ, A., RYSZKA, P., ANIELSKA, T., WALIGÓRSKI, P., BIAŁOŃSKA, D., GÓRALSKA, K., TSIMILLI-MICHAEL, M. & TURNAU, K. 2010. Optimization of culture conditions of Arnica L.: effects of mycorrhizal fungi and competing plants. Mycorrhiza 20(5): 293–306. KARST, J., MARCZAK, L., JONES, M. D. & TURKINGTON, R. 2008. The mutualism- parasitism continuum in ectomycorrhizas: a quantitative assessment using meta-analysis. Ecology 89(4): 1032–1042. KENRICK, P. & STRULLU-DERRIEN, C. 2014. The origin and early evolution of roots. Plant Physiol. 166(2): 570–580. KESSLER, M., JONAS. R., STRASBERG, D. & LEHNERT, M. 2010. Mycorrhizal colonizations of ferns and lycophytes on the island of La Réunion in relation to nutrient availability. Basic Appl. Ecol. 11: 329–336. KESSLER, M., JONAS, R., CICUZZA, D., KLUGE, J., PIATEK, K., NAKS, P. & LEHNERT, M. 2009. A survey of the mycorrhization of Southeast Asian ferns and lycophytes. Plant Biol. 12: 788–793. KESSLER, M., GÜDEL, R., SALAZAR L., HOMEIER J. & KLUGE J. 2014a. Impact of mycorrhization on the abundance, growth and leaf nutrient status of ferns along a tropical elevational gradient. Oecologia 175: 887–900. KESSLER, M., SALAZAR, L., HOMEIER J. & KLUGE, J. 2014b. Species richness- productivity relationships of tropical terrestrial ferns at regional and local scales. J. Ecol. 102: 1623–1633. LARA-PÉREZ, L.A., VALDÉS-BAIZABAL, M.D., NOA-CARRAZANA, J.C., ZULUETA-RODRÍGUEZ, R., LARA-CAPISTRÁN, L. & ANDRADE-TORRES, A. 2015. Mycorrhizal associations of ferns and lycopods of central Veracruz, Mexico. LEHNERT&KESSLER: MYCORRHIZAL RELATIONSHIPS 113

Symbiosis 65(2): 85–92. LEHNERT, M., KOTTKE, I., SETARO, S., PAZMINO, L. F., SUÁREZ, J. P. & KESSLER, M. 2009 (2010). Mycorrhizal associations in ferns from Southern Ecuador. Amer. Fern J. 99: 292–306. LEKBERG, Y., ROSENDAHL, S. & OLSSON, P.A. 2015. The fungal perspective of arbuscular mycorrhizal colonization in ‘nonmycorrhizal’plants. New Phytol. 205(4): 1399–1403. LESICA, P. & ANTIBUS, R. K. 1990. The occurrence of mycorrhizae in vascular epiphytes of two Costa Rican rain forests. Biotropica 33: 250–258. MARSH, A. S., ARNONE, J. A. A., III., BORMANN, B. T. & GORDON, J. C. 2000. The role of Equisetum in nutrient cycling in an Alaskan shrub . J. Ecol. 88: 999–1011. MEHLTRETER, K. 2010. Interactions of ferns with fungi and animals. Pp. 220–254. In: Mehltreter, K., Walker, L. R. & Sharpe, J. M. (Eds) Fern Ecology. Cambridge Univ. Press, Cambridge. MOTEETEE, A., DUCKETT, J. G. & RUSSELL, A. J. 1996. Mycorrhizas in the ferns of Lesotho. In: Camus, J. M., M. Gibby and R. J. Johns (Eds) Pteridology in perspective, pp. 621–631. Royal Botanic Gardens, Kew. MUTHUKUMAR, T. & PRABHA, K. 2012. Fungal associations in gametophytes and young sporophytic roots of the fern Nephrolepis exaltata. Acta Bot. Croat., 71(1): 139–146. MUTHUKUMAR, T. & PRABHA, K. 2013. Arbuscular mycorrhizal and septate endophyte fungal associations in lycophytes and ferns of south India. Symbiosis 59(1): 15–33. NEWMAN, E. I. & REDDELL, P. 1987. The distribution of mycorrhizas among families of vascular plants. New Phytol. 106: 745–751. NEWSHAM, K. K., FITTER, A. H. & WATKINSON, A. R. 1995. Arbuscular mycorrhiza protect an annual grass from root pathogenic fungi in the field. J. Ecol. 83(6): 991–1000. OGURA-TSUJITA, Y., HIRAYAMA, Y., SAKODA, A., SUZUKI, A., EBIHARA, A., MORITA, N., & IMAICHI, R. 2016. Arbuscular mycorrhizal colonization in field- collected terrestrial cordate gametophytes of pre-polypod leptosporangiate ferns (Osmundaceae, Gleicheniaceae, Plagiogyriaceae, Cyatheaceae). Mycorrhiza 26(2): 87–97. OGURA-TSUJITA, Y., SAKODA, A., EBIHARA, A., TOMOHISA YUKAWA, T. & IMAICHI. R. 2013. Arbuscular mycorrhiza formation in cordate gametophytes of two ferns, Angiopteris lygodiifolia and Osmunda japonica. J. Plant Res. 126 (1): 41– 50. OLSSON, P. A., ERIKSEN, B. & DAHLBERG, A. 2004. Colonization by arbuscular mycorrhizal and fine endophytic fungi in herbaceous vegetation in the Canadian High . Can. J. Bot. 82: 1547–1556. OLSSON, P. A., RAHM, J. & ALIASGHARZAD, N. 2010. Carbon dynamics in mycorrhizal symbioses is linked to carbon costs and phosphorus benefits. Fems Microbiol. Ecol. 72: 125–131. PAGE, C. N. 2002. Ecological strategies in fern evolution: a neopteridological overview. Rev. Palaeobot.Palyn. 119(1): 1–33. PAZMIÑO, L. 2006. Estudio preliminar de hongos que forman micorrizas en helechos de las familias Grammitidaceae y Polypodiaceae en el bosque de la ECSF y El Tiro, 114 FERN GAZ. 20(3):101-116. 2016

Loja, Ecuador. BSc thesis, Universidad Técnica Particular de Loja, Loja, Ecuador. pdf available upon request from the author. PEARSON, V. & READ, D.J. 1973. The biology of mycorrhiza in the Ericaceae: I. The isolation of the endophyte and synthesis of mycorrhizas in aseptic culture. New Phytol. 72(2): 371–379. PIROZYNSKI, K. A. & MALLOCH, D. W. 1975. The origin of land plants: A matter of mycotrophism. BioSystems 6: 153–164. PRESSEL, S., BIDARTONDO, M. I., LIGRONE, R. O. & DUCKETT, J. G. 2010. Fungal symbioses in bryophytes: new insights in the twenty first century. Phytotaxa 9(1): 238–53. RADHINKA, K. P. & RODRIGUES, B. F. 2007. Arbuscular mycorrhizae in association with aquatic and marshy plants in Goa, India. Aquatic Bot. 86: 291–294. READ, D. J. 1996. The structure and function of the ericoid mycorrhizal root. Ann. Bot. 77(4): 365–374. READ, D. J. & HASELWANDTER, K. 1981. Observations on the mycorrhizal status of some alpine plant communities. New Phytol. 88: 341–352. READ, D.J., DUCKETT, J.G., FRANCIS, R., LIGRONE, R. & RUSSELL, A. 2000. Symbiotic fungal associations in “lower” land plants. P. Trans. Roy. Soc. B 355: 815– 831. REMY, W., TAYLOR, T.N., HASS, H. & KERP, H. 1994. Four hundred-million-year- old vesicular arbuscular mycorrhizae. PNAS 91(25): 11841–11843. REYES–JARAMILLO, I., CAMARGO-RICALDE, S. L. & AQUIAHUATL-RAMOS, M.A. 2008. Mycorrhizal-like interaction between gametophytes and young sporophytes of the fern Dryopteris muenchii (Filicales) and its fungal endophyte. Rev. Biol. Trop. 56(3): 1101–1107. RIMINGTON, W.R., PRESSEL, S., DUCKETT, J.G. & BIDARTONDO, M.I. 2015. Fungal associations of basal vascular plants: reopening a closed book? New Phytol. 205(4): 1394–1398. SCHMID, E. & OBERWINKLER, F. 1994. Light and electron microscopy of the host– fungus interaction in the achlorophyllous gametophyte of Botrychium lunaria. Can. J. Bot. 72(2): 182–188. SCHMID, E. & OBERWINKLER, F. 1995. A light‐and electron‐microscopic study on a vesicular–arbuscular host‐fungus interaction in gametophytes and young sporophytes of the Gleicheniaceae (Filicales). New Phytol. 129(2): 317–324. SCHMID, E. & OBERWINKLER, F. 1996 Light and electron microscopy of a distinctive VA mycorrhiza in mature sporophytes of Ophioglossum reticulatum. Mycol. Res. 100: 843–849. SCHMID, E., OBERWINKLER, F. & GOMEZ L. D. 1995 Light and electron microscopy of a host-fungus interaction in the roots of some epiphytic ferns from . Can. J. Bot. 73: 991–996. SCHMIDT S. K., SOBIENIAK-WISEMAN, L.C., KAGEYAMA, S. A., HALLOY, S. R. P. & SCHADT, C. W. 2008. Mycorrhizal and dark-septate fungi in plant roots above 4270 meters elevation in the Andes and Rocky Mountains. Arct. Antarct. Alp. Res. 40: 576–583. SCHÜßLER, A., SCHWARZOTT, D. & WALKER, C. 2001. A new fungal , the Glomeromycota: phylogeny and evolution. Mycological Research 105: 1413–1421. SELOSSE, M. A. & ROUSSET, F. 2011. The plant-fungal marketplace. Science 333(6044): 828–829. LEHNERT&KESSLER: MYCORRHIZAL RELATIONSHIPS 115

SELOSSE, M. A., STRULLU‐DERRIEN, C., MARTIN, F. M., KAMOUN, S. & KENRICK, P. 2015. Plants, fungi and oomycetes: a 400‐million year affair that shapes the biosphere. New Phytol. 206(2): 501–506 SIDDIQUI, Z.A. & PICHTEL, J. 2008. Mycorrhizae: an overview. In: Siddiqui, Z. A., Akhtar, M. S. & Futai, K. (Eds) Mycorrhizae: Sustainable Agriculture and Forestry, pp. 1–35. Springer Science and Business Media B.V. SMITH, A. R., PRYER, K. M., SCHUETTPELZ, E., KORALL, P., SCHNEIDER, H. & WOLF, P.G. 2006. A classification for extant ferns. Taxon 55: 705–731. SMITH, S. E. & READ, D. J. 2008. Mycorrhizal Symbiosis. Academic Press; 3rd edition. SMITH, S. E. & SMITH, F. A. 2011. Roles of arbuscular mycorrhizas in plant nutrition and growth: New paradigms from cellular to ecosystem scales. Ann. Rev. Plant Biol. 62: 227–50. STRIBLEY, D.P., TINKER, P. B. & RAYNER, J. H. 1980. Relation of internal phosphorus concentration and plant weight in plants infected by vesicular-arbuscular mycorrhizas. New Phytol. 86: 261–266. STRULLU-DERRIEN, C. & STRULLU, D. G. 2007. Mycorrhization of and living plants. Comptes Rendus Palevol, 6(6), pp.483–494. STRULLU‐DERRIEN, C., KENRICK, P., PRESSEL, S., DUCKETT, J. G., RIOULT, J. P., & STRULLU, D. G. 2014. Fungal associations in Horneophyton ligneri from the Rhynie Chert (c. 407 million year old) closely resemble those in extant lower land plants: novel insights into ancestral plant–fungus symbioses. New Phytologist 203(3): 964–979. TAYLOR, T. N., REMY, W., HASS, H. & KERP, H. 1995. Fossil arbuscular mycorrhizae from the Early . Mycologia 87(4): 560–573. TAYLOR, A. F. & ALEXANDER, I. A. N. 2005. The ectomycorrhizal symbiosis: life in the real world. Mycologist 19(03): 102–112. TRAPPE, J. M. 1987. Phylogenetic and ecologic aspects of mycotrophy in the angiosperms from an evolutionary standpoint. In: Safir, G.R. (Ed.) Ecophysiology of VA mycorrhizal plants, pp. 2–25. CRC Press, Boca Raton. TURNAU, K., ANIELSKA, T. & JURKIEWICZ, A. 2005. Mycothallic/mycorrhizal symbiosis of chlorophyllous gametophytes and sporophytes of a fern, Pellaea viridis (Forsk.) Prantl (Pellaeaceae, Pteridales). Mycorrhiza 15(2): 121–128. VAN DER HEIJDEN, M. G., MARTIN, F. M., SELOSSE, M. A. & SANDERS, I. R. 2015. Mycorrhizal ecology and evolution: the past, the present, and the future. New Phytol. 205(4): 1406–1423. VEIGA, R. S. L, JANSA, J., FROSSARD, E. & VAN DER HEIJDEN M. G. A. 2011. Can arbuscular mycorrhizal fungi reduce the growth of agricultural weeds? PloS One 6: e27825. WANG, B. & QIU, Y. L. 2006. Phylogenetic distribution and evolution of mycorrhizas in land plants. Mycorrhiza 16: 299–363. WANG, B., YEUN, L., XUE, J. Y., LIU, Y., ANÉ, J. M. & QIU, Y. L. 2010. Presence of three mycorrhizal genes in the common ancestor of land plants suggests a key role of mycorrhizae in the colonisation of land by plants. New Phytol. 186(2): 514–525. WARREN, J. M., BROOKS, J. R., MEINZER, F. C. & EBERHART, J. L. 2008. Hydraulic redistribution of water from Pinus ponderosa trees to seedlings: evidence for an ectomycorrhizal pathway. New Phytol. 178(2): 382–394. WATKINS, J.E., HOLBROOK, N.M. & ZWIENIECKI, M.A. 2010. Hydraulic properties of fern sporophytes: consequences for ecological and evolutionary diversification. 116 FERN GAZ. 20(3):101-116. 2016

Amer. J. Bot. 97(12): 2007–2019. WILKINSON, D. M. 2001. Mycorrhizal evolution. Trends Ecol. Evol. 16: 64–65. WINTHER, J. L. & FRIEDMAN, W. E. 2007a. Arbuscular mycorrhizal associations in Lycopodiaceae. New Phytol. 177(3): 790–801. WINTHER, J. L. & FRIEDMAN, W. E. 2007b. Arbuscular mycorrhizal symbionts in Botrychium (Ophioglossaceae). Amer. J. Bot. 94(7): 1248–1255. WINTHER, J. L. & FRIEDMAN, W. E. 2009. Phylogenetic affinity of arbuscular mycorrhizal symbionts in Psilotum nudum. J. Plant Res. 122(5): 485 496. ZHAO, Z. W. 2000. The arbuscular mycorrhizas of pteridophytes in Yunnan, southwest China: evolutionary interpretations. Mycorrhiza 10: 145–149. ZUBEK, S., PIĄTEK, K., NAKS, P., HEISE, W., WAYDA, M. & MLECZKO, P. 2010. Fungal root endophyte colonization of fern and species from Celaque National Park in Honduras. Amer. Fern. J 100: 126–136. FERN GAZ. 20(3). 2016 117


Michael Kessler was born and grew up in Peru where he spent much of his childhood chasing birds across the forest and mountains of the country. He studied biology at the University of Göttingen, Germany from 1986 onwards, culminating with his PhD thesis on the taxonomy, ecology, and biogeography of the high-Andean tree genus Polylepis () in 1995. He then shifted his focus to the plant diversity of tropical montane forests, and, inspired by the work of Hanna Tuomisto in Amazonia, picked ferns as a study group. Over the years, ferns have become the main focus of his work, with research mainly on diversity patterns and ecology, but also numerous taxonomic studies, especially on Bolivian ferns. Since 2008, he has worked as scientific curator of the botanical garden and researcher at the University of Zurich, Switzerland. Currently, his fern work is mainly concerned with understanding the evolutionary and ecological processes that determine the richness and composition of fern assemblages, involving fieldwork in, e.g., Mexico, the Andes, Uganda, Indonesia, and New Guinea. Other projects involve studies on the diversification of bromeliads and palms in the neotropics and the flora of the canton of Zurich in Switzerland. FERN GAZ. 20(3). 2016 118

Marcus Lehnert was born on 1 October 1975 in Neutstadt/Ostholstein, Germany. He attended Göttingen University, where he received his Diplom (M.S. equivalent) in 2002 and his Ph.D. in 2007. His research was based first at the Albrecht-von-Haller Institute for Plant Sciences, Göttingen (2000–2007), with interludes at the Instituto de Ecología, Universidad Mayor de San Andres, La Paz, Bolivia (2002–2003) and the University of California, Berkeley (2006–2007). From 2008-2012, he was employed as a Postdoctoral Researcher in the Botany Department of the State Museum for Natural History in Stuttgart. Since 2013 he has been a research assistant at the Nees Institute for Plant Biodiversity, University Bonn. His research interest focuses on the taxonomy, phylogeny, and floristics of ferns but also includes mycorrhiza, general biogeography, and the systematics of several angiosperm families. For his fieldwork, he has visited among others Puerto Rico, , Peru, Argentina, Brazil, and several times Ecuador, Colombia, Bolivia and New Guinea. Marcus Lehnert is currently working on a large- scale projects focusing on the use of DNA barcoding for the rapid assessment of biodiversity (Accelaration of Biodiversity Assessment—ABA Ecuador and German Barcode of Life-GBoL). He has contributed his taxonomical expertise on scaly tree ferns (Cyatheaceae) to floral projects like “Catálogo de las Plantas Vasculares del Cono Sur” and “Ferns of Bolivia.” FERN GAZ. 20(3):119-132. 2016 119



1 Department of Botany, Faculty of Science, Istanbul University, Istanbul, Turkey. E-mail: [email protected] 2 Royal Botanic Garden Edinburgh, Edinburgh, UK. E-mail: [email protected]

Keywords: Adiantum, Allosorus, Anogramma, Cheilanthes, Cryptogramma, Paragymnopteris, Pteridaceae, Pteris, Cosentinia, Turkey.

ABSTRACT The last comprehensive study of Turkish Pteridaceae was made over 50 years ago. In this study the family Pteridaceae in Turkey is reviewed according to recent taxonomic and nomenclatural changes and a full description, synonyms and distribution information are given for each taxon and a key provided.

INTRODUCTION A generic classification of cheilanthoid ferns (Pteridaceae) has eluded taxonomists for more than 200 years (Grusz & Windham, 2013). Several taxonomic and nomenclatural updates have been made in recent decades, based on morphological and molecular methods (Gastony & Rollo, 1995; Sanchez-Baracaldo, 2004; Zhang et al., 2005; Smith et al., 2006; Kamau, 2007; Schuettpelz et al., 2007; Windham et al., 2009; Grusz & Windham, 2013; Schneider et al., 2013; Abotsi et al., 2015). The current family diversity is about 1000 species distributed in 53 genera (Schuettpelz et al., 2007; Christenhusz et al., 2011). The last comprehensive study of Turkish Pteridaceae was made over 50 years ago (Henderson, 1965) for the Flora of Turkey. A major project was recently initiated to illustrate and describe the flora of Turkey in the Turkish language and the first volume of Resimli Türkiye Florası [Illustrated Flora of Turkey] was published by Güner and Ekim (2014). The second volume, which will include the family Pteridaceae, is scheduled for publication in 2016. The aim of the present study has been to review Turkish Pteridaceae according to recent taxonomic and nomenclatural changes and to provide descriptions and distributions for each taxon. It is based on recent literature on Pteridaceae (e.g. Nardi & Reichstein, 1985; Benlioğlu et al., 1997; Kaynak et al., 2008; Christenhusz et al., 2011; Greuter & Raab-Straube, 2012; Jessen et al., 2012; Güner et al., 2012), and on herbarium specimens from Ankara University Faculty of Pharmacy (AEF), Ankara University Faculty of Science (ANK), Natural History Museum, London (BM), Royal Botanic Garden Edinburgh (E), Geneva (G), Gazi University Faculty (GAZI), Hacettepe University (HUB), Istanbul University Faculty of Pharmacy (ISTE) and Royal Botanic Gardens, Kew (K). All specimens listed below are from E, except where indicated otherwise.

PTERIDACEAE E.D.M.Kirchn., Schul-Bot.: 109 (1831). Synonym: Adiantaceae Newman, Hist. Brit. Ferns: 5 (1840), Sinopteridaceae Koidz., Acta Phytotax. Geobot. 3: 50 (1934). Cryptogrammaceae Pic.Serm., Webbia 17: 299 (1963). Cheilanthaceae B.K.Nayar, Taxon 19: 233 (1970). 120 FERN GAZ. 20(3):119-132. 2016

Key for Turkish Pteridaceae taxa 1. Fertile and sterile leaves or pinnae conspicuously dimorphic ...... 2 1. Fertile and sterile leaves or pinnae not, or weakly dimorphic ...... 4 2. Annual, sori on the lower surface of the leaf, indusium absent ...... Anogramma leptophylla 2. Perennial, sori marginal, covered by the recurved leaf margin...... 3 3. Lamina of sterile leaves up to 16 cm long, ovate to deltoid-ovate, spores <60μm long ...... Cryptogramma crispa 3. Lamina of sterile leaves up to 7 cm long, broadly deltoid, spores >60μm long ...... Cryptogramma bithynica 4. Pinnules fan-shaped; petiole blackish, glabrous, fine and wiry ...... Adiantum capillus-veneris 4. Pinnules not fan-shaped; petiole not as above...... 5 5. Fronds pinnate to pinnatisect, weakly dimorphic...... 6 5. Fronds 2-pinnate, not dimorphic ...... 7 6. Leaves 1-pinnate, lanceolate, >10 pinnae pairs ...... Pteris vittata 6. Lower pinnae pinnatisect, frond ovate-triangular, with 7 or fewer pinnae pairs...... Pteris cretica 7. Underside of pinnules glabrous or with glandular hairs...... 8 7. Underside of pinnules densely covered with scales or eglandular hairs ...... 10 8. Pseudoindusium entire, frond sparsely glandular below...... Allosorus tinaei 8. Pseudoindusium ciliate, frond eglandular below ...... 9 9. Pseudoindusium narrow, with dense long marginal hairs, spores <45 μm long ...... Allosorus persicus 9. Pseudoindusium broad, irregular, with short cilia, spores >45 μm long ...... Allosorus acrosticus 10. Upper surface of pinnules glabrous ...... Paragymnopteris marantae 10. Upper surface of pinnules white, woolly ...... Cosentinia vellea

Adiantum L., Sp. Pl. 1094 (1753). Short-creeping, scaly rhizome. Fronds dense or sparse; stipe dark brown or black shiny. Pinnae fan-shaped, without a midrib, sori borne on reflexed marginal lobes of lamina. A. capillus-veneris L., Sp. Pl. 2: 1096 (1753). Synonyms: Adiantum capillus Sw., J. Bot. (Schrader) (2): 83 (1801). A. formosum R.Br., Prodr. Fl. Nov. Holland 155 (1810). A. schaffneri Fourn., Bull. Soc. Bot. France 27: 328 (1880). A. modestum Underw., Bull. Torrey Bot. Club. 28: 46 (1901). A. michelii Christ, Bull. Geogr. Bot. Mans. 12 (1910). Lectotype: Herb. Linn. 1252.9 (LINN)! Rhizome densely covered with shiny, narrow lanceolate scales. Fronds bipinnate, (5– )15–30(–45) × (1.5–)4–6(–10) cm, glabrous; stipe (1.5–)8–12(–20) cm long, brown or black, shiny, glabrous. Pinnae 2–7 × 1–4 cm with stalk 0.3–3 cm long. Pinnules fan- shaped, (0.7–)1–2(–3.5) × (0.7–)1–2.4(–3) cm, cuneate at base, without midrib, 2–8 lobed with stalk (1–)2–5(–16) mm long. Sori borne on reflexed marginal lobes of lamina. Habitat: In crevices of limestone and serpentine or tufa and base-rich soil on shady banks, 1–1000 m. Distribution in Turkey: Adana: Feke; Göksu gorge, 800 m, 09.vii.1952, Davis 19855! Antalya: Kemer, Sumakşehiri Deresi, kalkerli G. Deresi değil vadisi, P. nigra-C. BONA & GIBBY: THE FERN FAMILY PTERIDACEAE IN TURKEY 121 sempervirens ormanı 500-700 m, 06.x.1977, H. Peşmen 3569 & Ş. Yıldırımlı HUB 36734! Alanya, 120 m, 04.viii.1982, Ş. Yıldırımlı 4451 HUB 36732! Aksu, Düden Çağlayanı, 06.ix.1988, Ş. Yıldırımlı 11596 HUB 36736! Artvin: Çoruh gorge, between Yusufeli and Artvin. 600 m, 01.viii.1966, Davis 47690! Demirkent nahiyesi, zwischen Tortum Gölü und Artvin, 620 m, 03.viii.1973, F. Holtz, P. Hanel & T. Kesercioğlu 991! Hopa, Kemalpaşa-Sarp arası, denize dik kayalıklar, 0-50 m, 19.v.1985, Ş. Yıldırımlı 7898 HUB 36740! Bursa: Uludağ, 25.vi.1946, A. Yılmak! Denizli: 13.vii.1947, Davis 13263! Pamukkale, 16.x.1980, Ş. Yıldırımlı HUB 36728! Düzce: Akçakoca, Edilli Köyü, kıyı kumul, orman, 50 m, 29.ix.2001, A. Doğru Koca 1489 (HUB)! Hatay: Dörtyol, Kuzuculu to Bülke on forest road, 1000 m, 04.iv.1965, M.J.E. Coode & B.M.G. Jones 416! Valley above Dörtyol, south bank, north facing, boulder clay and serpentine, 150 m, 12.viii.1969, J. Darrah 623! Valley above Yeşilkent, 305 m, 11.viii.1969, J. Darrah 505! Samadağ, Çevlik, Vespasiyanus ve Titus Tüneli boyunca, Beşikli mağarası ve taşlık maki, 10-150 m, 04.ix.1990, Ş. Yıldırımlı 13926 (GAZI)! Antakya, Harbiye, Şelale, 180 m, 25.iv.1978, M. Coşkun 332, B. Şener & F. Ilisulu AEF 6353! Kırıkhan, Delibekirli, nemli kayalıklar, 500 m, 21.iv.1977, M. Koyuncu AEF 5668! Musa Dağı, nemli yerlerde, 14.iv.1967, Y. Akman ANK 7943! İçel: Kanlıdivane, 23.iv.1974, H. Peşmen & S. Erik HUB 36733! Anamur, Nasrettin Köyü üstü, 21.iv.1975, M. Koyuncu & M. Coşkun AEF 4854! İzmir: Kemalpaşa, Next to a stream in shady ravine in macchia in the foothills of Nif Dağ, 06.vi.1966, R. Alava & C. von Regel 5040! Karamanmaraş: Andırın; 8 miles South of Çatak, 800 m, 17.v.1965, M.J.E. Coode & B.M.G. Jones 1147! Süleymanlı, Akdağ, nemli dere içi, 1500 m, 24.vii.1977, B. Yıldız 1555 HUB 36731! Konya: Ermenek, Kazancı kasabası, Yeşilköy, Göksu Nehri boyunca, 700 m, 12.x.1983, H. Sümbül 2536 HUB 36730! Ermenek, Göktepe Kasabası, Darcın Köyü, Ayaşlı mevkii, Göksu Nehri Boyunca, 600 m, 15.ix.1983, H. Sümbül 2477 HUB 36729! Malatya: Pütürge, Ferikhan Köyü, dere kenarı, ca. 650 m, 06.v.1981, Y Altan ANK 1252! Manisa: Manisa Dağı (Spil), Karaçay boyunca, 150-450 m, 23.iv.1984, Ş. Yıldırımlı 6014, HUB 36739! Mardin: Hessana at S foot of Cudi Dağ, 900 m, 10.v.1966, Davis 42776! Kasrık Boğazı, 11 km from Cizre to Şırnak, shady river bank near bridge, 400 m, 7.v.1966, Davis 42641! Dargeçit, Çelik Köyü, Umutlu mezrası, kaya üzeri, 520 m, S. Aslan 3308 et al. (GAZI)! Muğla: 13 km South of Köyceğiz, by road to Dalyan, just east of the village of Tepearası, 10–20 m, 19.v.1990, R. Lampinen 7492! Köyceğiz, Yangı Köyü Yangı Deresi, kalkerli, sarp derin vadi, kazmofit, 22.iii.1981, A. Güner 8421 et al. HUB 36725! Fethiye, Katrancı Adası, 0-20 m, 21.v.2011, Z. Aytaç 9830 (GAZI)! Boyobası Köyü, Saklayıcı mevkii, şistik arazi, nemli çakıllık, 120 m, 18.ii.1991, A. Güner 8103 et al. (GAZI)! Yatağan- Milas arası, su kenarı, 21.v.1970, N. Tanker AEF 4851! Rize: Rize-Çayeli arası, c. 10 m, yol kenarı, 19.viii.1985, A. Güner 6848 & M. Vural HUB 36726! Pazar-Hemşin, nemli kaya üzerinde, 150 m, 19.vi.1984, M. Vural 2913 HUB 36735! Sakarya: Adapazarı; Karasu to Söğütlü, 30 m, 07.viii.1962, Davis & Coode, D. 39101! Samsun: Samsun’un 10 km doğusu, asfalt kenarı, 10 m, 12.ix.1964, H. Kayacık & G. Elçin 2211! Seaside cliffs, 3 miles east of Samsun, 10 m, 19.v.1963, Tobey 133! Sinop: On south wall of bath, 10 m, 23.iv.1966, Tobey 1624! Ayancık, Bakırlı Köyü yol 122 FERN GAZ. 20(3):119-132. 2016 ayrımından Akgöl’e 2 km, 15.ix.2009, A. Dönmez 16205 & Z. Uğurlu (HUB)! Şanlıurfa: Urfa-Gaziantep c. 1 km from Urfa, 600 m, 11.ix.1956, McNeill 794! Trabzon: Maçka, 24 km south of Trabzon, 250 m, 28.vi.1960, Stainton & Henderson 5895! General Distribution: Widespread in temperate and subtropical regions of the New and Old World, absent from South America.

Anogramma Link, Fil. Spec. 137 (1841). Small, annual, delicate plants with short rhizome. Prothallus perennial and mostly persistent. Fronds dimorphic, mature fertile fronds bi-tripinnate, pinnae simple. Sori linear, indusium absent. A. leptophylla (L.) Link, Fil. Spec. 137 (1841). Synonyms: Polypodium leptophyllum L., Sp. Pl. 2: 1092 (1753); Acrostichum leptophyllum Lam. & DC., Fl. Franc. (de Candolle & Lamarck), ed. 3. 2: 565. (1805); leptophylla (L.) Sw. Syn. Fil. (Swartz) 23, 218, t. 1. f. 6. (1806); Gymnogramma leptophylla (L.) Desv. in Berl. Mag. 5: 305 (1811); Hemionitis leptophylla Lag. Gen. Sp. Pl. (Lagasca) 33: (1816). Lectotype: Herb. Linn. 1251: 56 (LINN)! Plant delicate, up to 15 cm high, annual, with short rhizomes. Prothallus perennial, reniform, bilobed, 7–10 mm long. Fronds yellow-green, glabrous, young fronds reniform and 3–5 lobed, mature fronds bipinnate 5–15 ×1–1.5 (–2) cm, stipe glabrous 2.5–7 cm long, dark brown-black below and yellow-green above. Pinnae glabrous, 0.5–2 × 0.5–1 cm, dichotomously veined, pinnae stipe 1–2 mm. Pinnule mostly bilobed or lanceolate. Sori linear along veins; no indusium. Habitat: Dry metamorphic rocks or moist shady serpentine rocks, 30–1000 m. Distribution in Turkey: Adana: Feke; Senean dere between Gurumze and Suphandere, 1000 m, 01.vii.1952, Davis 1961, Dodds & Çetik! Antalya: Rocks near the sea, 12.iv.1955, H. Walter 3891! Side, c. 40 m, 19.v.1994, K. Karamanoğlu, M. Koyuncu & M. Coşkun AEF 16! Kemer, Kesmeboğaz, kalker kayalığı, 200 m, 25.iii.1978, H. Peşmen 3649 & B. Yıldız HUB 36798! Kemer, Beycik-Delikin Tepesi arası, 500-700 m, kalker kayalığı, P. brutia maki karışık ormanı, 28.iv.1980, H. Peşmen 4818 HUB 36796! Hatay: Dörtyol; Rabat, vertical serpentine flush, c. 100 m, 05.v.1965, M.J.E. Coode & B.M.G. Jones! Isparta: Ş.Karaağaç, Kızıldağ Milli Parkı, Kuzgu Tepe, kaya üzeri, 1350 m, 24.vii.1994, B. Mutlu 1084 HUB 36794! İçel: Anamur, Nasrettin Köyü üstü, pınar civarı nemli yerlerde, 250 m, 21.iv.1975, M. Koyuncu & M. Coşkun HUB 36797! Kastamonu: Taşköprü, Kayabaşı Köyü yukarısı, yangın kulesine tırmanış, dağ bozkırı, P. nigra-P. sylvestris açıklığı, 04.viii.2010, A. Dönmez 17533 & Z. Uğurlu (HUB)! Muğla: behind Marmaris, 50–100 m, 14.iv.1965, Davis 41407! Marmaris, On soil amongst rocks in ravine forest near Hotel Lidya, 06.v.1984, E.C. Wallace! Marmaris, Pinetum brutiae, on serpentine, 30 m, 24.iii.1956, Davis 25258 & O. Polunin! Ortaca, Dalyan, Gökbel Köyü, Kızıl Çam ormanı, kalkerli arazi, 100-150 m, 14.iv.1991, A. Güner 8622 et al. HUB 36795! Köyceğiz, Ekincik Köyü, Sandallı tepe, makilik, kalkerli arazi, 10-500 m, 19.v.1991, A. Güner 9097, M. Vural & H. Şağban HUB 36799! Ortaca, Dalyan, Marmarlı çevresi, makilik arazi, 30 m, 19.iv.1991, A. Güner 8918 et al. (GAZI)! BONA & GIBBY: THE FERN FAMILY PTERIDACEAE IN TURKEY 123

General Distribution: Mediterranean area, Switzerland, W. France and Channel Islands, Crimea, N. America; scattered to S. Africa, India and Australasia.

Allosorus Bernh., Neues J. Bot. 1(2): 36 (1805). Rhizome short-creeping or tufted. Fronds persistent, bipinnate; stipe wiry. Sori terminal on veins, protected by a linear pseudoindusium, a recurved and modified leaf margin. A. acrosticus (Balbis) Christenh. Willdenowia 42(2):284. (2012) Synonyms: Pteris acrostica Balb. Elenco 98. (1801). Cheilanthes acrostica (Balbis) Tod. Giornale de Scienze Naturali ed Economiche de Palermo 1(10): 215. (1866). Lectotype: “Polypodium ilvense Bell. / puto esse potius novam plantam pteridis genus” [Balbis’s handwriting] / “est Polypodium fragrans L.” [not Balbis’s handwriting] (TO). Four plants. (see Nardi & Reichstein. 1985). Plant up to 20 cm, rhizome brown and covered with long linear, reddish brown scales. Fronds bipinnate, 3–20 × 1–2.5 cm, glabrous, green; stipe dark brown-black 1.5–11.5 cm with a few scales. Pinnae 0.5–3 × 0.3–1 cm, stalk sessile, 1–3 mm long at end of the fronds. Pinnule 0.5–2 mm broad, without midrib. Sori many; pseudoindusium broad, irregularly lobed, ciliate. Habitat: Crevices in limestone rocks, sea level up to 500 m. Distribution in Turkey: Antalya: Antalya-Burdur yolu, 33. km, 12.iv.1965, A. Baytop & T. Baytop 7926! Ģarma Dağ, 700ft., 17.vi.1958, E.R.B.Little! Aspendos Amphitheatre near Serik, c.50m. 02.viii.1975, C.R.Fraser Jenkins 4573. BM! Kemer Küçükburun, kasabanın doğusundaki küçük yarımada, P. brutia ormanı, 20 m, 21.iii.1949, H. Peşmen 4150 & A. Güner HUB 36705! Kemer, Kesmeboğaz, kalker kayalığı, 300 m, 25.iii.1978, H Peşmen & B. Yıldız HUB 36707! Kaş, Kekova, Alan-Kale Mahallesi, kalker kaya çatlağı, 10-20 m, 14.ii.2009, Z. Aytaç 9286 & H. Duman (GAZI)! Alanya, Hacımemetli Köyü, Bağderesi, 200 m, 21.iii.1993, M. Koyuncu 9595 & H. Duman (AEF)! Aydın: Söke Milet haraberleri, anfitiyatro duvarları üzerinde, 10.v.1965, Kayacık & Yaltırık ISTO 3545! Balıkesir: Ayvalık, Pınar Adası, 60 m, 10.v.1996, K. Alpınar ISTE 71797! Çorum: Alacahöyük Köyü, 01.vii.1995, U. Sönmez ISTE 68382! Denizli: Mt. above Pamukkale (Hierapolis) hot springs near Burdur, rock crevice, dry place on vertical earth bank with C. persica and Selaginella denticulata, 1000-1150m, 16.v.1973, Betty E.G. Molesworth Allen 8102. BM! Hatay: Castle of Payas, near Dörtyol, sea level, 03.iv.1965, M.J. Coode & B.M.G. Jones 394! Kırıkhan to Hamam, c. 100 m, 06.v.1965, M.J. Coode & B.M.G. Jones 533! İçel: Işıktepe (Seydüşıh) Köyü üstü, maki, kalker anakaya, 150-500 m, 19.iv.1985, Ş. Yıldırımlı 7594 HUB 36723! Mersin, Anamur çevresi, 09.v.1972, M. Koyuncu HUB 36697! Dibek Mağarası girişi, Cennet-Cehennem, 09.xi.1973, A. Baytop & E. Tuzlacı ISTE 27072! İzmir: Schuth hinter Friendhof von Ahmetbeli, ca. 100 m, 19.iii.1966, G. Bocquet 1523! Selçuk harabeleri, 09.iv.1971, A. Baytop & T. Baytop ISTE 19112! Konya: Bucakkışla, Dede Dağı, Kayalık, kurak yamaçlar, maki, ca.580 m, 25v.1979, M. Vural 1873 GAZI)! Muğla: ca. 4 km nw Muğla. Felsen in der Macchie, 03.iv.1974, K. Lewejohann (Tu-74- 137) & F. Holtz! Dereköy, between Marmaris and Muğla, 10 m, 26.iii.1956, Davis 25395 & O. Polunin! BM! Fethiye, 30 m, 27.iii.1956, Davis 25421 & O. Polunin! BM! Kişle to Kozağaç (Milas-Yatağan), 550 m, 13.iv.1965, Davis 41056! Marmaris, Söğüt to 124 FERN GAZ. 20(3):119-132. 2016

Bozburun, sea level–50 m, 15.iv.1965, Davis 41168! Dalyan-Sülüngür Gölü, Ortaca arası, 100-200 m, 14.iv.1981, A Güner 8571 et al. (HUB)! Bodrum, Türkbükü-Bodrum, 200- 500 m, 15.iii.1984, Ş. Yıldırımlı 5883 HUB 36696! Köyceğiz, Çandır Köyü, Kaunos Harabeleri, duvar arası, 80 m, 08.xi.1991, A. Güner 10323 (GAZI)! Köyceğiz, Yangı Köyü, Yangı Deresi, sarp ve derin vadi, kalkerli yamaçlar, 60-80 m, 24.v.1981, A. Güner 8324, M. Vural & H. Şağban (GAZI)! General Distribution: From Mediterranean area to West Himalayas. Note: There has been confusion in the past between Allosorus acrosticus and A. pteridioides (Reichard) Christenh. (see Nardi & Reichstein, 1985). Allosorus acrosticus is a tetraploid taxon, whereas A. pteridioides is diploid. This latter has a more western Mediterranean distribution and, so far, has not been recorded for Turkey, although there are records from Cyprus and Jordan in BM and from Syria and Palestine in E, and it may have been overlooked in Turkey. The two taxa have similar morphology but can be distinguished; A. acrosticus has a pseudoindusium with ciliate margins, with scales on rachis and costae, whereas A. pteridioides has pseudoindusium entire, and rachis and costae are ± glabrous. A. persicus (Bory) Christenh. Willdenowia 42(2): 284 (2012). Synonyms: Notholaena persica Bory in Bélanger & al., Voy. Indes Or.: 23 (1833); Cheilanthes szovitsii Fisch. & Mey. apud Hohen. in Bull. Soc. Nat. Mosc. 1838 No. 3: 241 (1838); Cheilanthes persica (Bory) Kuhn in Bot. Zeitung (Berlin) 26: 234 (1868). Lectotype: [Iran] La Perse, parmi les rochers, 1821, C. Bélanger s.n. P00607931 (P)! Plant up to 20 cm, rhizomes covered densely in narrow dark brown-black scales. Fronds bipinnate, 3–16 × 1–4 cm, green and glabrous above and with brown scales on rachis and costae, eglandular; stipe 1–8 cm, covered with dark brown-black and narrow lanceolate or linear long brown hairs. Pinna (0.3–) 1–3 × 0.2–2 cm; pinna stalk very short c. 1 mm, pinnules lobed, lobes lancolate. Sori many; pseudoindusium narrow, with many long marginal hairs that cover the lower surface of the segment. Habitat: Shady crevices in limestone or metamorphic rocks, 200–2300 m. Distribution in Turkey: Adana: Feke, Sencan Dere between Belenköy and Suphandere, 1000 m, 02.vii.1952, Davis 19586A, Dodds, Çetik! Kozan, (Anti-taurus), 300 m, 12.iv.1957, Davis 26614, Hedge! Osmaniye-Yarpuz, 15 km e Osmaniye, 800 m, 29.iv.1986, M. Nydegger HUB 36695! Ankara: mamak, Kıbrıs Köyü vadisi, Dipsiz gölü mevkii, kaya çatlağı, gölgeli yerler, 1000-1100 m, 26.vi.2005, S. Arslan 2352 (GAZI)! Antalya: Kumluca, Sazak Koyu, kalker kayalığı, 40 m, 29.iv.1980, H. Peşmen 4876 HUB 36704! Konyaaltı, 10 m, 23.v.1977, M. Koyuncu & M. Coşkun HUB 36703! Koslu Dere, n.of Bozburun Dağ, 1200 m, 27.vii.1949 Davis 15754. Aydın: 3 km westlich Ortaklar, Abangh am Ovacık Dag oberhalb der Strasse Selçuk- Ortaklar, 200 m, 22.v.1966, G. Bocquet 2472! Söke, piriyen, 400 m, 30.v.1972, N. Tanker, AEF 17! Denizli: Bozdağ above Geynam yaila, 16.vii.1947, Davis 13432! Erzincan: Kemaliye, Yaka Köyü, Sazak deresi, kazmofit, 850-1000 m, 18.xi.1980, Ş. Yıldırmlı 4186 HUB 36708! Isparta: Ş. Karaağaç, Kızıldağ Milli Parkı girişi, sedir ormanı, kalker, 1200-1300 m, 17.xi.1993, B. Mutlu 163 HUB 36701! Eğridir, Yaka Köy, 2 km güneyi, derin kalker vadisi, P. nigra-J. excelsa ormanı, 1800 m, 20.v.1973, Peşmen 3401, Erik, Pamukçuoğlu HUB 36709! Eğridir, Anamas Köyü, Kapız Deresi, kalkerli sarp ve derin vadi, 1250- BONA & GIBBY: THE FERN FAMILY PTERIDACEAE IN TURKEY 125

1450 m, 05.viii.1974, H. Peşmen & A. Güner 1836 HUB 36706! Eğridir, Aksu, Karacahisar Köyü, Başak mevkii, kalkerli vadi, 1000 m, 03.vii.1974, H. Peşmen & A. Güner 1653 HUB 36702! Konya: Ereğli, Aydos Dağı, Delimahmutlu, Kapız, kayalık, 1600 m, 21.vi.1977, S. Erik 2179 HUB 36718! Beyşehir, Kurucuova Köy, Kuzgun tepe, P. ningra ormanı, kalkerli K:B. yamaç kayalık arazi, 1300-1500 m, 24.iv.1974, H. Peşmen & A. Güner 1019 HUB 36710! Mardin: Cudi Dağ above Hessana (d. Silopi), 900–1150 m, 11.v.1966, Davis 42862A! Savur, 900 m, 24.v.1957, Davis 28568, Hedge! Dargeçit, Çelik Köyü, Umutlu mezrası, kaya üzeri, 520 m, S. Arslan 3297 (GAZI)! Muğla: Kara Dağ, 750 m, 18.v.1988, P. Ainsworth T27/88! Ortaca, Dalyan-Sülüngür Gölü arası, 10 m, kalkerli arazi, makilik dik yamaçlar, 12.ii.1991, A. Güner 7999 et al. (HUB)! Fethiye, Babadağı, batı etekleri, gıdırak mevkii, 100 m, 10.v.1984, E. Tuzlacı ISTE 53054! Niğde: Side valley W. of Maden, 30.vii.1969, J. Darrah 349! Samsun: Vezirköprü at Kızılırmak, 400 m, 06.iv.1969, Tobey 2446! Şanlıurfa: Yenice Köyü üstü, Nemrut Dağları, kalker kayalığı tabakalar, taşlık, bozkır, 475-675 m, 26.vi.1988, Ş. Yıldırımlı 10791a HUB 36700! Şırnak: Beytüşebap, Dağdibi Köyünden anayola, kalker kayalığı, 25.v.2004, A. Dönmez 11916 (HUB)! Siirt: Botan Çay, by the bridge 19 km from Siirt, 400 m, 17.v.1966, Davis 43220! Tunceli: Ovacık, Munzur Dağları, Karagöl vadisi, 1300-1500 m, 06.v.1979, Ş. Yıldırımlı 1388 & Ş. Kaplan HUB 36711! Yozgat: Sorgun to Çekerek, 1200 m, 27.v.1965, M.J.E. Coode & B.M.G. Jones 1598! General Distribution: Distributed in Europe, Asia, and Africa. A. tinaei (Tod.) Christenh. Willdenowia 42(2): 284 (2012). Synonyms: Cheilanthes tinaei Tod. in Giorn. Sci. Nat. Econ. Palermo 1: 217 (1866); C. duriensis Mendonça & Vasc., Anais Inst. Vinho Porto 15(4): 47 (1956); C. pteridioides subsp. tinaei (Tod.) O. Bolòs & al., Fl. Man. Països Catalans 1213 (1990). Holotype: [Sicily] “Messina, Cheilanthes odora var. stipites glabrato” Id. 9541 (PAL). Isotype: Id. 9542 (PAL) (see Nardi, Rasbach & Reichstein, 1979). Plant up to 25 cm, rhizome covered with brown long scales. Fronds bipinnate 5–25 × 1– 2.5 cm, shortly and sparingly pilose when young, later ± glabrate above, scattered 2-5-celled red glandular hairs beneath; stipe densely covered with long scales at base and with a few scattered scales further up when young then almost glabrous, brown- black, 1–10 cm. Pinnae 0.5–1.5 × 0.3–0.5 cm with very short stalks c. 1 mm. Pinnule 0.5–2 mm broad. Pseudoindusium with rounded, entire membranous margin. Habitat: Coastal, bare rocks, sea level to 1250 m. Distribution in Turkey: Istanbul: Büyükada, Eskibağ, kayalıklar, 17.08.1977, H. Demiriz, B. Tütel, A. Aydın (ISTE 31028!) General Distribution: N.W. Africa, Portugal, S. Europe.

Cryptogramma R.Br., Narr. Journey Polar Sea [Franklin] 767 (1823). Rhizome creeping to ascending covered with light brown to black long scales. Fronds dimorphic, tripinnate; sori terminal on veins protected by recurved membranous lateral margins of segments. C. bithynica S.Jess., L.Lehm. & Bujnoch, Fern Gaz. 19(2): 52 (2012). 126 FERN GAZ. 20(3):119-132. 2016

Holotype: Northwestern Turkey: Uludağ southeastern of Bursa: silicate scree near the roadway, NNE of the summit of Uludağtepe, 1770 m, 09.ix.2009, leg. S. Jesen & L. Lehmann (SJ-3820), JE. Isotype: 09.ix.2009, leg. S. Jesen & L. Lehmann (SJ-3820), ANK! Plant 10(–22) cm, - covered with light brown long scales. Fronds tripinnate, green, reddish at the end of the fronds, glabrous above, veins visible at and of the lobes in white- yellow colour. Stipe straw-coloured to greenish, glabrous to sparsely covered with light brown scales. Fertile fronds 8–10 × 3–4 cm; stipe 6–7 cm. Pinnae 2 × 1–2 cm; pinna stalks 1–3 mm. Pinnules linear-lobed, convolute at the edge of lobes. Vegetative fronds slightly thick and relatively tough, 8–10(–22) × 3–4.5 cm, broadly deltate; stipe 5–6 cm. Pinnae ovate to orbicular, 1.5–2.5 × 1–2.5 cm; pinna stalks 1–3 mm. Pinnules lanceolate- lobed. Sori terminal on veins protected by recurved margins of lobes. Habitat: Silicate scree near the cycle roadway, 1700–2300 m. Distribution in Turkey: Northwestern Turkey: Uludağ southeastern of Bursa: silicate scree near the roadway, NNE of the summit of Uludağtepe, 1770 m, 09.ix.2009, leg. S. Jesen & L. Lehmann (SJ- 3820), JE. Isotype: 09.ix.2009, leg. S. Jesen & L. Lehmann (SJ-3820)(ANK)! General Distribution: Endemic to Turkey. C. crispa (L.) R.Br. ex Hook., Gen. Fil. pl. 115B (1842). Synonyms: Osmunda crispa L., Sp. Pl. 1067 (1753); Allosorus crispus (L.) Bernh. in Schrad Jour. 1(2): 36 (1800). Type: Habitat in Anglia, Helvetia, Herb. Sloane 85: 128 (BM-SL). Plant to 30 cm, rhizome covered with brown-black long scales. Fronds thin, tripinnate, green, reddish at the end of the fronds, glabrous above, veins visible at and of the lobes in white-yellow colour. Stipe straw-coloured to greenish, glabrous. Fertile fronds 15–30 × 2.5–5 cm; pinna stalks 8–20 cm. Pinnae 2–5 × 1–1.5 cm; stipe 1–6 mm. Pinnules linear- lobed, convolute at the edge of lobes. Vegetative fronds ovate to deltoid–ovate, 15–20 × 3–4 cm; pinna stalks 4–11 cm. Pinnae narrowly ovate, 1.5–3 × 0.7–1.8 cm; pinna stalks 1–4 mm. Pinnules lanceolate-lobed. Sori terminal on veins protected by recurved margins of lobes. Habitat: Screes, 200–2400 m. Distribution in Turkey: Artvin: Arhavi, above Dikyamaç village, 2200 m, 26.vii.1974, M. Çoşkun AEF 19! Çoruh, Şaval Tepe above Murgul (igneus), 2000 m, 13.viii.1957 Davis 32393 & Hedge! Şavşat, Kocabey Kışlası, Camız patlatan mevkii, kayalık alanlar, 1900-1950 m, 14.v.2004, H. Altınözlü 3945 (HUB)! Kaçkal Dağı, Pırnallı Üst Yaylası, Cehennem Deresi Mevkii, 1750-3100 m, 12.vii.1984, Demirkuş 2641b HUB 36758! Arhavi, Dikyamaç Köyü yaylası, Arpaçay civarı, 2200 m, M. Coşkun AEF! Rize: Kaçkar Mountains, east of Elevit, north of road beyond village, 2365 m, 27.ix.2008! Çamlıhemşin, Amlakıt Yaylası, çayırlık, 1350 m, 10.viii.1984, A. Güner 6117 & M. Vural HUB 36755! Çamlıhemşin, Amlakıt Yaylası, Kayguttan Çovinovit yoluyla yayla, kalkerli arazi, alpinik step, 2200-2720 m, 19.vii.1974, A. Güner 1087 HUB 36753! Çamlıhemşin, Yukarı Amlakıt Yaylası-Kışyatağı arası, Rhododendron caucasicum çalılığı, granit anakaya, 2200-2350 m, 20.ix.1979, A. Güner 2190 HUB 36754! General Distribution: Europe and Caucasus.

Cosentinia Tod. Giorn. Sci. Nat. Econ. Palermo 1: 219 (1866). Rhizomes short-creeping to erect with long linear brown scales. Fronds pinnate, both BONA & GIBBY: THE FERN FAMILY PTERIDACEAE IN TURKEY 127 surfaces of the frond covered, densely below, with long whitish multicellular hairs. C. vellea (Aiton) Tod. Giorn. Sci. Nat. Econ. Palermo 1: 220 (1866). Synonyms: Acrostichum velleum Aiton Hort. Kew. 3: 457 (1789). Notholaena vellea (Aiton) Desv. in J. Bot. Appl., 1:92 (1813) non R. Br. (1810); N. lanuginosa (Desf.) Desv. ex Poir., Fragenta Phytographiae Australiae 5 (1865); Cheilanthes vellea (Aiton) F. Muell. Domin Bibl. Bot. 85. 133 nota. (1913); Acrostichum catanense Cosent., Atti Accad. Gioenia Sci. Nat. Catania 1/2: 207 (1827); Cheilanthes catanensis (Cosent.) H.P.Fuchs, Brit. Fern Gaz. 9(2): 45 (1961). Lectotype: Portugal, 1777, F. Masson s.n. BM000829683 (BM)! Plant 10–20 cm, rhizomes brown with long linear pale brown scales. Fronds pinnate, 8– 16 × 1.5–4 cm, green above and brownish below, both surfaces of the frond covered, densely below, with tangled long whitish multicellular hairs; stipe brown, 1–4.5 cm, densely covered with hairs when young, becoming glabrous when mature, young fronds almost sessile, lobed, 0.5–2 × 0.3–0.8 cm. Sori not discrete, sporangia spread along veins from leaf margin, ± protected by recurved but not modified leaf-margin. Habitat: Dry limestone, calcareous rocks, sea level to 300 m. Distribution in Turkey: Ankara: Beynam, Karaçam ormanı, 1300-1500 m, 15.v.1986, Ş. Yıldırımlı 9221 HUB 36713! Antalya: Kumluca, Adrasan Köyü kuzeyi, serpantin arazi, 300 m, 29.ii.1980, H. Peşmen 4791 & A Güner HUB 36721! Aydın: Priene, 100 m, 09.iv.1965, Davis 40788! Milet harabeleri, R. Lampinen 7441 ISTE 63662! Bolu: Eğerci’den Yedigöller’e 4. km, Yağmurca Köyü ilerisi, 193 m, 03.xi.2001, A. Dönmez 10387 & S. Işık (HUB)! Kıbrıscık, Kıbrıscık’tan Beypazarı’na 5 km, Paliurus spina-christi açıklığı, 1110 m, 08.viii.2010, A. Dönmez 17616 & Z. Uğurlu (HUB)! Çankırı: Eldivan, Seydiköy üstü, step, 1300-1350 m, 09.viii.1984, Ş. Yıldırımlı 7409 HUB 36716! Hatay: İskenderun, Musa Dağı, Kale ilerisi, P. brutia ormanı, 200 m, 30.iii.1995, A. Dönmez 4423 (HUB)! Isparta: Ş.Karaağaç, Kızıldağ Milli Parkı girişi, Sedir ormanı açıklığı, kalker, 1200-1300 m, 18.xi.1993, B. Mutlu 180! İçel: Anamur’un üstü, Kayalıklarda 200 m, 06.iii.1977, M. Koyuncu HUB 36722! Kastamonu: Hanönü, Karapınar Köyü’nden, Derencik Köyü’ne gidiş, köprü çevresi, çakıllı yamaçlar, 26.ii.2010, A. Dönmez 16603 & Z. Uğurlu (HUB)! Kayseri: Yılanlı Dağ, 1200 m, 12.vi.1975, M. Coşkun & N. Çelik HUB 36719! Muğla: Marmaris-Emecik, 5 m, 25.iii.1956, Davis 25349 & O. Polunin! Marmaris, Hisarönü to Turgut, 20 m, 15.iv.1965, Davis 41147! Ören, 50–100 m, 10.iv.1965, Davis 40855! Köyceğiz, Yargı Köyü, Yargı Deresi, 60-80 m, sarp ve derin vadi, kalkerli kayalık yamaçlar, kazmofit, 24.v.1981, A. Güner 9322, M. Vural & H. Şağban HUB 36720! Köyceğiz, Sultaniye, Kersele Deresi, dere yatağı, metamorfik arazi, 15-30 m, 21.v.1991, A. Güner 9195, M. Vural & H. Şağban HUB 36715! General distribution: Macaronesia, Mediterranean region including S. Europe and North Africa and east to Turkey, Egypt, Yemen and Oman. Note: Cosentinia vellea has two subspecies, a tetraploid C. vellea subsp. vellea and a diploid C. vellea subsp. bivalens (Reichstein) Rivas Mart. & Salvo. On macromorphological characters the two are indistinguishable, but can be separated by comparison of spore size and stomatal length (see Haou et al., 2011). Of four Turkish 128 FERN GAZ. 20(3):119-132. 2016 specimens for which spores have been measured, three correspond with subsp. vellea [64-(67)-71µm: Davis 25349,40855, 41147] and one with subsp. bivalens [55-(57)-62 µm: Davis 40788], although cytological verification is still lacking.

Paragymnopteris K.H. Shing, Indian Fern J. 10(1–2): 227 (1994) [1993]. Rhizome short-creeping, covered with long, linear scales. Fronds pinnate, linear- lanceolate scales along the midrib above and covered with lanceolate brown scales below. P. marantae (L.) K.H. Shing, Indian Fern J. 10(1–2): 229 (1994) [1993]. Synonyms: Acrostichum marantae L., Sp. Pl. 2: 1071 (1753). Ceterach marantae (L.) DC., Fl. Franc. ed. 3. 2: 567 (1805); Cheilanthes marantae (L.) R.Br., Prodr. Fl. Nov. Holland. 145 (1810); Notholaena marantae (L.) R.Br., Prod. Fl. N. Holl. 146 (1810); Cincinalis maranthae (L.) Desv., Berl. Mag. 5. 312 (1811); Gymnogramma marantae (L.) Mett., Fil. Hort. Bot. Lips. 43 (1856); Gymnopteris marantae (L.) Ching, Acta Phytotax. Sin. 10: 303 (1965); Paraceterach marantae (L.) R.M.Tryon, Amer. Fern J. 76(4): 186 (1986). Lectotype: Pierre Magnol s.n. Herb. Linn. 1245.12 (LINN)! Plant 10–30 cm. Rhizome covered with long, linear light brown scales. Fronds pinnate- pinnatisect, 10–30 × 1.2–4.5 cm, linear-lanceolate scales only along the midrib above, lower frond surface densely covered with lanceolate brown scales; stipe 4–20 cm, with scattered brown scales. Pinnae 1–2.5 × 0.5–1.2 cm, 3–7 lanceolate or broad triangular lobed, main lobe almost lyre-shaped, edge of pinnae undulate or simple; pinna stalks 1– 2 mm or sessile. Sori not discrete, sporangia spread along veins from leaf margin, ± protected by slightly recurved and modified leaf-margin. Habitat: Serpentine rocky slopes, sea level to 1400 m. Distribution in Turkey: Adana: Karsantı, Kumbükü, Hızar yolu, ca. 880 m, 05.v.1972, E Yurdakul ANK 1768! Feke: Sencan Dere between Belankey and Suphandere, metamorphic rocks, 100 m, 02.vii.1952, Davis 19586B, Dodds, Cetik! Bahçe (N. Amanus), Dumanli Dağ above Haruniye, shady metamorphic rocks, 1200 m, 19.iv.1957, Davis 26845, Hedge! Ardahan: In Felsritzen etwa 10-15 km östlich von Kosor zw. Göle und Olta, 22.iv.1957, E. Sauer 306/57! Artvin: Yusufeli, Altıparmak Köyü üzeri, P. sylvestris-P. orientalis ormanı, 1250 m, 20.v.1983, A. Güner 4829 & M. Vural (GAZI)! Erzincan: Erzincan’ın 30 km batısında serpantin boğazı, ca. 1500 m, 18.vii.1958, H. Birand ANK 10909! Keşiş dağ above Cimin, among igneous rocks, 2700 m, 28.vii.1957, Davis 31652, Hedge! Giresun: Şebinkarahisar, Arslanyurdu Deresi, ca. 1380-1460 m, 28.viii.1976, K.P. Buttler ANK 21819! Hatay: Hassa to Dörtyol, mountain road, slopes above Dörtyol, 1100 m, A.J. Byfield & D. Pearman B 2620 ISTE 73362! İçel: Mersin, Fındıkpınar, Demirışık, P. brutia altı, nemli yerler, 24.vi.1971, T. Uslu ANK 526! Kahramanmaraş: Andırın, Elma Dağı, orman açıklığı, 1550 m, 22.vi.1979, E. Tuzlacı & M. Saraçoğlu ISTE 42386! Kastamonu: Tosya, Sekiler Köyü, Seki Deresi, kayaların arası, ca. 1100 m, 28.ix.1977, M. Kılınç ANK 6866! Konya: 2 km from Çamlık village, Akseki-Beyşehir area, 1360–1370 m, 03.vi.2003, R.D. Reeves 2693 & N. Adıgüzel! BONA & GIBBY: THE FERN FAMILY PTERIDACEAE IN TURKEY 129

Muğla: Marmaris, 50 m, 13.iv.1965, Davis 41021! Marmaris, Pinetum brutiae, on serpentine, shady rocks, 30 m, 24.iii.1956. Davis 25,255, O.Polunin! Nordwestlich Köyceğiz oberhalb Ağla, Felsspalte im Kiefernforst, 950 m, 30.vi.1973, F. Holtz 428, P. Hanel, T. Kesercioğlu! Köyceğiz, Sultaniye, Kersele Deresi, dere yatağı, metamorfik arazi, 5-30 m, 21.v.1991, A. Güner 9195, M. Vural, H. Şağban HUB 36715! Sandras Dağı, Çiçekbaba Tepesinin güney yamaçları, 1700 m, 14.vii.1978, N. Özhatay & E. Özhatay ISTE40635! Samsun: Ladik Istasyon, Karadağ, 1050 m, 08.v.1965, Tobey 947! Vezirköprü at Kızılırmak, 400 m, 20.iv.1969, Tobey 2451! Zonguldak: Dirgine to Devrek, cliff edges (serpentine), 180m, 18.vii.1962. Davis 37906, Coode, Yaltirik,! General Distribution: Mediterranean area, northern Austria and Caucasus. C. & S. Europe, Macaronesia, N. Africa.

Pteris L., Sp. Pl. 2: 1073 (1753). Fronds pinnate to bipinnate, green and glabrous, midrib and secondary veins distinct. P. cretica L., Mant. Pl. 130 (1767). Synonyms: none. Lectotype: Pietro Arduino s.n. Herb. Linn. 1246.7 (LINN)! Plant 30–70 cm. Rhizomes short-creeping dark brown with dark brown scales. Fronds pinnate to bipinnate, green and glabrous, midrib and secondary veins distinct. Pinnae ≤ 7 pairs, often with a long basal segment. Fertile fronds serrate only at the end, convolute at the edge of lobes, 40–70 × 15–20 cm; stipe 20–40 cm, pinnae 10–15 cm and sessile or with c. 1 mm stalk. Vegetative fronds serrate at margin, 30–70 × 10–20 cm; stipe glabrous, 20–35 cm. Pinnae 5–20 cm, sessile or with 1–3 mm stalk. Sori submarginal, in a continuous line protected by scarious recurved leaf-margin. Habitat: Shady banks and shady damp places in forests, sea level to 800 m. Distribution in Turkey: Artvin: 10 km south of Hopa on road to Artvin, 120 m, 25, 25.ix.2005, B. Güner et al., TUDI 46! Arhavi, Dikyamaç village, 800 m, 15.vii.1994, M. Coşkun AEF 20! Çoruh, 1 km südwestlich von Hopa neben Küstenstrabe steiler, 05.viii.1973, F. Holtz (01.056), P. Hanel & T. Kesercioğlu! Çoruh, Borçka, Hopa, 300 m, 21.vi.1957, Davis & Hedge 29832! Çoruh, deciduous forest, 2 ft. 6 ins. high, 18.viii.1966, Tong 496! Çoruh, Hopa, near sea level, 22.vi.1957, Davis & Hedge 29876! Hopa, Sarp Köyü, 5 m, 11.iv.1982, Ş. Yıldırımlı 4414 HUB 36746! Hopa, Kemalpaşa-Sarp arası denize dik kayalıklar, 0-50 m, 19.v.1985, Ş. Yıldırımlı 7895 HUB 36751! Hopa, orman altı, 0-50 m, 25.01.1992, D. Osmanoğlu & Z. Aytaç 4473 (GAZI)! Murgul, Meratit Köyü, 02.viii.1959, R. Çetik & Davis ANK 20500! Giresun: Denize yakın çalılık ve ıslak yerlerde, 07.vii.1958, H. Birand, A 10692a! Gengene Köyü, Fagus ormanı, 24.vi.1977, Y. Akman ANK 6977! Ordu: Fatsa, rocks above Kayalar Dere, 60 m, 23.iv.1963, C. Tobey 46! Rize: 2 km south east of Rize, 100 m, 04.viii.1974, J.R. Akeroyd 173! Shady banks, 100– 200 m, 21.viii.1952, Davis 20785 & Dodds! Çamlıhemşin, Aşağı Vice Mahallesi üstü, karışık orman ve nemli çayırlık, 500-600 m, 13.vii.1979, A. Güner 2095 HUB 36750! Çamlıhemşin, Boğazıçik Köyü, Pogina çevresi, karışık orman, 650 m, 04.ix.1978, A. Güner 1820 HUB 36748! Alipaşa Köyü civarı, 29.vii.1956, K. Karaosmanoğlu HUB 36749! Trabzon: Valley above Çaykara, 700 m, 05.viii.1957, Davis & Hedge 32107! Sürmene, 130 FERN GAZ. 20(3):119-132. 2016

Gökçesu Mahallesi, İdrum tepe çevresi, 350-550 m, 04.vii.1997, H. Duman 6464 (GAZI)! General Distribution: S. Europe, W. Syria, Caucasus, subtropical Asia, E. and S. Africa, N. America, Hawaii. P. vittata L., Sp. Pl. 1074 (1753). Synonyms: P. ensifolia Poir. in Lam, Encycl. 5: 711 (1804); P. diversifolia Sw. Syn. Fil. 96: 288 (1806); P. costata Bory, Sp. Pl. 5: 367 (1810); P. inaequilateralis Poir. Encycl. 4: 601 (1816); Pycnodoria vittata (L.) Small, Ferns Florida 89 (1931). Lectotype: Herb. Linn. 1246.3 (LINN)! Plant 10–70 cm. Rhizomes short-creeping, brown with fine long scales. Fronds pinnate, 10–70 × 5–20 cm, green, long thin scales along with midrib; stipe 1.5–10 cm, yellowish brown with thin long scales. Pinnae (2–)6–10 × 0.3–1 cm, ≥10 pairs, longer at apex of frond, reducing in length towards frond base, narrowly lanceolate, undivided, serrate, almost sessile. Sori submarginal, in a continuous line protected by scarious recurved leaf-margin. Habitat: Shady rocks or serpentine. Distribution in Turkey: Antalya: Kemer-Ulupınar arası, 200 m, 08.vii.1979, H. Peşmen 4380 & A. Güner HUB 36744! Kemer, Götnük Deresi, Kalker vadisi, 50-200 m, 22.iii.1979, H. Peşmen 4199 & A. Güner HUB 36743! Alanya, P. brutia altı, c. 300 m, 04.viii.1982, Ş. Yıldırımlı 4452 HUB 36741! Düden Şelalesi, 23.iv.1976, N. Kızılgül AEF 5714! Hatay: Valley above Dörtyol, S. bank, N. facing, 150 m, 12.viii.1969, J. Darrah 534, 634, 620! Antakya, Harbiye, şelale civarı, 180 m, M. Coşkun 321, B. Şener & F. Ilisulu! Mersin: Silifke-Anamur arasında Değirmenönü, köy değirmeni yakını, 50 m, 09.ix.1962, H. Kayacık 2958! General Distribution: Europe and Asia.

CONCLUSION In the first volume of the Flora of Turkey (Henderson, 1965) the family currently recognised as Pteridaceae was represented by nine species from five genera and classified in five different families. Since this publication, further new records of Cheilanthes tinaei (Davis, 1988) and Cryptogramma bithynica (Jessen et al., 2012), have been published. In this revision the single family Pteridaceae in Turkey includes 11 species from seven different genera. Families Adiantaceae, Cryptogrammaceae, Gymnogrammaceae and Sinopteridaceae have been combined in the family Pteridaceae (see Christenhusz et al., 2011). The names of the genera Anogramma, Adiantum, Cryptogramma and Pteris have not changed since 1965. However, Turkish Cheilanthes species are now placed in three different genera: Allosorus, Cosentinia and Paragymnopteris. Cheilanthes marantae is a synonym of Paragymnopteris marantae and Cheilanthes catanensis is a synonym of Cosentinia vellea, whilst Cheilanthes persica, Cheilanthes acrostica and Cheilanthes tinaei are now placed within Allosorus (Christenhusz et al., 2011; Greuter & Raab- Straube, 2012). ACKNOWLEDGEMENTS We are grateful to Prof. Adil Güner for his help in accessing resources on Pteridaceae, to Prof. Gönül Kaynak who sent images of Allosorus tinaei and to Miss Alison Paul and Dr Fred Rumsey for reviewing the manuscript. This study was supported financially by the Edinburgh Botanic Garden (Sibbald) Trust to M.B. BONA & GIBBY: THE FERN FAMILY PTERIDACEAE IN TURKEY 131

REFERENCES ABOTSI, K. E., RADJI, A. R., ROUHAN, G., DUBUISSON, J. Y. & KOKOU, K. 2015. The Pteridaceae family diversity in Togo. Biodivers. Data J. 3: e5078. BENLIOĞLU, O., KAYNAK, G. & TARIMCILAR, G. 1997. Ferns of the Black Sea region of Turkey: chorological and ecological studies. Fern Gaz. 15(5): 169-192. CHRISTENHUSZ, M.M., ZHANG, X. & SCHNEIDER, H. 2011. A linear sequence of extant families and genera of lycophytes and ferns. Phytotaxa 19: 7-54. DAVIS, P.H. 1988. Flora of Turkey and The East Aegean Islands, vol. 10. Edinburgh University Press, Edinburgh. GASTONY, G. & ROLLO, D. 1995. Phylogeny and generic circumscriptions of Cheilanthoid ferns (Pteridaceae: Cheilanthoideae) inferred from rbcL nucleotide sequences. Am. Fern J. 85(4): 341-360. GREUTER, W. & RAAB-STRAUBE, E.V. 2012. Euro+ Med Notulae, 6. Willdenowia, 42: 283-285. GRUSZ, A.L. & WINDHAM, M.D. 2013. Toward a monophyletic Cheilanthes: The resurrection and recircumscription of Myriopteris (Pteridaceae). PhytoKeys 32: 49-64. GÜNER, A., ASLAN, S., EKIM, T., VURAL, M. & BABAÇ, M.T. 2012. Türkiye Bitkileri Listesi (Damarlı Bitkiler). Nezahat Gökyiğit Botanik Bahçesi ve Flora Araştırmaları Derneği Yayını. GÜNER, A. & EKIM, T. 2014. Resimli Türkiye Florası, Vol. 1. Ali Nihat Gökyiğit Vakfı, Flora Araştırmaları Derneği and Türkiye İş Bankası Kültür Yayınları, İstanbul. HAOU, S., DE BÉLAIR, G. & VIANE, R.L.L. 2011. Inventory of the ferns (filicopsida) of Numidia’s (North-Eastern Algeria). Int. J. Biodiv. Cons. 3(6): pp. 206-223. HENDERSON, M.D. 1965. Filicales. In: Davis, P.H. (Ed.), Flora of Turkey and the East Aegean Islands, Vol. 1, pp. 38-63. Edinburgh: Edinburgh University Press. JESSEN, S., LEHMANN, L. & BUJNOCH, W. 2012. Cryptogramma bithynica spec. nov. (Pteridaceae, Pteridophyta) - A new fern species from Northwestern Anatolia/Turkey. Fern Gaz. 19(2) 47-54. KAMAU, P.W. 2007. Systematic Revision of Pteris L. in Tropical Africa and Ecology of Ferns and Lycophytes in Lowland Tropical . PhD Thesis. Universität Koblenz-Landau, Germany. KAYNAK, G., TARIMCILAR, G., DAŞKIN, R. & YILMAZ, Ö. 2008. Chorological and Ecological investigations on Cheilanthoid ferns in Turkey. J. Env. Biol. 2(6) 83-88. NARDI, E., RASBACH, H. & REICHSTEIN, T. 1979. Cheilanthes tinaei Tod., an earlier name for C. corsica Reichstein et Vida and related species in Sicily. Webbia, 33: 449-456. NARDI, E. & REICHSTEIN, T. 1985. Nomenclatural notes on Cheilanthes pteriodioides (Reichard) C. Chr. (Sinopteridaceae, Pteridophyta). Webbia 39(1): 135-139. SANCHEZ-BARACALDO, P. 2004. and biogeography of the neotropical fern genera Jamesonia and Eriosorus (Pteridaceae). Am. J. Bot. 91(2): 274-284. SCHNEIDER, H., HE, L., HENNEQUIN, S. & ZHANG, X. 2013. Towards a natural classification of Pteridaceae: inferring the relationships of enigmatic pteridoid fern species occurring in the Sino-Himalaya and Afro-. Phytotaxa 77(4): 49-60. SCHUETTPELZ, E., SCHNEIDER, H., HUIET, L., WINDHAM, M. & PRYER, K. 2007. A molecular phylogeny of the fern family Pteridaceae: Assessing overall 132 FERN GAZ. 20(3):119-132. 2016

relationships and the affinities of previously unsampled genera. Mol. Phyl. Evol. 44(3): 1172-1185. SMITH, A.R., PRYER, K.M., SCHUETTPELZ, E., KORALL, P., SCHNEIDER, H. & WOLF, P.G. 2006. A classification for extant ferns. Taxon 55(3) 705-731. WINDHAM, M.D., HUIET, L., SCHUETTPELZ, E., GRUSZ, A.L., ROTHFELS, C., BECK, J., YATSKIEVYCH, G. & PRYER, K.M. 2009. Using plastid and nuclear DNA sequences to redraw generic boundaries and demystify species complexes in cheilanthoid ferns. Am. Fern J. 99: 128-132. ZHANG, G., ZHANG, X. & CHEN, Z. 2005. Phylogeny of cryptogrammoid ferns and related taxa based on rbcL sequences. Nord. J. Bot. 23: 485-493. FERN GAZ. 20(3). 2016 133


HENNIPMAN, E. 1966. Pteridoblechnum, a new genus of blechnoid ferns from Australia. Blumea Supplement, Leiden 13: 397-403. HENNIPMAN, E. & TOUW, A. 1966. Report on the Thai-Dutch Botanical Expedition 1965/1966. Nat. Hist. Bull. Siam Soc., 21: 269-281. HENNIPMAN, E. 1967. Some notes on Dutch Cladoniae (Lichenes). Persoonia 4: 427- 428. HENNIPMAN, E. & WIEFFERING, J. 1967. Ophioglossaceae. Dansk Botanisk Arkiv 25: 68-70. HENNIPMAN, E. 1968. incrassata Floerke, een nieuw licheen voor ons land. Gorteria 4: 28-29 (in Dutch). HENNIPMAN, E. 1968. The mucilage secreting hairs on the young fronds of some leptosporangiate ferns. Blumea 16: 97–103. HENNIPMAN, E. 1968. A new Todea from New Guinea with remarks on the generic limitation in recent Osmundaceae (Filices). Blumea 16: 105–108. HENNIPMAN, E. 1969. De Nederlandse Cladonia's. KNNV (in Dutch). HENNIPMAN, E. 1970. Electron & light microscopical observations on the perine of the spores of some species (Filices). Acta Bot. Neerl. 19: 671-680. HENNIPMAN, E. 1970. Some novelties in Bolbitis from Asia & the Pacific (Filices). Blumea 18: 147-149. JONCHEERE, G.J.DE & HENNIPMAN, E. 1970. Two new species of Platycerium & the identification of P. grande (Fée) Presl. Br. Fern Gaz. 10: 113–118. HENNIPMAN, E. 1974. The lectotypification of Acrostichum acuminatum Willd. & the reinstatement of the name Photinopteris speciosa (Bl.) Presl. Blumea 22: 51-52. HENNIPMAN, E. 1975. New combinations in American Bolbitis species. Am. Fern J. 65: 30. HENNIPMAN, E. 1975. A re-definition of the gymnogrammoid genus Austrogramme Fournier. Fern Gaz. 11: 61–72. HENNIPMAN, E. 1976. A second species of Pteridoblechnum (Blechnaceae) formerly referred to Leptochilus acuminatus C.T.White & Goy. Proc. Roy. Soc. Queensl. 87: 95–98. HENNIPMAN, E. 1977. A monograph of the fern genus Bolbitis (Lomariopsidaceae). Leiden Bot. Ser. 2: 1–331. HENNIPMAN, E. 1978. Bolbitis. In: HOLTTUM, R.E. Lomariopsis-group. Flora Malesiana II,1 4. The Hague Boston, London, M. Nijhoff / W. Junk. HENNIPMAN, E., SIPMAN, H. J. M. & VIJSMA, E. 1978. De Nederlandse Cladonia's Lichenes. 2nd Ed., KNNV in Dutch. SEN, U. & HENNIPMAN, E. 1981. Structure & ontogeny of stomata in Polypodiaceae. Blumea 27: 175-201. HENNIPMAN, E. & ROOS, M.C. 1982. A monograph of the fern genus Platycerium (Polypodiaceae). Amsterdam, Oxford, New York, North-Holland Publishing Company. HENNIPMAN, E. & ROOS, M.C. 1983. Phylogenetic systematics of the Polypodiaceae (Filicales). Verh. Naturwiss. Ver. Hamburg 26: 321–342.

1 An obituary for Bert Hennipman by Peter Hovenkamp was published in the British Pteridological Society Bulletin, Volume 8(1), 2014. 134 FERN GAZ. 20(3). 2016

HENNIPMAN, E. & HETTERSCHEID, W.L.A. 1984. Emendation of the fern genus Christiopteris, including the transference of two taxa to the microsorioid Polypodiaceae. Bot. Jahrb. Syst. 105: 1-10. HETTERSCHEID, W.L.A. & HENNIPMAN, E. 1985. Venation patterns, soral characteristics, and shape of the fronds of the Microsoroid Polypodiaceae. Bot.Jahrb.Syst. 105: 11-47. UFFELEN, G.A.VAN & HENNIPMAN, E. 1985. The spores of Pyrrosia Mirbel (Polypodiaceae), a SEM study. Pollen et spores 27: 155–198. HENNIPMAN, E. 1986. Notes on the ant-ferns of Lecanopteris sensu stricto in Sulawesi, with the description of two new species. Kew Bull. 41: 781–788. RAVENSBERG, W.J. & HENNIPMAN, E. 1986. The Pyrrosia species formerly referred to Drymoglossum and Saxiglossum. Leiden Bot. Ser.. Leiden, Brill. 9: 281-310. HOVENKAMP, P., GITTENBERGER, E., HENNIPMAN, E., DE JONG, R., ROOS, M.C., SLUYS, R. & ZANDEE, M. (Eds). 1987. Systematics and evolution: a matter of diversity. Utrecht, Utrecht University, 341 pp. HENNIPMAN, E. & VERDUYN, G.P. 1987. A taxonomic revision of the genus Lecanopteris Polypodiaceae in Sulawesi, Indonesia. Blumea 32: 313–319. BAAYEN, R.P. & HENNIPMAN, E. 1987. The paraphyses of the Polypodiaceae Filicales. I. General part. Beitr. Biol. Pfl. 62: 251–315. BAAYEN, R. & HENNIPMAN, E. 1988. The paraphyses of the Polypodiaceae Filicales. II. Taxonomic part. Beitr. Biol. Pfl. 62: 317–347. HENNIPMAN, E. 1990. The significance of the SEM for character analysis of spores of Polypodiaceae (Filicales). In: CLAUGHER, D. (Ed.). Scanning Electron Microscopy in Taxonomy and Functional Morphology. Syst. Ass. Spec. Oxford, Clarendon Press. 41: 23-44. HENNIPMAN, E., VELDHOEN, P., KRAMER, K.U. & PRICE M.G. 1990. Polypodiaceae. In: KRAMER, K. U. & GREEN, P.S. (Eds). The Families and Genera of Vascular plants. 1. Pteridophytes and Gymnosperms. Berlin etc., Springer: 203-214. KRAMER, K.U., CHAMBERS, T.C. & HENNIPMAN, E. 1990. Blechnaceae. In: KRAMER, K.U. & GREEN, P.S. (Eds). The Families and Genera of Vascular plants. 1. Pteridophytes and Gymnosperms. Berlin etc., Springer: 60–68. HENNIPMAN, E. 1996. Scientific consensus classification of Pteridophyta. In: CAMUS, J.M., GIBBY, M. & JOHNS, R.J. (Ed.) Pteridology in perspective. Proceedings of the Holttum memorial pteridophyte symposium. Kew, 1995. Royal Botanic Gardens, Kew. 730pp. HENNIPMAN, E. & HOVENKAMP, P.H. 1998. Lecanopteris. In: HOVENKAMP, P. H. (Ed.) Polypodiaceae. Flora Malesiana II 3: 59-72. Leiden, Rijksherbarium/Hortus Botanicus. HENNIPMAN, E. 2000. Sustainable exploitation in ornamental horticulture, an example: Hippeastrum (Amaryllidaceae). Acta Hortic. 541, 67-73. HAUFLER, C.H., GRAMMER, W.A. HENNIPMAN, E., RANKER, T.A., SMITH, A.R. & SCHNEIDER, H. 2003. Systematics of the ant-fern genus Lecanopteris (Polypodiaceae): Testing phylogenetic hypotheses with DNA sequences. Syst. Bot. 282: 217-227. FERN GAZ. 20(3). 2016 135

PhD Theses supervised

ROOS, M.C. 1985. Phylogenetic systematics of the Drynarioideae (Polypodiaceae). Utrecht, Universiteit Utrecht. HOVENKAMP, P.H. 1986. A monograph of the fern genus Pyrrosia. Leiden Botanical Series 9. Brill, Leiden. RÖDL-LINDER, G. 1990. A monograph of the fern genus . Blumea 342: 277-423. BOSMAN, M.T.M. 1991. A monograph of the fern genus Microsorum. Leiden Botanical Series 14, Rijksherbarium/Hortus Botanicus. UFFELEN, G.A. VAN 1993. Morphogenesis & evolution of the spore wall of Polypodiaceae (Filicales). Leiden, Rijksherbarium/Hortus Botanicus.



BARRY, D.H. & JERMY, A.C. 1953. Observations on Najas marina. Trans. Norf. Norw. Nat. Soc. 17: 294-297. JERMY, A.C. 1959. A preliminary census list of British pteridophytes. Br. Fern Gaz. 9(1): 4-15. JERMY, A.C. 1960. A revised preliminary census list of British Pteridophytes Br. Fern Gaz. 9(1) (Suppl.): 1-12. EVANS, G.B. & JERMY, A.C. 1962. Notes on the history of British Fern species I: confusion in the filmy ferns. Br. Fern Gaz. 9(3): 81-84. JERMY, A.C. 1963. Name changes in the British fern list. Br. Fern Gaz. 9(4): 119. CRABBE, J.A., JERMY, A.C. & LOVIS, J.D. 1964. Asplenium L. In: TUTIN, T.G. et al. (Eds) Flora Europaea 1: 14-17. Cambridge University Press. JERMY, A.C. 1964. Athyrium Roth, Cyclosorus Link, Diplazium Swartz, Gymnocarpium Newman, Isoetes L., Thelypteris Schmidel. In: TUTIN, T.G. et al. (Eds) Flora Europaea 1: 5-6, 13-14, 18, 22. Cambridge University Press. JERMY, A.C. & FUCHS, H.P. 1964. Cheilanthes Swartz. In: TUTIN, T.G. et al. (Eds) Flora Europaea 1: 10. Cambridge University Press. WALKER, S. & JERMY, A.C. 1964. Dryopteris assimilis S.Walker in Britain. Br. Fern Gaz. 9(5): 137-140. JERMY, A.C. 1964. Two species of Diplazium in Europe. Br. Fern Gaz. 9(5): 160-162. JERMY, A.C. & LOVIS, J.D. 1964. Asplenium majoricum Litardière. Br. Fern Gaz. 9(5): 163-167. PICHI-SERMOLLI, R.E.G. with BALLARD, F., HOLTTUM, R.E., ITÔ, H., JARRETT, F.M., JERMY, A.C., SCHELPE, E.A.C.L.E., TARDIEU-BLOT, M.-L. & TRYON, R.M. 1965. Index filicum: Supplementum quartum (pro annis 1934-1960), xiv, 370 pp. Firenze. CRABBE, J.A., JERMY, A.C. & MATTHEWS, G.A. 1965. Two forms of at Braunton Burrows. Proc. Bot. Soc. Br. Isl. 6: 40-42. JERMY, A.C. 1965. Isoetes in DAVIS, P.H. (Ed.) Flora of Turkey and the East Aegean Islands, Vol. 1, pp. 37-38. Edinburgh University Press, Edinburgh. JERMY, A.C. 1966. British Museum (Natural History) and University of Newcastle-upon-Tyne Expedition to New Guinea, 1964-1965. London. 52 pp. JERMY, A.C. 1967. [Fern] Abstracts . Br. Fern Gaz. 9(8): 362. JERMY, A.C. 1967. Carex section Carex (= Acutae Fr.). Proc. Bot. Soc. Br. Isl. 6(4): 375-379. JERMY, A.C., JONES, K. & COLDEN, C. 1967. Cytomorphological variation in Selaginella. J. Linn. Soc., Bot. 60: 147-158. JERMY, A.C. & SAYERS, C.D. 1967. An expedition to the Finisterre mountains of Eastern New Guinea. J. R. Hort. Soc. 92(3): 117-128. JERMY, A.C. 1968. Two new hybrids involving Dryopteris aemula. Br. Fern Gaz. 10(1): 9-12. JERMY, A.C. 1968. A new Selaginella from Zambia. Br. Fern Gaz. 10(1): 30-32. JERMY, A.C. & TUTIN, T.G. 1968. British sedges. Botanical Society of the British Isles, London. 199 pp.

1 An obituary for Clive Jermy by Alison Paul was published in the British Pteridological Society Bulletin, Volume 8(1), 2014. FERN GAZ. 20(3). 2016 137

JERMY, A.C. 1970. Selaginellaceae; Isoetaceae. In: EXELL, A.W. & LAUNERT, E. (Eds) Flora Zambesiaca: Pteridophyta. 22-29; 29-32. Crown Agents for Overseas Governments and Administrations. London. CRABBE, J.A., JERMY, A.C. & WALKER, S. 1970. The distribution of Dryopteris assimilis S.Walker in Britain. Watsonia 8: 3-15. CHAMBERS, T.C., JERMY, A.C. & CRABBE, J.A. 1971. A collection of ferns from Espiritu Santo, New Hebrides. Br. Fern Gaz. 10(4): 175-182. JERMY, A.C. & HARPER, L. 1971. Spore morphology of the Cystopteris fragilis complex. Br. Fern Gaz. 10(4): 211-213. JERMY, A.C. 1972. Distribution of British pteridophytes. Br. Pterid. Soc. Newsl. 10: 32. JERMY, A.C. & WORLAND, A.J. 1972. The code of conduct: A list of rare plants. Br. Pterid. Soc. Newsl. 10: 33-34. BROWNSEY, P.J. & JERMY, A.C. 1973. A fern collecting expedition to Crete. Br. Fern Gaz. 10(6): 331-348. CRABBE, J.A. & JERMY, A.C. 1973. Seven new species of Selaginella from tropical South America. Am. Fern J. 63(3): 135-144. JERMY, A.C., CRABBE, J.A. & THOMAS, B.A. (Eds). 1973. The phylogeny and classification of the ferns. Academic Press for The Linnean Society of London, London. xiii, 284 pp. HOLMAN, J.H. & JERMY, A.C. 1973. An international directory of Pteridologists. The British Pteridological Society. London. 31 pp. JERMY, A.C. & STOTT, P.A. (Eds). 1973. Chalk grassland - studies on its conservation and management in South-East England. Kent Trust for Nature Conservation. Maidstone. pp. 59. JERMY, A.C. & STOTT, P.A. 1973. Appendix 1:Select bibliography; Appendix 2: chalk grassland reserves and SSSI’s of S.E. England. pp. 48-50; 51-59 in JERMY, A.C. & STOTT, P.A. (Eds). Chalk grassland - studies on its conservation and management in South-East England. Kent Trust for Nature Conservation. Maidstone. pp. 59. BRITTON, D.M. & JERMY, A.C. 1974. The spores of Dryopteris filix-mas and related taxa in North America. Can. J. Bot. 52(8): 1923-1926. PETTITT, J.M. & JERMY, A.C. 1974. The surface coats on spores. Biol. J. Linn. Soc. 6(3): 245-257. BROWNSEY, P.J. & JERMY, A.C. 1975. Asplenium × khaniense, a new wild hybrid from Crete. Candollea 30: 21-28. CRABBE, J.A., JERMY, A.C. & MICKEL, J.T. 1975. A new generic sequence for the pteridophyte herbarium. Fern Gaz. 11(2&3): 141-162. JERMY, A.C. 1975. Cystopteris, Dryopteris, Woodsia, Gymnocarpium. pp. 112-118, 120. In STACE, C.A. (Ed.) Hybridization and the flora of the British Isles. Academic Press & Botanical Society of the British Isles. London. 626 pp. JERMY, A.C. 1975. The tropical rain forest: an ecosystem on resource. Proc. 13th Pacific Sci. Congr. (Abstract of paper read). JERMY, A.C. & WALKER, T.G. 1975. Lecanopteris spinosa - a new ant-fern from Indonesia. Fern Gaz. 11(2&3): 165-176. MOORE, J.A., JERMY, A.C. & MULLIN, J.M. 1975. Lamprothamnium papulosum, a new record for Ireland. Irish Nat. J. 18(8): 233-237. PETTITT, J.M. & JERMY, A.C. 1975. Pollen in hydrophilous angiosperms. Micron 5: 138 FERN GAZ. 20(3). 2016

377-405. CRABBE, J.A. & JERMY, A.C. 1976. Six new species of Selaginella from tropical South America. Fern Gaz. 11(4): 255-262. FRASER-JENKINS, C.R. & JERMY, A.C. 1976. Nomenclatural notes on Dryopteris Adans. Taxon 25(5&6): 659-676. JERMY, A.C. & WILSON, R.A.I. 1976. Guide to reserves and other sites managed by the Trust. Kent Trust for Nature Conservation, Maidstone. pp. 68. WIDÉN, C.-J., LOUNASMAA, M., JERMY, A.C., JOSEF, V.E. & REICHSTEIN, T. 1976. Die Phloroglucide von zwei Farnhybriden aus England und Schottland, von authentischen “Aspidium remotum” A.Braun und von Dryopteris aemula (Aiton) O.Kuntze aus Irland. Helv. Chim. Acta 59(5): 1725-1744. FRASER-JENKINS, C.R. & JERMY, A.C. 1977. Nomenclatural notes on Dryopteris: 2. Fern Gaz. 11(5): 338-340. GIBBY, M., JERMY, A.C., RASBACH, H, RASBACH, K., REICHSTEIN, T. & VIDA, G. 1977. The fern genus Dryopteris in the Canary Isles and Azores and the description of two new tetraploid species. Bot. J. Linn. Soc.: 74: 251-277. HOLLIS, D., JERMY, A.C. & LINCOLN, R.J. 1977. Biological collecting for the small expedition. Geogrl J. 143(2): 249-265. JERMY, A.C. & WALKER, T.G. 1977. A note on the cytology of Botrychium lanuginosum and the occurrence of the genus in Malesia. Gard. Bull. Straits Settlem. 30: 293-298. SCHELPE, E.A.C.L.E. & JERMY, A.C. 1977. Selaginellaceae; Isoetaceae. pp. 23-31, 31-32. In SCHELPE, E.A.C.L.E.. Conspectus Florae Angolensis, vol. Pteridophyta. Junta de Investigações Cientificas do Ultramar, Lisbon. pp. vi,142. BANGERTER, E.B., CANNON, J.F.M. & JERMY, A.C. 1978. Ferns and their allies (chap. 12). In JERMY, A.C. & CRABBE, J.A. (Eds). The island of Mull, a survey of its flora and environment. British Museum (Natural History), London. 631pp. JERMY, A.C. 1978. General description and topography (chap. 3); Climate (chap. 6). In JERMY, A.C. & CRABBE, J.A. (Eds). The island of Mull, a survey of its flora and environment. British Museum (Natural History), London. 631pp. JERMY, A.C. & CRABBE, J.A. (Eds) 1978. The island of Mull, a survey of its flora and environment. British Museum (Natural History), London. 631pp. JERMY, A.C., HIBBERD, D.J. & SIMS, P.A. 1978. Brackish and freshwater ecosystems (chap. 9). In JERMY, A.C. & CRABBE, J.A. (Eds). The island of Mull, a survey of its flora and environment. British Museum (Natural History), London. 631pp. JERMY, A.C., JAMES, P.W. & EDDY, A. 1978. Terrestrial ecosystems (chap. 10). In JERMY, A.C. & CRABBE, J.A. (Eds). The island of Mull, a survey of its flora and environment. British Museum (Natural History), London. 631pp. WOOLLEY, A.R. & JERMY, A.C. 1978. Geology (chap. 4). In JERMY, A.C. & CRABBE, J.A. (Eds). The island of Mull, a survey of its flora and environment. British Museum (Natural History), London. 631pp. JERMY, A.C., ARNOLD, H.R., FARRELL, L. & PERRING, F.H. (Eds). 1978. Atlas of ferns of the British Isles. Botanical Society of the British Isles & British Pteridological Society. London. 101 pp. FRASER-JENKINS, C.R. & JERMY, A.C. 1978. Nomenclatural Notes on Dryopteris Adans. [3] Taxon 27(1): 129-131. FERN GAZ. 20(3). 2016 139

FRASER-JENKINS, C.R. & JERMY, A.C. 1979. Dryopteris hybrids: a correction. Fern Gaz. 12(1): 56. HANBURY-TENISON, A.R. & JERMY, A.C. 1979. The RGS Expedition to Gunong Mulu, Sarawak 1977-78. Geogrl J. 145: 175-191. JERMY, A.C. & PAGE, C.N. 1980. Additional field characters separating the subspecies of Asplenium trichomanes in Britain. Fern Gaz. 12(2): 112-113. JERMY, A.C. 1980. Notulae et novitates Muluenses: No. 1. Bot. J. Linn. Soc. 81(1): 1-46. JERMY A.C. 1980. Biosystematic studies of Dryopteris. Acta Phytotax. Sin. 18(1):37-44. JERMY, A.C. 1980. Eriophorum. pp. 280-281. In TUTIN, T.G. et al. (Eds) Flora Europaea Vol. 5. Cambridge University Press. xxxvi, 452 pp. JERMY, A.C. 1980. Diphasiastrum (Lycopodium) issleri (Rouy) Holub in Gloucestershire. Gloucs. Nats Soc. 31(11&12): 438-439. ALSTON, A.H.G., JERMY, A.C. & RANKIN, J.M. 1981. The genus Selaginella in tropical South America. Bull. Brit. Mus. (Nat. Hist.) Bot. 9(4): 233-330. JERMY, A.C. 1981. Asplenium cuneifolium Viv. erroneously recorded in the British Isles. Watsonia 13(4): 322-323. JERMY, A.C. 1981. Youth and Exploration: Tropical rain forests. BP International Limited and Royal Geographical Society. London. 27 pp. ANDERSON, J.A.R., JERMY, A.C. & CRANBROOK, Earl of. 1982. Gunung Mulu National Park: A management and development plan. (1979 Report to the Sarawak Government) Royal Geographical Society. London. JERMY, A.C. 1982. Gunung Mulu National Park: the 1977-78 survey. Sarawak Mus. J. 30 (51 new ser.): 1-15. JERMY, A.C. & KAVANAGH, K.P. (Eds). 1982. Gunung Mulu National Park, Sarawak. An account of its environmemt and biota being the results of the RGS/Sarawak Government expedition and survey 1977-78. Sarawak Mus. J. 30 (51 new ser.) Special Issue No. 2, Pt 1: 1-279. JERMY, A.C. PARRIS, B.S. & RANKIN, J.M. 1982. A preliminary list of Pteridophytes recorded in Gunung Mulu National Park. App. IV in ANDERSON, J.A.R., JERMY, A.C. & CRANBROOK, Earl of. Gunung Mulu National Park: A management and development plan. (1979 Report to the Sarawak Government) Royal Geographical Society. London. JERMY, A.C. & SCHELPE, E.A.C.L.E. 1982. Isoetaceae. In New species and new combinations in African Pteridophyta and . Contr. Bolus Herb. 10:150-151. WALKER, T.G. & JERMY, A.C. 1982. The ecology and cytology of Phanerosorus (Matoniaceae). Fern. Gaz. 12(4): 209-213. JERMY, A.C., CHATER, A.O. & DAVID, R.W. 1982. Sedges of the British Isles. BSBI Handbook No l, 2nd edition. Botanical Society of the British Isles, London. 268 pp. JERMY, A.C. 1983. Gunung Mulu National Park, Sarawak. Oryx. 17: 6-14. JERMY, A.C. 1984. Origin and distribution of Pteridophytes in the Mediterranean area. Webbia 38(1): 397-416. JERMY, A.C. 1984. The scaly male fern – problem fern number one. Pteridologist 1(1): 10-12. JERMY, A.C. (Ed.) 1984. Studies on the flora of Gunung Mulu National Park, Sarawak. Forest Department, Sarawak. Kuching. xv, 233 pp. 140 FERN GAZ. 20(3). 2016

JERMY, A.C. 1984. Introduction. pp. v-xv. In JERMY, A.C. (Ed.). Studies on the flora of Gunung Mulu National Park, Sarawak. Forest Department, Sarawak. Kuching. xv, 233 pp. JERMY, A.C., PARRIS, B.S., CAMUS, J.M. & PAUL, A.M. 1984. The Pteridophyta of Gunung Mulu National Park. pp. 145-233. In JERMY, A.C. (Ed.). Studies on the flora of Gunung Mulu National Park, Sarawak. Forest Department., Sarawak. Kuching. xv, 233 pp. JERMY A.C. 1985. Cytotaxonomic studies of the ferns of Trinidad 1. The climate, geology, and vegetation of Trinidad with particular reference to the ecology of ferns. Bull. Br. Mus. Nat. Hist. (Bot.) 13(2): 133-147. JERMY A.C. & WALKER, T.G. 1985. Cytotaxonomic studies of the ferns of Trinidad 3. Descriptions of new species and hybrids and a new combination. Bull. Br. Mus. Nat. Hist. (Bot.) 13(2): 251-147. GIVEN, D.R. & JERMY, A.C. 1985. Conservation of pteridophytes: a postscript. Proc. Roy. Soc. Edinburgh. Section B. Biol. Sci. 86: 435-437. JERMY, A.C. 1986. Subgeneric names in Selaginella. Fern Gaz. 13(2): 117-118. JERMY, A.C. 1986. Two new Selaginella species from Gunung Mulu National Park, Sarawak. Kew Bull. 41: 547-559. DERRICK, L.N., JERMY, A.C. & PAUL, A.M. 1987. Checklist of European pteridophytes. Sommerfeltia 6: xx, 94 pp. FITZGERALD, R. & JERMY, A.C. 1987. Equisetum ramosissimum in Somerset. Pteridologist 1(4): 178-181. JERMY, A.C. & WALKER, T.G. 1987. Adiantum × spurium, a new name for A. × villosolucidum. Fern Gaz. 13(3): 188. JERMY, A.C. & WOODHAMS, J.R. 1987. Ferns at Kew Garden - A review. Pteridologist 1(4): 159-161. VIANE, R., MAYOR LÓPEZ, M. & JERMY, A.C. 1987. The systematics and ecology of the pteridophytes of northern Spain. Excursion guide. XIV International Botanical Congress. Berlin. 54pp. JERMY, A.C. 1988. Responsibilities for plant conservation: past, present and future. Plants Today. November-December 1988: 175-177. JERMY, A.C. 1989. The history of Diphasiastrum issleri (Lycopodiaceae) in Britain and a review of its taxonomic status. Fern Gaz. 13(5): 257-265. SHEFFIELD, E., WOLF, P.G., HAUFLER, C.H., RANKER, T. & JERMY, A.C. 1989. A re‐evaluation of plants referred to as Pteridium herediae (Colmeiro) Löve & Kjellqvist. Bot. J. Linn. Soc. 99(4): 377-386. CAMUS, J.M. & JERMY, A.C. 1990. Ferns of woods and moorland. Br. Wildl. 1(6): 350-353. JERMY, A.C. 1990. Conservation of Pteridophytes, p. 14, In K.U. Kramer & P.S. Green (Eds.) The families and genera of vascular plants. Vol.1. Pteridophytes and Gymnosperms. Springer-Verlag, Berlin, 1. JERMY, A.C. 1990. Isoetaceae. pp. 26-31, In K.U. Kramer & P.S. Green (Eds.) The families and genera of vascular plants. Vol.1. Pteridophytes and Gymnosperms. Springer-Verlag, Berlin, 1. JERMY, A.C. 1990. Selaginellaceae. pp. 39-45, In K.U. Kramer & P.S. Green (Eds.) The families and genera of vascular plants. Vol.1. Pteridophytes and Gymnosperms. Springer-Verlag, Berlin, 1. JERMY, A.C. & CAMUS, J.M. 1991. The illustrated field guide to ferns and allied FERN GAZ. 20(3). 2016 141

plants of the British Isles. Natural History Museum Publications, London. xiv, 194 pp. CAMUS, J.M., JERMY, A.C. & THOMAS, B.A. 1991. A world of ferns. Natural History Museum Publications, London. 112 pp. JERMY, A.C. & JARVIS, C.E. 1992. The lectotypification of Asplenium trichomanes-ramosum L., the correct name for A. viride Hudson. pp. 319-321. In Newton, M.E. Flora Europaea: notulae systematicae ad floram Europaeam spectantes. Ser. 2 No. 5. Bot. J. Linn. Soc. 109(3): 319-328. JERMY, A.C. & VIANE, R. 1992. New subgenera within Asplenium L.. pp. 321-322. In Newton, M.E. Flora Europaea: notulae systematicae ad floram Europaeam spectantes. Ser. 2 No. 5. Bot. J. Linn. Soc. 109(3): 319-328. IDE, J.M., JERMY, A.C. & PAUL, A.M. (Eds). 1992. Fern horticulture: past, present and future perspectives. Intercept, Andover. xii, 308 pp. JERMY, A.C. 1992. What’s in a name?: Nomenclature, of fern ‘varieties’. pp. 87-96. In: IDE, J.M., JERMY, A.C. & PAUL, A.M. (Eds) Fern horticulture: past, present and future perspectives. Intercept, Andover. xii, 308 pp. CRABBE, J. A. & JERMY, A.C. 1993. Cystopteris Bernh. In: TUTIN, T.G., BURGESS, N.A., CHATER, A.O. et al. Flora Europaea, vol. 1: Psilotaceae to Platanaceae, pp. 24-24. JERMY, A.C., TITTLEY, I. & GEORGE, J.D. 1992. A brief biological survey of the proposed pipeline route across Walney Island. A report for British Gas Exploration and Production Limited. Natural History Museum. London. 13pp. JERMY, A.C. 1993. British clubmosses. Br. Wildl. 4(4): 216-220. JERMY, A.C. 1993. Athyrium Roth, Deparia Hooker & Grev., Holub, Diplazium Swartz, Gymnocarpium Newman. In: TUTIN, T.G., BURGESS, N.A., CHATER, A.O. et al. Flora Europaea, vol. 1: Psilotaceae to Platanaceae, pp. 24, 26. JERMY, A.C. & AKEROYD, J. R. 1993. Isoetes L. In TUTIN, T.G., BURGESS, N.A., CHATER, A.O. et al. Flora Europaea, vol. 1: Psilotaceae to Platanaceae, pp. 6-7. JERMY, A.C. & CHAPMAN, R. 1993. Tropical forest expeditions. 4th edition. Royal Geographical Society Expedition Advisory Centre. London. 104 pp. JERMY, A.C., FUCHS, H.P. & PAUL, A.M. 1993. Cheilanthes Swartz. In TUTIN, T.G., BURGESS, N.A., CHATER, A.O. et al. Flora Europaea, vol. 1: Psilotaceae to Platanaceae, p. 12. JERMY, A.C. & PAUL, A.M. 1993. Christella Léveillé, Cosentinia Tod., Notholaena R.Br., Oreopteris J. Holub, Phegopteris (C.Presl) Fée, Stegnogramma Blume, Thelypteris Schmidel. In TUTIN, T.G., BURGESS, N.A., CHATER, A.O. et al. Flora Europaea, vol. 1: Psilotaceae to Platanaceae, pp. 12-13, 17-18. ROTHMALER, W. & JERMY, A.C. 1993. Diphasiastrum J. Holub, Lycopodiella J. Holub, Lycopodium L. In TUTIN, T.G., BURGESS, N.A., CHATER, A.O. et al. Flora Europaea, vol. 1: Psilotaceae to Platanaceae, pp. 3-5. VALENTINE, D.H., JERMY, A.C. & AKEROYD, J.R. Polypodium L. In TUTIN, T.G., BURGESS, N.A., CHATER, A.O. et al. Flora Europaea vol. 1: Psilotaceae to Platanaceae, pp. 15-16. VIANE, R., JERMY, A.C. & LOVIS, J.D. 1993. Asplenium. In TUTIN, T.G., BURGESS, N.A., CHATER, A.O. et al. Flora Europaea, vol. 1: Psilotaceae to Platanaceae, pp. 18-23. VOGEL, J.C., JESSEN, S., GIBBY, M., JERMY, A.C. & ELLIS, L. 1993. Gametophytes of Trichomanes speciosum Willd. (Hymenophyllaceae, 142 FERN GAZ. 20(3). 2016

Pteridophyta) in Central Europe. Fern Gaz. 14: 227-232. TITTLEY, I., HUXLEY, R. & JERMY, A.C. 1993. Investigation of the effects of sewage spills on the vegetation of Walthamstow Marshes. A report to Thames Water Utilities. Natural History Museum. London. 72pp. MORAN R.C., ZIMMER, B. & JERMY A.C. 1995. Adiantum. In: Moran, R.C. & Riba, R. (Eds). Flora Mesoamericana. Volumen 1. Psilotaceae a Salviniaceae. pp. 106-117. Univ. Nac. Autónoma de México. México D. F., México. WORKMAN, C., GIMINGHAM, A. & JERMY, A.C. (Eds) 1995. Environmental responsibility for expeditions – A guide to good practice. British Ecological Society and Young Explorers’ Trust. JERMY, A.C., LONG, D., SANDS, M.J.S., STORK, N.E. & WINSER, S. 1996. Biodiversity assessment review: a guide to good practice. HMSO Publications Centre. JERMY, A.C. 1996. British Equisetum - horsetails. Br. Wildl. 8(1): 37-41. JERMY, A.C. 1998. Fern recording and monitoring: a programme to take the BPS into the next century. Pteridologist 3(3): 5-12. RICH, T.C.G. & JERMY, A.C. 1998. Plant crib. Bot. Soc. Br. Isl., London. 392 pp. RUMSEY, F.J., JERMY, A.C. & SHEFFIELD, E. 1998. The independent gametophytic stage of Trichomanes speciosum Willd. (Hymenophyllaceae), the Killarney Fern and its distribution in the British Isles. Watsonia 22(1): 1-19. STEWART, N.F., FITZGERALD, R., JERMY, A.C. & LANSDOWN, R.V. 1999. The conservation of pillwort globulifera in England. Interim Report No. 1. Plantlife Report No. 130. Plantlife, London. JERMY, A.C. & CHAPMAN, R. 2002. Tropical forest expeditions. 5th edition. RGS-IBG Expedition Advisory Centre. London. 197 pp. DEAN, M., HUTCHEON, K., CAYOUETTE, J., JERMY, A.C. & ASHTON, P.A. 2005. Carex salina - a new species of sedge for Britain. BSBI News (99): 17-19. JERMY, A.C., SIMPSON, D.A., FOLEY, M.J.Y. & PORTER, M.S. 2007. Sedges of the British Isles. BSBI Handbook No. 1, 3rd edition. Bot. Soc. Br. Isl., London. 554 pp. SIMPSON, D.A. & JERMY, A.C. 2007. The status of Trichophorum cespitosum nothosubsp. foersteri (Cyperaceae). Kew Bull. 62(1): 165.

Also abstracts of presentations, obituaries and c. 50 reviews of books in a number of biological and geographical journals.




Species-rich eastern India includes the states of Sikkim, West Bengal, Arunachal Pradesh, Meghalaya, Assam, Tripura, Manipur, Nagaland and Mizoram (Ghosh et al. 2004). Rejection of records of a number of new species and new distributional records from this region by Fraser-Jenkins (2008, 2012) necessitate a revision to the treatment by Ghosh et al. As a part of ongoing revision of the ferns of Eastern India, Adiantum incisum Forssk. (Pteridaceae) and Pteris interrupta Willd. (current name: Cyclosorus interruptus (Willd.) H.Ito, Thelypteridaceae) are retypified.

Adiantum incisum Forssk., Fl. Aegypt.-Arab. 187. 1775. Lectotype (designated here): Yemen. Al Hadiyah [‘Hadîe’], 1763, P. Forsskål s.n. (C10001579, Isolectotype C10001580 images!).

The name Adiantum incisum was proposed by Linnaeus’s disciple Peter Forsskål (1775) based on his collection from Yemen. The Botanical Garden, University of Copenhagen, Denmark (C) stores two specimens: C10001579 (http://www.daim.snm.ku.dk/digitized-type-collection-details- simple?catno=C10001579) and C10001580 (http://www.daim.snm.ku.dk/digitized-type-collection-details-simple?catno=C10001580). C10001579 is a complete plant with Forsskål’s field label “Adianthum *incisum Hadîe”, thus undoubtedly original material and type of Adiantum incisum. C10001580 is single isolated frond. It does not have Forsskål’s annotation but resembles C10001579 and is most probably from same gathering. During a revision of the ferns of tropical East Africa the renowned botanist the late Dr. Bernard Verdcourt selected the specimen in C with the original field label (i.e. C10001579) as lectotype (see Verdcourt 2002: 55). Although his lectotype selection is taxonomically correct, it unfortunately did not fulfill requirements specified in the Melbourne Code (McNeill et al. 2012). Lectotypification statements published on or after 1 Jan 2001 must include the phrase “designated here” (hic designatus) or an equivalent (McNeill et al. 2012 Art. 7.10). Verdcourt’s lectotypification was published in 2002 without such statement. Here it is republished to fix the application of the name A. incisum.

Pteris interrupta Willd., Phytographia 13 t. 10 f. 1. 1794. [Sep 1794]. Lectotype (designated here): Willdenow (1794), Phytographia t. 10, f. 1. [Icon]. Epitype (designated here): India. Tamil Nadu [“Madras”], Aug 1794, Rottler ex Klein s.n. (BW19770011, Isoepitype BW19770012).

Pteris interrupta (Cyclosorus interruptus) is the type of the name Cyclosorus Link. Fosberg & Sachet (1972) considered a specimen (BW19770011; http://herbarium.bgbm.org/object/BW19770011) in Herb. Willdenow from Herbarium Berolinense, Berlin (B) as the type of Pteris interrupta and this has been considered to be a lectotype. Holttum (1973) also treated it as such. The name Pteris interrupta was published in September 1794 by Willdenow, but the 144 FERN GAZ. 20(3). 2016 supposed type sheet (BW19770011) was annotated by Willdenow as “Klein Ind. 1797. W.” This indicates that Willdenow received this specimen in 1797 from Johann Gottfried Klein, a missionary and botanist from South India. Thus this specimen is not original material, rather a later collection. The putative type (BW19770011) also carries the annotation “Pteris sp. n.? Aug. 1794. Auf den Reise nach Madras” written by Johan Peter Rottler, a missionary and botanist from South India (Dr. Frits Adema pers. comm. 2015). However the annotation “(Klein)” was written by Willdenow. So this specimen was actually collected by Rottler in August 1794 during a trip through Madras (now Tamil Nadu, India) and reached Willdenow through Klein. It is unlikely that Rottler’s collection (in August 1794) was subsequently studied and published by Willdenow within short period of one month (in September 1794). This sheet does not carry Willdenow’s name Pteris interrupta, but Willdenow’s annotation “Asp. obtusatum 1a.” The name Aspidium obtusatum Sw. was published in 1801 by Swartz. As explained by Fosberg & Sachet (1972: 8) Willdenow sent a pinna from BW19770011 to Swartz. The pinna, now in the Natural History Museum, London (BM), was identified by Swartz as Aspidium obtusatum and he informed Willdenow of this. Another sheet (BW19770012; http://herbarium.bgbm.org/object/BW19770012) exists in Herb. Willdenow in B carrying two isolated fronds and Willdenow’s annotation “Asp. obtusatum 1b.” These two specimens resemble BW19770011 and were most probably collected by Rottler from same individual or population. Thus no type specimen for Pteris interrupta is known to exist. The illustration “t. 10 f. 1.” of Pteris interrupta in the protologue (Willdenow 1794) is the only original material and I select it as lectotype and BW19770011 as epitype.

ACKNOWLEDGEMENTS I sincerely thank Dr. J. McNeill, Royal Botanic Garden Edinburgh for constructive revision and Dr. F. Adema, Naturalis Biodiversity Center, Leiden for helpful advice.

REFERENCES FORSSKÅL, P. 1775. Flora Aegyptiaco-Arabica etc. Post mortem auctoris edidit Carsten Niebuhr. ex officina Mölleri, Copenhagen. FOSBERG, F.R. & SACHET, M.H. 1972. Three Indo-Pacific Thelypteroid species reinterpreted and a new African species described. Smiths. Contr. Bot. 8: 1–10. FRASER-JENKINS, C.R. 2008. Taxonomic revision of three hundred Indian subcontinental Pteridophytes with a revised Census- List. Bishen Singh Mahendra Pal Singh, Dehra Dun. FRASER-JENKINS, C.R. 2012. Rare and Threatened Pteridophytes of Asia 2. Endangered Species of India—the Higher IUCN Categories. Bull. Natl. Mus. Nat. Sci., Ser. B, 38: 153–181. GHOSH, S.R., GHOSH, B., BISWAS, A. & GHOSH, R.K. 2004. Flora of India, Series 4: The Pteridophytic flora of eastern India. Vol. 1, Botanical Survey of India, Kolkata. HOLTTUM, R.E. 1973. The identity of three type specimens in the Willdenow Herbarium. Amer. Fern J. 63: 81–84. MCNEILL, J., BARRIE, F.R., BUCK, W.R., DEMOULIN, V., GREUTER, W., HAWKSWORTH, D.L., HERENDEEN, P.S., KNAPP, S., MARHOLD, K., PRADO, J., PRUD’HOMME VAN REINE, W.F., SMITH, G.F., WIERSEMA, J.H., FERN GAZ. 20(3). 2016 145

TURLAND, N.J. (Eds.) 2012. International Code of Nomenclature for algae, fungi, and plants (Melbourne Code): adopted by the Eighteenth International Botanical Congress Melbourne, Australia, July 2011. Regnum Vegetabile 154, 1–274. SWARTZ, O. 1801. Genera et species filicum ordine systematico redactorum. J. Bot. (Schrader) 1800: 1–136. VERDCOURT, B. 2002. Adiantaceae. In: Flora of Tropical East Africa. Beentje, H.J. (Ed.), A.A. Balkema Publishers, Rotterdam. WILLDENOW, C.L. VON 1794. Phytographia seu descriptio rariorum minus rognitarum plantarum etc. Sumptibus Wolfgangi Walther, Alemania. (http://bibdigital.rjb.csic.es/ing/Libro.php?Libro=6074).

J. MAZUMDAR Department of Biological Sciences, Burdwan Town School, Burdwan-71301, West Bengal, India. Email: [email protected] 146 FERN GAZ. 20(3). 2016


Ferns and Fern-allies of Nepal, Volume 1. Fraser-Jenkins C.R., Kandel D.R. & Pariyar S. 2015. pp 508. ISBN: 978-9937-2-9496-6 : National Herbarium and Plant Laboratories, Department of Plant Resources, Ministry of Forests and Soil Conservation, Kathmandu, Nepal.

Christopher Fraser-Jenkins has been studying Himalayan pteridophytes for over 30 years and for much of this time also living in Nepal. He is the pre-eminent expert of ferns in Himalayan region and also possesses vast expertise on the ferns of wider Asia and Europe. In this volume (the first of 2) the results of his studies of Nepalese ferns and lycophytes are summarised. The richness of the flora is highlighted by the fact that 580 species and subspecies will be included once the second volume has been completed. By comparison, England and Wales occupying an area of similar size if not topography, has about 80 taxa including naturalised introductions. The introduction to this volume includes interesting and useful summaries on phytogeography, collectors, collections and publications pertaining to Nepal. Then follows a section outlining the classification utilised (basically that of Smith et al. (2006)), and a longer section on the apparent deficiencies of recent molecular-based phylogenies. Fraser-Jenkins is well known for his scepticism of the utilisation of small sections of DNA as a basis for classification. His frequent strongly stated views and objections of studies that don’t coincide with his ideas based on traditional morphological taxonomies are presented again here. Next are short sections with notes on the history and progress of the current volume and the format of the species accounts. This is followed with a list of accepted species and subspecies included in both Volume 1 and 2. The list of taxa to be published in Volume 2 includes taxonomic and nomenclatural notes on various species as well as new combinations, new species and even a new genus, Katoella (Davalliaceae). This latter example highlights one of the inconsistencies of his morphological approach. Davalliaceae is split into a number of small genera whereas the much larger and morphologically diverse family Thelypteridaceae contains a single genus, Thelypteris. Recent molecular studies are starting to unravel the phylogeny of this family, but will they be acceptable to Fraser-Jenkins? Maybe we will find out when volume 2 is published. Intriguingly, in this list, Dryopsis is lumped back in to Ctenitis without explanation, which seems to be at odds with both morphological and molecular evidence. Hopefully this decision will also be explained in more detail in the second volume. The main bulk of the book is taken up with the taxonomic accounts of 20 families including those of the Lycophyta and the early branching fern orders through to the Pteridaceae in the (Vittariaceae remains separated and is the concluding family to Volume 1). The amount of information and knowledge collated here is astounding. For example, a page opened at random revealed the treatment of Aleuritopteris bicolor. It starts with a list of synonyms and misapplied names. Then follows a short overview of its confused history, cytology, ethnobotany and a brief comparative description. Next is a list of dozens of examined specimens listed under the different Nepalese regions and finally a note on its total distribution and status in Nepal. It should be emphasised that this is not an identification manual as such and no keys are provided. To confidently identify one of the many different Pteris, Onychium or Aleuritopteris species without prior experience would be difficult. However for smaller genera, such as the difficult Coniogramme, accurate identification should be possible FERN GAZ. 20(3). 2016 147 from the brief descriptions. Incidentally under Coniogramme and several other genera, Fraser-Jenkins, with some justification, draws attention to the many errors of taxonomy and nomenclature contained within the recent Flora of China Pteridophyta treatment. As we have come to expect, his opinion is strongly presented, finishing with a radical proposal to resolve the problems. In regard to Coniogramme I have spent some time trying to sort out the species in cultivation and in my view his ideas make far more sense than those found in the Flora of China account. As is often the case in Fraser-Jenkins’ works, many interesting comments and observations are concealed under various species accounts, including unexpected notes on species far away from Nepal. For example, both and Pteris longifolia of the New World are reduced to subspecies of and Pteris vittata respectively. Several important accounts clarifying the taxonomy and distribution of difficult genera are included. Onychium is comprehensively revised for the region, with descriptions of two new species and a new hybrid. Selaginella and Pteris are the two largest genera in Volume 1 with 23 and 27 species respectively. I noticed that the name Pteris longipes is considered to have been misapplied and is actually an earlier name for the well-known P. wallichiana. Fraser-Jenkins favours rejection of the former name and I suspect a formal proposal is in the pipeline. Other complex genera such as Huperzia, Adiantum, Aleuritopteris and Microlepia are very fully treated and the latter, in particular, includes much new and useful information. One important name change affects the widespread and common fern, Dennstaedtia scabra. The rules of priority will mean D. zeylanica now takes precedence. At the end of the book are 138 colour plates that show many of useful discriminating characters for a range of species. Finally there is an appendix explaining in detail the confused identity and nomenclature of Athyrium setiferum and the index. I noted very few printing or spelling mistakes. Only one error stood out, Sambucus edulis (page 270), presumably meaning S. nigra (Elder). This is an example that demonstrates the author’s intimate knowledge of Asian ferns; he notes that living fronds of Coniogramme procera smell strongly of Elder when crushed. I doubt that this small but useful piece of information has ever been published previously. Rather frustratingly there is no bibliography in the present volume and presumably this will be included in Volume 2. Also a separate and complete list of new taxa and combinations would be most useful. Although they are included in bold type in the index of this volume I noticed at least one had been overlooked. The book is reasonably well produced although the binding leaves much to be desired and my copy is already showing signs of distress. In summary, Chris Fraser-Jenkins and his co-authors are to be warmly congratulated on producing this landmark in Asian pteridology. It will provide a detailed baseline of data that will be used widely throughout the Himalayan region and beyond. I and many other pteridologists, both professional and amateur, will eagerly await the arrival of the second volume.

REFERENCES SMITH, A.R., PRYER, K.M., SCHUETTPELZ, E., KORALL, P., SCHNEIDER, H & WOLF, P.G. 2006. A classification for extant ferns. Taxon 55(3): 705-731.

T. PYNER 148 FERN GAZ. 20(3). 2016


PAPERS should not usually exceed 20 printed pages and are generally expected to be considerably shorter. As well as reports of original research, review articles and short notes, e.g. new records, are encouraged. The senior author should supply an email address to facilitate correspondence. REVIEWS should cover recent developments in any aspect of research on ferns and lycopods. This can include unpublished research by the author, and/or the possible direction of future research. They should be aimed at the general reader who may not be an expert in the chosen topic. Proposals for a review should be discussed first with the review editors. MANUSCRIPTS should be submitted in English (British) in electronic format in 10-point Times New Roman font and double spaced. Electronic versions of text and tables should be compatible with WORD, with figures as pdf or jpg files, and sent as email attachments or CDroms. All manuscripts will be refereed. THE TITLE should reflect the content of the paper and be in BOLD CAPITALS (11-point) and centrally aligned. Generic and specific names should be in italics and any title containing a generic or specific name should be followed by the family name in brackets e.g. TRICHOMANES SPECIOSUM (HYMENOPHYLLACEAE) IN SOUTHERN SPAIN AUTHOR ABBREVIATIONS should follow Pichi Sermolli’s (1996) Authors of scientific names in Pteridophyta, Royal Botanic Gardens, Kew. MAIN HEADINGS: should be in BOLD CAPITALS (10-point) and centrally aligned. SUBSIDIARY HEADINGS: should be in bold, the first letter of each word in capitals, the rest in lower case and left-aligned. AUTHORS’ NAMES AND FULL ADDRESSES (including email address): follow the title and are centrally aligned. KEY WORDS: up to ten. ABSTRACT: should reflect the content of the paper. FIGURES: there is no distinction between photographs and line drawings in numbering. All should be presented in a form ready for reproduction, colour, TIF format and 300 dpi (please contact editor with queries), with a scale bar where appropriate. Lettering or numbers (Arabic) should be in the bottom left using uppercase Times Roman. Figure captions should be on a separate sheet. TABLES: can be printed in either portrait or landscape format. Authors should consider this when preparing tables. Authors should ensure that tables fit the printed page size in a legible form. PAGESIZE: The Gazette has a useable page size of 123 × 204 mm. This can be used in portrait or landscape mode but any supplied artwork should not be larger than this. MEASUREMENTS: follow the metric system. CHECKLISTS: follow the format of Baksh-Comeau, Fern Gaz. 16(1, 2): 11- 122. REFERENCES: follow the style of a recent issue of The Fern Gazette, e.g.:- HOOKER, W.J. 1864. Species Filicum, 5. Dulau & Co., London. MORTON, C.V. 1947. The American species of Hymenophyllum, section Sphaeroconium. Contr. U.S. Natl. Herb. 29(3): 139-201. STEVENSON, D.W. & LOCONTE, H. 1996. Ordinal and familial relationships of pteridophyte genera. In: CAMUS, J.M., GIBBY, M. & JOHNS, R.J. (Eds) Pteridology in perspective, pp. 435-467. Royal Botanic Gardens, Kew. JOURNAL ABBREVIATIONS: should follow Botanico Periodicum Huntianum & Supplements. Alterations from the original text at proof stage will be charged for unless they are minor points of detail. A pdf will be provided free to the senior author. FERN GAZ. 20(4):149-155. 2016 149



1 Nishio Nursing College, Togasaki, Nishio City, Aichi Prefecture 445-0074, Japan. email: [email protected] 2 Aichi University of Education, Hirosawa, Kariya, Aichi Prefecture, 448-8542, Japan. email:[email protected] 3 Biology Division, Hovd State University, Hovd, Hovd Province 213500, Mongolia. 4 Department of Biology, Aichi University of Education, Kariya City, Aichi Prefecture 448-8542, Japan. email: [email protected] 5 Department of Plant Systematics and South-Siberian Botanical Garden, Altai State University, Barnaul 656099, Russia. email: [email protected] 6 The Research Institute of Evolutionary Biology, Tokyo University of Agriculture, 2-4-28, Kamiyoga, Setagaya, Tokyo 158-0098, Japan. email: [email protected] *Author for correspondence, email:[email protected]

Key words: Cystopteris fragilis, decaploid, meiosis, pentaploid, sporogenesis

ABSTRACT Germinating spores were obtained from a pentaploid Cystopteris fragilis collected in Mongolian Altai. DNA content of nuclei in gametophytes showed for the first time that gametophytes that developed from spores of the pentaploid sporophyte had a larger number of genomes than those of the mother sporophyte, and some appeared to be decaploid. Fertile decaploid (10n) spores may be produced in the pentaploid C. fragilis and derived from monad spores observed in sporogenesis. Antheridia were produced on the 10n gametophytes, although antherozooids with swimming ability were not observed. Archegonia were not observed. Apogamous sporophytes with 10n genome content were induced from decaploid gametophytes. The monad spores produced may contribute to the formation of higher polyploid sporophyte series in ferns.

INTRODUCTION Cystopteris fragilis (L.) Bernh. collected in Mongolian Altai showed three cytotypes, tetraploid, pentaploid and hexaploid, and although numerous univalent and bivalent chromosomes were observed in the spore mother cells of the pentaploid C. fragilis (2n = 5x = ca. 210), some germinating spores were obtained from the sporophyte (Kawakami et al., 2010). In the present paper, therefore, we aimed to investigate the genome contents of germinating spores produced and also to determine how these viable spores are produced in the pentaploid sporophyte. From the results of DNA contents of nuclei in gametophytes raised from spores of the pentaploid sporophyte, it was shown for the first time that the gametophytes produced had not the same but a larger number of genomes than those of the mother sporophyte, and surprisingly, some gametophytes had decaploid 150 FERN GAZ. 20(4):149-155. 2016

(10n) genome contents. The results might indicate that 10n spores are produced in the sporophyte and that they derive from monad spores observed in sporogenesis. In the present study, not only decaploid spore formation but also other viable spore formation processes are discussed. Furthermore, since polyploidy is one of the outstanding features in ferns (Manton, 1950; Lovis, 1977; Walker, 1979; Takamiya, 1996; Kato, 1997), we also investigated the gametophytes cultivated on agar to determine whether or not these spores could contribute to the formation of higher polyploid sporophyte series in C. fragilis.

MATERIALS AND METHODS Cystopteris fragilis was collected in Mongolia, west of Hovd Province, Erdeneburen sum, N 48º 38’69”, E 091º 07’74”, alt. 2450 m (Kawakami et al., 2010), and then cultivated in Japan. Spores collected were used for axenic culture. Gametophytes were cultivated on 1/4 strength of Murashige and Skoog (1962) (MS) medium supplemented with 0.75 % sucrose and 0.7 % agar. For the apogamous sporophyte formation, gametophytes were transplanted on 1/4 strength of MS medium supplemented with 3 %

Figure 1. Sporogenesis in pentaploid Cystopteris fragilis, A: sporangia on the underside of the frond; B: meiotic chromosome separation; C: two nuclei formed by chromosome separation; D: dyad spores produced in a sporangium; E: young spores produced in a sporangium, arrow indicates a monad spore; F: four spores in a tetrad; G: sterile spores produced in one sporangium; H: a large round spore with spiked ornamentation produced in a sporangium; I: spores collected from the pentaploid sporophyte, arrow indicates a fertile spore. Scale bars: A: 1 mm; B, C: 10 μm; D, E: 50 μm; F: 25 μm; G, H, I: 100 μm. KAWAKAMI ET AL: CYSTOPTERIS FRAGILIS 151 sucrose, 0.1 % casamino acid and 0.8 % agar. Cultures were maintained at 25 ℃ and illuminated by two fluorescent lamps (NEC FL 15BR) to keep 800 lux at the surface. Meiotic chromosomes were observed by fixing sporangia with 3:1 ethanol-acetic acid for 30 min at 5 ℃ and squashing them in 2 % aceto-orcein solution. The DNA contents of nuclei in fronds were estimated by flow cytometry using a Partec Ploidy Analyzer PA (Partec, Münster, Germany) (Kawakami et al., 2003).

RESULTS The pentaploid Cystopteris fragilis produced sporangia on the underside of the frond (Figure 1A). In spore mother cells, numerous univalent and bivalent chromosomes were observed at meiotic metaphaseⅠ. By meiotic chromosome separation (Figure 1B) two nuclei were formed (Figure 1C), and dyad spores were observed frequently in sporangia (Figure 1D). Some of these consisted of both large and small spores. Monad spores (Figure 1E) and spores in tetrads (Figure 1F) were more rarely observed. Mostly, young spores produced in sporangia did not mature and the sporangia did not develop (Figure 1G). In a few expanded sporangia, a few large spores with spiked ornamentation were observed (Figure 1H). Although spores obtained were mostly abortive, some were able to germinate (Figure 1I). DNA contents of nuclei in 13 gametophytes derived from spores of the pentaploid sporophyte were investigated. Their genome contents were greater than those of the donor pentaploid sporophyte (Figure 2A). Two gametophytes had approximately 8n genome content (Figure 2B), another two had approximately 9n and nine gametophytes had approximately 10n genomes (Figure 2C). Eight out of nine 10n gametophytes produced antheridia (Figure 3A), however, swimming antherozooids were not observed. Archegonia were not observed on any gametophytes during three years of culture. Apogamous sporophytes were induced from two 10n gametophytes after one year of culture (Figure 3B). They grew to approximately 10 mm in height but then died.

DISCUSSION Manton (1950) made a cytological study of the pteridophyta, and following her, similar investigations on pteridophyta were carried out by many researchers (e.g. Wagner, 1954; Lovis, 1964; Sleep, 1966, 2014*; Reichstein, 1981; Pinter, 1995; Ekrt & Koutecky, 2016). In these studies, various suggestions have been proposed about differentiation and development in ferns through observations of meiosis in hybrid species. Generally speaking, meiosis of sporophytes with many univalent chromosomes is irregular and spores produced are mostly abortive. If fertile spores could be obtained from those sporophytes, one might consider whether they are reduced spores with aneuploid chromosome numbers, as reported in triploid Osmunda regalis L. (Manton, 1950) or unreduced spores with the same chromosome number as the mother plant, produced by the pathway of Döpp-Manton or Braithwaite (Manton, 1950; Braithwaite, 1964; Walker, 1979; Kato, 1997; Kawakami et al., 2003). In the present study, however, the genome contents of gametophytes developed from spores of the pentaploid C. fragilis were greater than those of the donor pentaploid sporophyte and some gametophytes had * Sleep examined Polystichum and proposed her ideas on the development of this genus. Her studies were not limited to Polystichum but also extended to other ferns. Because of her early death, this study (Sleep, 2014) was published posthumously. 152 FERN GAZ. 20(4):149-155. 2016

10n contents that double the genome contents of the donor sporophyte. Since gametophytes develop directly from spores, their genome content must be considered to be the same as that of the spore from which an individual is derived. The results, therefore, suggest that spores with a higher ploidy level than the mother sporophyte are, surprisingly, produced in the pentaploid C. fragilis. The formation of gametophytes with genomes doubled that of the donor sporophyte is reported here for the first time. From the observation of meiosis, it might be considered that spores with 10n genome

Figure 2. Relative DNA content of gametophytes derived from spores of the pentaploid Cystopteris fragilis, A: distribution of relative DNA content of 13 gametophytes; B: sample of gametophytes with approximately 8n genome contents; C: Sample of gametophytes with approximately 10n genome contents. KAWAKAMI ET AL: CYSTOPTERIS FRAGILIS 153 contents are from monad spores produced in sporangia. They might be produced without meiotic cell division from the spore mother cells with doubled genome contents. When monad spores are produced, restitution nuclei might occur; and if some chromosomes are lost when they occur, monad spores with genomes smaller than 10n, for example, 9n or 8n, might be produced. However, in the formation of spores with 9n or 8n genome contents, especially in the case of 8n spore formation, another pathway might be considered. Since in sporogenesis dyad spores consisting of large and small spores were observed in the present study, the larger spore with 8n might be fertile, though the smaller spore with 2n might be sterile. Although dyad spores are well known to be produced in many ferns, they are unreduced spores with the same genome contents as the donor sporophyte (Walker, 1979; Kato, 1997; Kawakami et.al., 2003). Why fertile 5n spores were not obtained in this study is unknown. Further studies of fertile spores may be required to understand sporogenesis of the pentaploid sporophyte. The present study revealed the monad spore formation in the pentaploid C. fragilis. It is well known that polyploidy is one of the outstanding cytological features in ferns (Manton, 1950; Wagner, 1954; Lovis, 1968; Walker, 1979; Kato, 1997). The highest polyploid fern in Japan is 10x (Nakato, 1987; Takamiya, 1996) and the highest polyploid in the world is reported to be 16x (Walker, 1979). The evolutionary process of how polyploid ferns such as 10x or 16x are produced has not been investigated in detail. Since unreduced spores are well known to play a very important role for polyploid formation (Gastony, 1986; Kato, 1997), the pathway of the decaploid sporophyte (10x) formation could well be that they are produced by fertilization of pentaploid gametes produced from pentaploid gametophytes derived from unreduced spores produced in pentaploid plants. The present study, however, may well indicate another method of decaploid sporophyte formation: the pentaploid sporophytes produce decaploid spores and from decaploid gametophytes developed from spores, decaploid sporophytes could be induced apogamously. Furthermore, if antherozooids with an ability of fertilization could be produced from the decaploid gametophytes, though these were not observed in the present study, it might be possible to produce plants with higher polyploid levels than decaploid, such as 12x plants, by fertilization between decaploid male gametes and female diploid gametes derived from tetraploid sporophytes. Whether the fertile spores produced in pentaploid sporophytes can play a role in the formation of higher polyploid

Figure 3. An antheridium with antherozooids (A) and apogamous sporophytes (B) produced on gametophytes with approximately 10n genome content in Cystopteris fragilis. Scale bars: A: 25 μm; B: 1 mm. 154 FERN GAZ. 20(4):149-155. 2016

C. fragilis in nature or not is quite intriguing. If decaploid C. fragilis were discovered in Mongolian Altai, it might have been produced from decaploid gametophytes apogamously. To understand the evolution of polyploidy in these ferns, further studies on C. fragilis in Mongolian Altai may be necessary. .

ACKNOWLEDGEMENT The study was supported by a Grant-in-Aid for Scientific Research Program (A) 19255004 (the representative: Katsuhiko Kondo) from the Japan Society for the Promotion of Science.

REFRENCES BRAITHWAITE, A.F. 1964. A new type of apogamy in ferns. New Phytol. 63: 293-305. EKRT, L. & KOUTECKY, P. 2016. Between sexual apomictic: unexpectedly variable sporogenesis and production of viable polyploids in the pentaploid fern of the Dryopteris affinis agg. (Dryopteridaceae). Annals of Botany 117:97-106. GASTONY, G.J. 1986. Electrophoretic evidence for the origin of a fern species by unreduced spores. Am. J. Bot. 73:1563-1569. KATO, M. 1997. Diversity and evolution of land plants. Shokabo Press, Tokyo, pp. 75- 104. KAWAKAMI, S. M., KAWAKAMI, S., KONDO, K., KATO, J. & ITO, M. 2003. Sporogenesis in haploid sporophytes of Osmunda japonica (Osmundaceae). J Plant Sci. 164:527-534. KAWAKAMI, S.M., KAWAKAMI, S., KATO, J., KONDO, K., SMIRNOV, S.V. & DAMDINSUREN, O. 2010. Cytological study of a fern Cystopteris fragilis in Mongolian Altai. Chromo. Bot. 5:1-3. LOVIS, J.D. 1964. Autoplyploidy in Asplenium. Nature 203: 324-325. LOVIS, J.D. 1968. Artificial reconstruction of a Species of fern, Asplenium adulterinum. Nature 217: 1163-1165. LOVIS, J.D. 1977. Evolutionary patterns and processes in ferns. Adv Bot Res 4: 229- 415. MANTON, I. 1950. Problems of cytology and evolution in the Pteridophyta. Cambridge University Press, Cambridge. MURASHIGE, T. & SKOOG, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 473-497. NAKATO, N. 1987. Chromosome numbers of three endemic species of the fern genus Blechnum in Japan. J. Jap. Bot. 62: 261-267. PINTER, I. 1995. Progeny studies of the fern hybrid Polystichum ×bicknellii (Dryopteridaceae: Pteridophyta). Fern Gaz. 15(1): 25-40. REICHSTEIN, T. 1981. Hybrids in European Aspleniaceae (Pteridophyta). Bot. Helv. 91: 89-139. SLEEP, A. 1966. Some cytotaxonomic problems in the fern genera Asplenium and Polystichum. Unpublished Ph.D. thesis, University of Leeds. SLEEP, A. 2014. Hybridization in Polystichum (Dryopteridaceae: Pteridophyta). Fern Gaz. 19(8): 319-341. TAKAMIYA, M. 1996. Index to chromosomes of Japanese pteridophyta (1910-1966). Japan Pteridological Society. WAGNER, W.H. 1954. Reticulation evolution in the Appalachian Asplenium. Evolution 8: 103-118. KAWAKAMI ET AL: CYSTOPTERIS FRAGILIS 155

WALKER, T.G. 1979. Experimental biology of ferns. In: DYER, A.F. (ed.) The cytogenetics of ferns. Academic Press, London, pp. 87-132. WALKER, T.G. 1984. Chromosomes and evolution in Pteridophytes. In: SHARMA, A.F. & SHARMA, A. (eds) Chromosomes in evolution of eukaryotic groups. CRC, Boca Raton, pp. 103-141. 156 FERN GAZ. 20(4). 2016



The Ferns of Thailand website (http//rbg-web2.rbge.org.uk/thaiferns/Pages/thai-fern- list.htm), updated 23 June 2016, has one wrongly named set of images and one species that has been re-identified amongst the grammitid ferns (Polypodiaceae). The on-line image of Prosaptia celebica is not of that species, but belongs to Ctenopterella blechnoides: the voucher specimen is Middleton et al. 5332 (E00700990!). The red-brown rhizome scales characteristic of most species of Ctenopterella can be seen in one of the images; in detail they are concolorous and glabrous. The rhizome scales of Prosaptia celebica are subclathrate to clathrate, medium to dark red-brown and ciliate. Thailand material formerly identified as Prosaptia alata (Middleton 4400 E00700991!, Middleton E00690558!) has been re-identified as P. serriformis. The former has the rachis prominent and concolorous on the adaxial surface and sori in cylindrical pouches, while the latter has the rachis slightly prominent to slightly sunken and concolorous to darker on the adaxial surface and sori in hemispherical or conical pouches. Peninsular Malaysian material of P alata has also been re-identified as P. serriformis.

ACKNOWLEDGEMENTS I wish to thank the Curator of the herbarium, Royal Botanic Garden Edinburgh for permission to examine specimens of Grammitids in July 2016.

Barbara S. Parris Fern Research Foundation, 21 James Kemp Place, Kerikeri, Bay of Islands, New Zealand 0230 Email: [email protected] FERN GAZ. 20(4):157-162. 2016 157



1 Universidad de la Amazonia, Programa de Biología, Calle 17 diagonal 17 con carrera 3F, Barrio el Porvenir, Florencia-Caquetá, Colombia; 2 Current address: Instituto de Ciências Biológicas, Departamento de Botânica, Universidade Federal de Minas Gerais, Caixa Postal 486, 30123-970, Belo Horizonte, Minas Gerais, Brazil. Email: [email protected]; 3 Herbario Nacional de Bolivia, Instituto de Ecología, Universidad Mayor de San Andrés, Cotacota, calle 27, Campus universitario, La Paz, Bolivia. Email: [email protected].

Key words: Inconspicuous species, Caldas, Caquetá, ferns, distribution.

ABSTRACT Despite its wide distribution, Psilotum nudum has been recorded previously only from the western slopes of the Occidental Andean Cordillera of Colombia, particularly in the Chocó Region, and in the Golf of Urabá. As a result of recent exploration, we here expand the distribution of P. nudum in Colombia by reporting two new records from the Central and Oriental Andean Cordilleras.

INTRODUCTION Psilotum nudum (L.) Beauv. is a remarkable fern taxon and the simple aerial axis was widely regarded as a possible ancestral shoot design for sporophyte land plants (Figure 1) (Bierhorst, 1977; Gensel, 1977; Schulte et al., 1987). The relationships among Ophioglossales, Marattiales, Psilotales and extinct taxa (Cladoxylales, Zigopteridales, Iridopteridales, Stauropteridales) remain highly ambiguous (Gensel, 1977; Bateman et al., 1998). However, based on more recent molecular analyses, it is currently classified in the family Psilotaceae as a of Ophioglossaceae, with both included in the Psilotopsida (Smith et al., 2006). These molecular results are supported by evidence from morphology, for example, both families have cylindrical subterranean, non-green, mycorrhizal gametophytes and possess sporangia that are born on the adaxial surface of the leaf (Moran, 2004). Psilotum nudum has a wide distribution, which is principally pantropical (Tryon & Tryon, 1982; Mickel & Smith, 2004), but it can be distributed in the subtropics of Australia and New Zealand (AVH, 2016) and in the more temperate zones of Spain (Molesworth-Allen, 1966; Galan et al., 1996). It can be found from sea level up to 1840m (Idárraga-Piedrahita et al., 2014; Murillo et al., 2016; TROPICOS, 2016). In America, it has been recorded from the south of the United States of America, Mexico, Central America, Colombia, Venezuela, Guayanas, Ecuador, Peru, Bolivia, Brazil, Paraguay, Argentina and in the Antilles (Mickel & Smith, 2004; Murillo et al., 2008; Moran 2011). It can be found either in pristine forest or urban zones (Moran, 2011). The taxon may have few records in Colombia due to the fact that it does not look like a fern (Moran, 2004) and it prefers to inhabit places in the shade (Galan et al., 1996). Both decrease the probability of its discovery, especially for the non-trained botanist. In 158 FERN GAZ. 20(4):157-162. 2016

Colombia, until now, it was recorded only from four localities in the Occidental Cordillera and in the Caribbean Golf of Urabá, principally in the Chocó Region (Table 1, Figure 2). As a result of a botanic exploration in the Magdalena Medio region and in the Andean Amazonian foothills, it was discovered growing as an epiphytic fern in two new locations (Table 1, Figure 2). However, the habit of the plant can be epiphytic, terrestrial or

Figure 1. Psilotum nudum. A: Habit of the plant, scale bar (2 cm). B: Stem and leaves, scale bar (5 mm). C: Sporangia, scale bar (1 mm). D: Ramification of the rhizome, scale bar (5 mm). All from D. Sanín & J. L. Peña 6046 (COL). Photos by J. L. Peña. SANÍN & TORREZ: PSILOTUM NUDUM 159 lithophytic (Galan et al., 1996; Windisch, 1997). Interestingly, this taxon can be found erect or pendulous, which may depend on the environmental conditions of the locality (see TROPICOS, 2016). A most suitable place to find this species is in the axes of the leaves of palm trees (Windisch, 1997; Moran, 2004), and we have found the new records on Bactris sp. or Elaeis guineensis Jacq. (Figure 1A). It was suggested that reports of this taxon from Antioquia, Colombia reflected the fact that it was cultivated here (Idárraga-Piedrahita et al., 2014), but these new records provide supporting evidence that it can grow wild here as proposed by Moran (2004).

Figure 2. Distribution of Psilotum nudum in Colombia. The black dots are previous records: 1) Apartadó, 2) Medellín, 3) Chocó, 4) Valle del Cauca and 5) Cauca. The asterisks are the new records in: the Central and Oriental Andean Cordillera of Colombia from 6) Norcasia-Caldas and 7) Florencia-Caquetá. 160 FERN GAZ. 20(4):157-162. 2016 Bibliography Idárraga et al. (2014). TROPICOS (2016). Ramírez & Macias-Pinto (2007). Murillo et al. (2016). Current record Current record Collection date 2 Dic. 2011 14 Jul. 2015 Not provided 14 Jan. 1992 2003 16 Nov. 1939 Apr. 05 Collection D. Sanín, A. F. Bohórquez- A. F. D. Sanín, V. O., J. Ramírez-G., Betancur & J. P. Ramírez-V. 5539 (HUA) D. Sanín & J.L. Peña 6046 (COL) J. Santa 378 (HUA) A. Gómez et al. 500 (MO) B. R. Ramírez P. 17461 B. R. Ramírez P. (CAUP) Killip 35510 (COL) E. P. 0 5 0 269 224 0-1500 in Colombia. Altitude (m) Psilotum nudum Records of Localities Table 1. Table Antioquia: Apartadó y Antioquia: Medellín. Caldas: Caldas, Norcasia, Quiebra de Roque, Vereda Reserva Riomanso, desviación a mano izquierda de la Quebrada Tostada. Caquetá: Florencia, Jardín Botánico Uniamazonia, Anaconda. Sendero de la Chocó: Nuqui-Pangui. Playa La Olimpica. Cauca: Guapi, Orillas del río Guapi: Población de Guapí. del Cauca: Valle Buenaventura, Bahía de Buenaventura. SANÍN & TORREZ: PSILOTUM NUDUM 161

The discovery of these populations of Psilotum nudum in Caldas and Caquetá department expand its distribution to the Central and Oriental Andean Cordilleras of Colombia (Figure 2), and suggest that the plant may have a wider distribution in Colombia. However, due the difficulties of recording it, its real distribution is still not well known.

ACKNOWLEDGEMENTS DS would like to thank the University of the Amazonia for the funding support. DS is indebted to the Biology program, especially to Professor Alexander Velasquez-Valencia and the staff of the Natural History Museum of the University of the Amazonia for providing infrastructure to develop the research. The photographs were kindly provided by J.L. Peña. We thank Professor E. Trujillo Trujillo, for generously providing literature and support in the initial phases of this research. We are grateful to Professor Mary Gibby for her kind comments that improved our manuscript.

REFERENCES AVH. 2016. Australia’s Virtual Herbarium, Council of Heads of Australasian Herbaria, , accessed <11 May. 2016>. BATEMAN, R.M., CRANE, P.R., DIMICHELE, W.A., KENRICK, P.R., ROWE, N.P., SPECK T. & STEIN, W.E. 1998. Early evolution of land plants: Phylogeny, physiology and ecology of the primary terrestrial radiation. Annual Rev. Ecol. Syst. 29: 263–92. BIERHOST, D.W. 1977. The systematic position of Psilotum and Tmesipteris. Brittonia 29 (1): 3–13. GALAN DE MERA, A., VICENTE ORELLANA, J.A., GONZALEZ, J.L. & FERNANDEZ LUNA J.C. 1996. New populations of Psilotum nudum in SW Europe (Psilotaceae: Pteridophyta). Fern Gaz. 15(3): 109–112. GENSEL, P.G. 1977. Morphologic and taxonomic relationships of the Psilotaceae relative to evolutionary lines in early land vascular plants. Brittonia 29 (1): 14–29. IDÁRRAGA-PIEDRAHITA, A., ORTIZ, R.D.C., CALLEJAS POSADA, R. & MERELLO, M. 2014. Listado de las plantas vasculares del departamento de Antioquia. Pp. 127-939. In: IDÁRRAGA-PIEDRAHITA, A., ORTIZ, R.D.C., CALLEJAS POSADA, R. & MERELLO, M. (Eds.) Flora de Antioquia: Catálogo de las plantas vasculares. Vol II. Universidad de Antioquia y Jardín Botánico de Misouri & Gobernación de Antioquia. Editorial D’Vinni, Bogotá, Colombia. MICKEL, J.T. & SMITH, A.R. 2004. The pteridophytes of Mexico. Mem. New York Bot. Gard. 88: 1–1054. MOLESWORTH-ALLEN, B. 1966. Psilotum nudum in Europe. Brit. Fern Gaz. 9: 249- 251. MORAN, R.C. 2004. Natural History of Ferns. Timber Press. 301 Pp. MURILLO-P., M. T., MURILLO-A., J.C. & LEÓN-P, A. 2016-07-29. Psilotum nudum (L.) P.Beauv. En: BERNAL, R., GRADSTEIN, S.R. & CELIS, M. (Eds.). 2015. Catálogo de plantas y líquenes de Colombia. Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Bogotá. http://catalogoplantasdecolombia.unal.edu.co MURILLO-P., M.T., MURILLO-A., J.C. & LEÓN-P., A. 2008. Los Pteridofitos de Colombia. Biblioteca José Jerónimo Triana No.18. Universidad Nacional de Colombia. Arfo Editores e Impresos LTDA. 162 FERN GAZ. 20(4):157-162. 2016

RAMÍREZ PADILLA, B.R. & MACÍAS PINTO, D.J. 2007. Catálogo de helechos y plantas afines del Departamento del Cauca. Universidad del Cauca, Popayán, 215 p. SCHULTE, P.J., GIBSON, A.C. & NOBEL, P.S. 1987. anatomy and hydraulic conductance of Psilotum nudum. Amer. J. Bot. 74 (9): 1438–1445. SMITH, A. R., K. M. PRYER, E. SCHUETTPELZ, P. KORALL, H. SCHNEIDER & P. G. WOLF. 2006. A classification for extant ferns. Taxon 55 (3): 705–731. TROPICOS. 2016. Missouri Botanical Garden. Accessed: 29 Jul 2016 TRYON, R.M. & TRYON, A.F. 1982. Ferns and Allied Plants. Springer-Verlag. New York, Heidelberg, Berlin, p. 783–786. WINDISCH, P.G. 1997. Pteridófitas do Estado de Mato Grosso: Psilotaceae. Bradea 8 (10): 57–60. FERN GAZ. 20(4). 2016 163



Two new combinations in Asian-Pacific Grammitid ferns are proposed: Polypodium hornei (Baker) Parris and Radiogrammitis setulifera (Alderw.) Parris. A lectotype is chosen for Polypodium hornei Baker. Herbarium abbreviations follow Thiers (2016).

Prosaptia hornei (Baker) Parris, comb. nov., based on Polypodium hornei Baker, J. Bot. 17: 298 (1879). Type: Fiji, Viti Levu between Nadrau & Babuca, c. 1000 ft alt., Horne 369 (lectotype K! barcode 001044319, chosen here). Syn. Ctenopteris hornei (Baker) Brownlie, Pacific Sci. 14: 402 (1960), Tomophyllum hornei (Baker) Parris, Kew Bull. 65(1): 124 (2010). Baker’s description mentions two localities for the species, “Nadrau to Babuca, Viti Levu”, and “mountains of Ovalau”. The type sheet at Kew bears two plants; the left hand one (K001044319) is well pressed and bears a label beneath it reading “on trees Nadrau to Babuca”. This plant is chosen as the lectotype. The right hand plant (K000604741) has rolled up fronds with the pinnae pressed together to obscure their abaxial surfaces and it has no label indicating its provenance. Baker (1879) and Brownlie (1977: 359) both describe the rhizome scales of the species as ciliate, a character common to Prosaptia and some species of Tomophyllum. The type material of Polypodium hornei has a dorsiventral rhizome with stipes in two rows and articulated to prominent phyllopodia, and it also lacks hydathodes on the vein endings on the adaxial surface of the lamina, all of which are characters of Prosaptia. Brownlie’s illustration of part of the frond (1977: 355, pl. XLI, 3, as Ctenopteris hornei) shows the crenulate pinna margin often found in Tomophyllum, but very rarely in Prosaptia, and material collected by him in CHR has the radial stipe arrangement of Tomophyllum. Nakamura & Kokubo (2008: 256, pl. 99, 1) illustrate (as Ctenopteris hornei) a plant that is obviously a species of Tomophyllum; it has the evident hydathodes on the vein endings on the adaxial surface of the lamina characteristic of Tomophyllum, the same slightly crenulate lamina margin that Brownlie illustrated, and rhizome scales with a single apical hair that are found in numerous species of Tomophyllum. Clearly, two species have been confused under the name Ctenopteris hornei; Polypodium hornei = Prosaptia hornei, and an un-named species of Tomophyllum. The confusion is partly due to the fact that Prosaptia hornei is one of a small group of Prosaptia species that have superficial sori (like Tomophyllum), as opposed to sori sunken in steep-walled depressions on the abaxial surface of the lamina or in marginal to submarginal pouches that are typical of Prosaptia.

Radiogrammitis setulifera (Alderw.) Parris, comb. nov., based on Polypodium setuliferum Alderw., Bull. Jard. Bot. Buitenzorg, sér. 2, 16: 32 (1914). Syntypes: Malacca, Matthew 408 (BO); Sumatra, Barisan Mts, Matthew 651 [657 in K] (BO, K000501091!), Jan. 1913, Matthew s. n. (E00194034! p. p.). Syn. P. pumilum Brause. Bot. Jahrb. Syst. 49: 38 (1912) non Cockayne (1909). Xiphopteris setulifera (Alderw.) Parris, Kew Bull. 41(1): 69 (1986); Oreogrammitis setulifera (Alderw.) Parris, Gardens’ Bull. 58(2): 268 (2007). 164 FERN GAZ. 20(4). 2016

Detailed examination of the rhizomes of Matthew s. n in E and Matthew 657 in K shows that they are radial rather than dorsiventral, thus the species belongs to Radiogrammitis, rather than Oreogrammitis. The species is very similar in frond indumentum and dissection to R. subpinnatifida (Blume) Parris, but differs in having fewer and much shorter stipes arranged in a series of looser spirals or whorls on the rhizome.

ACKNOWLEDGEMENTS I wish to thank the Keeper of E for loan of material, the Keeper of K for permission to examine the holdings of Asian and Pacific Grammitids during July 2016, and Ewen Cameron (AK) for expediting the loan from E.

REFERENCES ALDERWERELT VAN ROSENBURGH, C. R. W. K. 1914. New or interesting Malayan ferns 6. Bull. Jard. Bot. Buitenzorg, sér. 2, 16: 1-60. BAKER, J. G. 1879. On a collection of ferns gathered in the Fiji Islands by Mr. John Horne, F.L.S. J. Bot. 17: 292-300. BROWNLIE, G. 1977. The Pteridophyte Flora of Fiji. Nova Hedwigia, Beih. 55. J. Cramer, Vaduz. NAKAMURA, T. & KOKUBA, K. 2008. Polypodiaceae. In KATO, M., NAKAMURA, T., MATSUMOTO, S. & EBIHARA, A. (Eds) Illustrated Flora of Ferns & Fern Allies of South Pacific Islands, pp. 297. National Museum of Nature and Science, Tsukuba. THIERS, B. (2016). Index Herbariorum. New York Botanical Gardens, on-line.

Barbara S. Parris Fern Research Foundation, 21 James Kemp Place, Kerikeri, Bay of Islands, New Zealand 0230 Email: [email protected] FERN GAZ. 20(4). 2016 165


FERN NAMES AND THEIR MEANINGS - 2nd EDITION. J.W. Dyce, revised and expanded by J. Edgington. 2016. 59 pp. ISBN O-9509806-1-7. Published by The British Pteridological Society. Price £5.

The scientific names of plants are often puzzling and their meanings frequently obscure. Many users of these names find their translation and origin fascinating. Finding information regarding the names of ferns and particularly the myriad of applications is especially difficult. J.W (Jimmy) Dyce remedied this situation with the publication of this small book in 1988. Proving to be very popular it was reprinted in 1994 but has been difficult to obtain for several years. It is therefore a great pleasure to receive this new edition, greatly expanded and enhanced by Professor John Edgington.

Compared with the first edition, Dyce’s original text is mostly retained unaltered, however there are significant additions that considerably improve the utility of the book. Starting with the introduction Dyce gives a brief explanation of history of using Latin for the naming of plants and why it is still used in the modern era. He also alludes to the particular problem of fern cultivar nomenclature, which even today does not seem to be resolved to the satisfaction of all. (The problem arises because many fern cultivar names are actually brief descriptions or polynominals, and were abandoned in other areas of botanical nomenclature centuries ago). The next section, on the etymology of British fern generic names, has been fully revised and expanded by Professor Edgington. It now includes a brief statement regarding the origin of the name as well as the meaning. (Lycophytes were ignored by Dyce in the first edition but are happily now included despite making the book’s title slightly imprecise). 166 FERN GAZ. 20(4). 2016

The next chapter is completely new, an etymology of non-British genera including all those found in Europe and many that are found in cultivation. I found this part particularly interesting. As in the previous section, both the meaning and origin of the name is given. The next two sections, both helpful and informative, listing the word elements at the beginning and end of fern names, are left unrevised.

The next chapter, again new, comprehensively covers most of the specific names of European ferns including hybrids. This is a most useful addition and many obscure names are included, the exceptions being a large number of Asplenium hybrids, many only having been found once.

In 1984 Dyce published an updated list of British ferns and their current names and this has been included here, suitably revised taking into account recent research. Under the same heading, Fern nomenclature – old and new, are two new lists. The first covers non-native species that have recorded growing in the wild in the British Isles and the second is a comprehensive list of synonyms of British ferns and lycophytes. Both these lists are of great interest and will prove to be very useful.

There then follows the largest section of the book, a glossary of fern varietal names. This formed the major part of the first edition and is included here unchanged. Many of the terms are also widely used in species names so it is not restricted to horticultural varieties. Finally there is a new, brief glossary of technical terms and an updated list of references.

As I hope will be appreciated from this review this is a much improved publication where John Edgington has managed to retain the content and spirit of Jimmy Dyce’s original but also sought to include much additional relevant and interesting information. He has achieved this most successfully and with the book priced at just £5 it must rank as one of the bargains of the year.

T. Pyner FERN GAZ. 20(4). 2016 167 168 FERN GAZ. 20(4). 2016


PAPERS should not usually exceed 20 printed pages and are generally expected to be considerably shorter. As well as reports of original research, review articles and short notes, e.g. new records, are encouraged. The senior author should supply an email address to facilitate correspondence. REVIEWS should cover recent developments in any aspect of research on ferns and lycopods. This can include unpublished research by the author, and/or the possible direction of future research. They should be aimed at the general reader who may not be an expert in the chosen topic. Proposals for a review should be discussed first with the review editors. MANUSCRIPTS should be submitted in English (British) in electronic format in 10-point Times New Roman font and double spaced. Electronic versions of text and tables should be compatible with WORD, with figures as pdf or jpg files, and sent as email attachments or CDroms. All manuscripts will be refereed. THE TITLE should reflect the content of the paper and be in BOLD CAPITALS (11-point) and centrally aligned. Generic and specific names should be in italics and any title containing a generic or specific name should be followed by the family name in brackets e.g. TRICHOMANES SPECIOSUM (HYMENOPHYLLACEAE) IN SOUTHERN SPAIN AUTHOR ABBREVIATIONS should follow Pichi Sermolli’s (1996) Authors of scientific names in Pteridophyta, Royal Botanic Gardens, Kew. MAIN HEADINGS: should be in BOLD CAPITALS (10-point) and centrally aligned. SUBSIDIARY HEADINGS: should be in bold, the first letter of each word in capitals, the rest in lower case and left-aligned. AUTHORS’ NAMES AND FULL ADDRESSES (including email address): follow the title and are centrally aligned. KEY WORDS: up to ten. ABSTRACT: should reflect the content of the paper. FIGURES: there is no distinction between photographs and line drawings in numbering. All should be presented in a form ready for reproduction, colour, TIF format and 300 dpi (please contact editor with queries), with a scale bar where appropriate. Lettering or numbers (Arabic) should be in the bottom left using uppercase Times Roman. Figure captions should be on a separate sheet. TABLES: can be printed in either portrait or landscape format. Authors should consider this when preparing tables. Authors should ensure that tables fit the printed page size in a legible form. PAGESIZE: The Gazette has a useable page size of 123 × 204 mm. This can be used in portrait or landscape mode but any supplied artwork should not be larger than this. MEASUREMENTS: follow the metric system. CHECKLISTS: follow the format of Baksh-Comeau, Fern Gaz. 16(1, 2): 11- 122. REFERENCES: follow the style of a recent issue of The Fern Gazette, e.g.:- HOOKER, W.J. 1864. Species Filicum, 5. Dulau & Co., London. MORTON, C.V. 1947. The American species of Hymenophyllum, section Sphaeroconium. Contr. U.S. Natl. Herb. 29(3): 139-201. STEVENSON, D.W. & LOCONTE, H. 1996. Ordinal and familial relationships of pteridophyte genera. In: CAMUS, J.M., GIBBY, M. & JOHNS, R.J. (Eds) Pteridology in perspective, pp. 435-467. Royal Botanic Gardens, Kew. JOURNAL ABBREVIATIONS: should follow Botanico Periodicum Huntianum & Supplements. Alterations from the original text at proof stage will be charged for unless they are minor points of detail. A pdf will be provided free to the senior author. FERN GAZ. 20(5):169-179. 2017 169



MASAHIRO KATO Department of Botany, National Museum of Nature and Science, Tsukuba 305-0005, Japan ([email protected])

Key words: adaptive strategy, flood zone, heterochrony, Osmunda, recurrent adaptations, gametophyte, streamlined leaf

ABSTRACT Rheophytes grow in the zone on river beds and banks that are regularly flooded after heavy rains. In the tropics, this zone is of sufficient height (from the lowest to the highest water level) that different rheophytes can occur in the lower and upper subzones. Rheophytic species are obligate or facultative and some woody plants, rarely climbers, are paedorheophytic. I enumerate 141 rheophytes and possible rheophytes in 18 families and subfamilies of rheophytic ferns and lycophytes assigned to a variety of families, suggesting recurrent adaptations. The rheophyte morphology is adapted to the habitat and shared among the lineages, and especially the leaves or leaflets are streamlined with cuneate bases and glabrous, entire, tough blades. In leaf development the mesophyll cells and intercellular spaces, together with the epidermal cells, are smaller in size during a shortened period of ontogeny. The gametophyte of rheophytes, which is independent of the sporophyte, has a different adaptive strategy from the sporophyte: a shortened duration of generation and subtle morphological modification.

ECOLOGY Plants include 300,000‒330,000 or more species of bryophytes, lycophytes, ferns, gymnosperms and angiosperms (Evert and Eichhon, 2013; Mauseth, 2014). They grow in a wide range of habitats other than terrestrial, including alpine, arid, water, swamp, epiphytic, lithophytic (e.g., on limestone or ultramafic rocks), and other habitats (Crawford, 2008). The habitats are characterized by abiotic factors (temperature, precipitation, light, etc.) and associated organisms. Remarkable organismal associations are seen in, for example, symbiotic plants (Paracer and Ahmadjian, 2000; Evert and Eichhorn, 2013). This paper reviews our understanding of the diversity and adaptations of ferns that grow in rivers. A special habitat for plants is provided by rivers. Rivers are often subject to flash floods after heavy rains, even as frequently as once a week in the tropics, and plants called rheophytes grow exclusively on banks and beds of rivers and streams (van Steenis, 1981, 1987; Mehtreter, 2008). Van Steenis (1981) defined rheophytes in ecological terms as follows: rheophytes are “confined to the bank and bed and growing there up to flood level, but not beyond the reach of regularly occurring flash floods” (Figure 1). Rheophytes are largely tropical, but a few extend into temperate zones, e.g., Hokkaido, in northern Japan (43°N) (Iwatsuki, 1992). Most rheophytes live in the air much longer than under relatively brief flooded conditions; this is because water levels return to the 170 FERN GAZ. 20(5):169-179. 2017 more normal levels shortly after rains stop or abate. A few other rheophytes are hydrophytic and always grow submerged, as in e.g., Hymenasplenium obliquissimum (Hayata) Sugim. & Sa. Kurata. Similar habitats are present in seasonally inundated pools, where semiaquatic ferns, such as Platyzoma microphylla R.Br. (=Pteris platyzomopsis Christenh. & H.Schneid.) (Tryon, 1964) and Marsilea species (Kornas, 1988) grow, and in inundated riparian and marshland forests, which are one of the habitats preferred by, e.g., Osmunda regalis L. (Lellinger, 1985; Page, 1997). However, the lands are covered by still or slow-running water. The height of the flood zone, from the normal low level of river water to the highest flood level reached, differs in different climates -- up to 3 metres in humid tropical Borneo (Figure 1; M. Kato, unpubl. data). There are different preferences of rheophytes for the tropical flood zone. Some rheophytes, e.g., Ctenitis vilis (Kunze) Ching, grow in the higher zone, which is subject to rarer and shorter floods, while others, e.g., Microsorum pteropus (Blume) Copel., prefer the lower zone, which is subject to more frequent and longer floods. Microsorum paucijugum (Alderw.) K.Iwats. & M.Kato prefers the further lower zone. Rheophytes are herbaceous or woody. In herbaceous rheophytes, whole plants are submerged when flooded, while in tall woody rheophytes only the basal part of the plant is submerged while flooded. In tall paedorheophytes, seedlings and low juveniles are rheophytic and the adults are emergent and thus typically not rheophytic. esmeraldensis R.C.Moran of the tree-fern family Cyatheaceae is regarded as a rheophyte (Moran, 1995), but it may not be paedorheophytic because of the short (up to 80 cm) trunk. Van Steenis (1987) considered species as paedorheophytic, because

Figure 1. Habitat segregation of rheophytes and land species on river bank in West Kalimantan, Indonesia. Rheophytic Dipteris lobbiana occupies the flood zone, and land species such as D. conjugata Reinw. and Blechnum orientale L. grow above the zone KATO: DIVERSITY AND ADAPTATIONS OF RHEOPHYTIC FERNS 171 the juvenile creeping plants are rheophytic and only later climb trees. Further study is necessary to reveal paedorheophily of ferns. Rheophytes may be obligate or facultative. Usually, obligate-rheophytic species comprise only rheophytic populations, while facultative rheophytes comprise land- dwelling populations and rheophytic populations. Examples of facultative rheophytic ferns are (C.Presl) Ching in C.Chr. and B. sinuata (C.Presl) Hennipman. Ampelopteris prolifera (Retz.) Copel. and (Retz.) Sw. may also belong to this category.

ADAPTIVE MORPHOLOGY The impact of flash floods to plants is considerable because non-rheophytic land plants cannot invade the flood zone (Figure 1). Rheophytes have gained particular morphologies to resist or reduce pressure of flooding, although their leaves may be damaged by flooding (Sharpe, 1997). The shoot-branches and leaf-petioles are flexible or tough with developed bark in, e.g., Salix or with dense networks of cell walls in Osmunda (Imaichi and Kato, 1992). The roots adhere tightly to the river bank and bed. The most remarkable feature of rheophytes is leaf morphology (van Steenis, 1981). The leaves or leaflets are streamlined (usually narrow-lanceolate) with cuneate bases (Figure 2), entire or only minutely toothed at the margins, and hairless or sparsely hairy. Cuneate leaf-bases are particularly important because the bases are under pressure from currents. Thelypteridaceae, a large family generally characterized by having hairy leaves, has the largest number of rheophytes in ferns (Appendix), but the hairs of rheophytic species in this family are usually sparse, short, or antrorse (ascending or appressed toward the distal end of the lamina or pinnae). In paedorheophytes, only the leaves of juvenile plants are streamlined. Sharpe (1997) found that in the Puerto Rican rheophyte Meniscium angustifolium Willd. the leaf growth rate is faster than in other tropical land ferns, and the leaf life-span is shorter. The faster growth rate would reduce exposure of the fragile uncoiling leaves

Figure 2. Pinnules. A. Land-living Osmunda japonica. B. Rheophytic O. lancea. 172 FERN GAZ. 20(5):169-179. 2017 to flood, and the shorter life span would allow escape from stresses of flooding. The leaf phenology of rheophytic ferns should be further investigated to clarify the various adaptations (see also section “Adaptations”).

DIVERSITY Rheophytic ferns are most abundant in tropical areas with heavy rainfall, and van Steenis (1981, 1987) enumerated 67 rheophytic ferns and lycophytes. Kato et al. (1991) found about 40 species of rheophytic ferns from Borneo. It indicates that the ever-wet Borneo is remarkably species-rich for rheophytes. At present 141 rheophytic fern taxa are known in the world, and these are assigned to 18 families and subfamilies (Appendix 1). There are other species that require further study with respect to whether they are rheophytic or not. In many fern genera, only one or a few rheophytic species are known, while there are multiple rheophytes in other genera. This trend is obvious also in flowering-plant rheophytes, believed to be about 800 (0.3% of the total number) species belonging to 68 (17%) families (van Steenis, 1981, 1987). The scattered distribution of rheophytes over many lineages suggests that derivation of rheophytes from land-growing ancestors is markedly recurrent. Some rheophytes are secondarily derived from other rheophytic species in the same genus. I suggest that Microsorum paucijugum (Alderw.) K.Iwats. & M.Kato growing in the lower flood zone is derived from another rheophytic M. pteropus. Kato (1984) considered that two rheophytes, Deparia biserialis (Baker) M.Kato and D. confluens (Kunze) M.Kato, are derived from D. petersenii (Kunze) M.Kato, although molecular evidence to support these relationships is not available (Kuo et al., 2016). Rheophytic fern species are distributed in a wide range of ferns (PPG I, 2016) from the primitive Osmundaceae through the derived Polypodiaceae, and even in the eusporangiate Ophioglossaceae and the lycophytic family Isoëtaceae. Most fern families with rheophytic members are large (> 200 spp.) and tropical (for example, Thelypteridaceae). The most species-rich genus is Bolbitis (14 spp.), which prefers rock surfaces in wet valleys. I suggest that the evolution of rheophytes happened by chance in the tropics where the rheophytic habitats are abundant. However, the basal leptosporangiate families Osmundaceae and Dipteridaceae are small and comprise fewer than 20 species.

ADAPTATIONS Narrow streamlined leaves are characteristic of rheophytes (van Steenis, 1981; Vasco et al., 2013). Kato and Imaichi (1992a) examined 32 tropical rheophytic ferns and related land species, and found a strong correlation between leaf morphology and anatomy. The mesophyll cells and intercellular spaces are smaller in the narrow leaves than in the broad leaves of related species. Correlated with this, the epidermal cells are also smaller (Kato and Imaichi 1992a). In a comparative developmental study, Imaichi and Kato (1992, 1997) showed that the mesophyll cells at the pre-expansion stage are cubical and equal-sized and then expand in both Osmunda japonica Thunb. and O. lancea Thunb. (Figure 3A, E). However, the expansion time-span is less and the mesophyll cell arms are shorter, surrounding the smaller intercellular spaces, in O. lancea (Figure 3). In this study, we interpreted the evolution of the narrow leaves as heterochronic. Kato and Imaichi (1992b) found a broad-leaved variant of the rheophytic Tectaria lobbii (Hook.) Copel.in East Kalimantan. This variant grew together with the normal form and differed only in the broad pinnae with irregular lobes (Figure 4A, C). The broad-leaved variant KATO: DIVERSITY AND ADAPTATIONS OF RHEOPHYTIC FERNS 173 may have been called T. jacobii Holttum (Holttum, 1991). It is anatomically very similar to land-growing species (Figure 4B, D), suggesting atavism. Cells of petioles of the rheophytic Osmunda lancea, like those of the lamina, are smaller and denser, the cuticular layer is thicker, and the epicuticular wax deposits are denser than in O. japonica (Imaichi and Kato, 1992, 1993). This leaf construction increases mechanical strength and repels water (Juniper and Walker, 1985). Exceptional flooding (to a height of 10 metres above the normal level of water) rarely reached terrestrial ferns growing well above the flood zone and if this happens, it may decay their leaves (M. Kato, unpublished field observations). This contrasts with rheophytes, which have high resistance to regular flooding in fast-moving, sometimes violent, currents containing dense microbes and soil/rock particles. There is a clear habitat separation between rheophytes and land plants at the uppermost reaches of flood zones (Figure 1). Land-growing species cannot invade the flood zone, due to their non-adaptive morphology. It is likely that the habitat separation is established in a certain stage of life cycle. Imaichi and Kato (1993) showed that juvenile plants of Osmunda japonica grow together with the rheophytic O. lancea near the water, and morphological and anatomical differences become prominent at an early ontogenic stage (plants having 5th or 6th youngest leaves). We suggested that O. japonica

Figure 3. Cross sections of pinnules at different stages of development (A‒C, E, F) and paradermal sections of mature pinnules (D, G). A‒D. Osmunda japonica. A, B. Young pinnules (A, 12 mm long; B, 43 mm). C. Mature pinnule (71 mm). E‒G. O. lancea. E. Young pinnule (12 mm). F. Mature pinnule (45 mm). Arrowheads indicate veins. Osmunda japonica in young stage (A) has as many unexpanded mesophyll cells between veins as has O. lancea (E) (Imaichi and Kato, 1992). The mature stage of O. lancea (F) is similar to an immature stage of O. japonica (B). Intercellular spaces are surrounded by armed mesophyll cells (D, G). 174 FERN GAZ. 20(5):169-179. 2017 was selected and excluded early from the flood zone and the two species eventually become separated in space and habitat. In contrast, O. lancea cannot extend its habitat above the flood zone. This restriction may be caused by the narrowed leaf area and reduced intercellular space in the mesophyll layer, which may reduce photosynthetic power. This anatomical and the suggested physiological character may be a trade-off. A reduced photosynthetic rate was found in the rheophyte japonicum var. luchuense (Masam.) Kitam. () (Nomura et al., 2006), although it has a different mesophyll anatomy from fern rheophytes (see below). The isolation of the flood zone from the land zone appears strong, but this is not always strong. The diploid hybrid of O. japonica and O. lancea [= O. ×intermedia (Honda) Sugimoto] occurs in the interface between both habitats and can reproduce F2 or later offspring (Yatabe-Kakugawa et al., 2013). Ferns and lycophytes are characterized by the independence of the sporophyte and gametophyte. It is likely that the two generations have different adaptive strategies. The gametophyte generation is much smaller, simpler, and shorter-lived. There is little morphological difference between gametophytes of O. japonica and O. lancea (Hiyama et al., 1992). Hiyama et al. (1992) discovered that in the rheophytic O. lancea the life span of gametophytes is shorter and they interpreted that its gametophyte generation is shortened, relative to land ferns, showing a different strategy from the sporophyte. Compared with the ferns investigated, the leaves of rheophytes show different anatomy. Narrow leaves of the facultative-rheophyte Farfugium japonicum var.

Figure 4. Rheophytic Tectaria lobbii. A, B. Normal plants. C, D. Broad-leaved plants. In C, arrowhead indicates normal plants. B, D. Cross sections of pinnules. KATO: DIVERSITY AND ADAPTATIONS OF RHEOPHYTIC FERNS 175 luchuense have fewer mesophyll cells than the broad-leaved form (Usukura et al., 1994). Similar anatomy was found in other flowering plant rheophytes (Tsukaya, 2002; Setoguchi and Kajimaru, 2004; Okada and Tsukaya, 2010, 2013; Vermeulen and Tsukaya, 2011; Ohga et al., 2012; Matsui et al., 2013). However, a rheophytic orchid ecotype shows fern-type anatomy (Yorifuji et al., 2015).

EVOLUTION The pattern in which rheophytic ferns are scattered in various genera and families indicates that similar modes of adaptations produced rheophytes in a number of independent lineages (Kato et al., 1991). However, the phylogenetic relationships of rheophytic ferns have been clarified in only a few cases (e.g., Osmunda, Tsutsumi et al., 2012; Deparia, Kuo et al., 2016). Most rheophytes are obligate-rheophytic species, while some others are distinguished at varietal rank, e.g., Deparia lancea var. rheophila M.Kato, Darnaedi & K.Iwats., Lindsaea ensifolia var. rheophila K.Iwats. Still others, e.g., Bolbitis heteroclita and B. sinuata (C.Presl) Hennipman, are facultative and grow in both habitats. Little is known about the evolutionary process of rheophytes. One question to solve is whether facultative rheophytes or rheophytic varieties are transitional toward obligate rheophytes. Another is whether speciation of rheophytes is gradual or abrupt.

ACKNOWLEDGEMENTS I thank A. R. Smith for reviewing the manuscript and useful information, and A. Salino, N. Murakami, R. C. Moran and B. Parris for providing data of rheophytes.

REFERENCES CRAWFORD, R. M. M. 2008. Plants at the margin: ecological limits and climatic change. Cambridge University Press, Cambridge. EVERT, R. G. & EICHHORN, S. E. 2013. Raven Biology of Plants, 8th ed. W. H. Freeman and Company Publisher, New York. HIYAMA, T., IMAICHI, R. & KATO, M. 1992. Comparative development of gametophytes of Osmunda lancea and O. japonica (Osmundaceae): adaptation of rheophilous fern gametophyte. Bot. Mag. Tokyo 105: 215–225. HOLTTUM, R. E. 1991. Tectaria group. Fl. Males. Ser. II, 2(1): 1–132. IMAICHI, R. & KATO, M. 1992. Comparative leaf development of Osmunda lancea and O. japonica (Osmundaceae): heterochronic origin of rheophytic stenophylly. Bot. Mag. Tokyo 105: 199–213. IMAICHI, R. & KATO, M. 1993. Comparative leaf morphology of young sporophytes of rheophytic Osmunda lancea and dryland O. japonica. J. Plant Res. 106: 37–45. IMAICHI, R. & KATO, M. 1997. Speciation and morphological evolution in rheophytes. In: IWATSUKI, K. & RAVEN, P. H. (Eds) Evolution and diversification of land plants, pp. 309–318, Springer-Verlag, Tokyo. IWATSUKI, K. 1992. Ferns and Fern Allies of Japan. Heibonsha, Tokyo. (In Japanese) JUNIPER, B. E. & JEFFREE, C. E. 1983. Plant surfaces. Edward Arnold, London. KATO, M. 1984. A taxonomic study of the athyrioid fern genus Deparia with main reference to the Pacific species. J. Fac. Sci. Univ. Tokyo, Sect. III, 13: 371–430. KATO, M., DARNAEDI, D. & IWATSUKI, K. 1991. Fern rheophytes of Borneo. J. Fac. Sci. Univ. Tokyo, Sect. III, 15: 91–110. KATO, M. & IMAICHI, R. 1992a. Leaf anatomy of tropical fern rheophytes with its 176 FERN GAZ. 20(5):169-179. 2017

evolutionary and ecological implications. Canad. J. Bot. 70: 165–174. KATO, M. & IMAICHI, R. 1992b. A broad-leaved variant of the fern rheophyte, Tectaria lobbii. Int. J. Pl. Sci. 153: 212–216. KUO, L.-Y., EBIHARA, A., SHINOHARA, W., ROUHAN, G., WOOD, K. R., WANG, C.-N. & CHIOU, W.-L. 2016. Historical biogeography of the fern genus Deparia (Athyriaceae) and its relation with polyploidy. Molec. Phylogenet. Evol. 104: 123– 134. LELLINGER, D. B. 1985. A field manual of the ferns & fern-allies of the United States & Canada. Smithsonian Institution Press, Washington, DC. MATSUI, M., TAKEI, S., OHGA, K., HAYAKAWA, H., YOSHIDA M., ITO, K., ARAKAWA, R., MASUMOTO, T. & FUKUDA, T. 2013. Morphological and anatomical variations in rheophytic ecotype of violet, Viola mandshurica var. ikedaeana (Violaceae). Amer. J. Pl. Sci. 4(4): 859–865. MAUSETH, J. D. 2014. Botany; an introduction to plant biology, 5th ed. Jones and Bartlett Learning. Burlington, Massachusetts. MEHLTRETER, K. 2008. Phenology and habitat specificity. In: RANKER, T. A. & HAUFLER, C. H. (Eds) Biology and evolution of ferns and lycophytes, pp. 201– 221. Cambridge University Press, Cambridge. MORAN, R. C. 1995. Five new species and two new combinations of ferns from Ecuador (Polypodiopsida). Nord. J. Bot. 15: 49–58. NOMURA, N., SETOGUCHI, H. & TAKASO, T. 2006. Functional consequences of stenophylly for leaf productivity: comparison of the anatomy and physiology of a rheophyte, Farfugium japonicum var. luchuense, and a related non-rheophyte, F. japonicum (Asteraceae). J. Pl. Res. 119: 645–656. OHGA, K., MUROI, M., HAYAKAWA, H., YOKOYAMA, J., ITO, K., TEBAYASHI, S., ARAKAWA, R. & FUKUDA, T. 2012. Comparative morphology and anatomy of non-rheophytic and rheophytic types of triphylla var. japonica (). Amer. J. Pl. Sci. 3: 805–809. OKADA, H. & TSUKAYA. H. 2010. A new species of Piptospatha (: Schismatoglottideae) from West Kalimantan, Indonesian Borneo. Acta Phytotax. Geobot. 61: 87–92. OKADA, H. & TSUKAYA. H. 2013. A new species of Aridaram, Schimatoglottideae, Araceae, from West Kalimantan, Indonesian Borneo. Acta Phytotax. Geobot. 63:71– 75. PAGE, C. N. 1997. The ferns of Britain and Ireland. 2nd ed. Cambridge University Press, Cambridge. PARACER, S. & AHMADJIAN, V. 2000. Symbiosis: an introduction to biological associations. 2nd ed. Oxford University Press, Oxford. PPG I. 2016. A community-derived classification for extant lycophytes and ferns. J. Syst. Evol. 54: 563–604. SETOGUCHI, H. & KAJIMARU, G. 2004. Leaf morphology of the rheophyte, Rhododendron indicum f. otakumi (Ericaceae). Acta Phytotax. Geobot. 55: 45–54. SHARPE, J. M. 1997. Leaf growth and demography of the rheophytic fern Thelypteris angustifolia (Willdenow) Proctor in a Puerto Rican . Pl. Ecol. 130: 203– 212. TSUKAYA, H. 2002. The leaf index: heteroblasty, natural variation, and the genetic control of polar processes of leaf expansion. Pl. Cell Physiol. 43:372–378. USUKURA, M., IMAICHI, R. & KATO, M. 1994. Leaf morphology of a facultative KATO: DIVERSITY AND ADAPTATIONS OF RHEOPHYTIC FERNS 177

rheophyte, Farfugium japonicum var. luchuense (Compositae). J. Pl. Res. 107: 263– 267. VAN STEENIS, C. G. G. J. 1981. Rheophytes of the world. Sijthoff & Noordhoff, Alphen aan den Rijn. VAN STEENIS, C. G. G. J. 1987. Rheophytes of the world: supplement. Allertonia 4(5): 267–330. VASCO, A., MORAN, R. C. & AMBROSE, B. A. 2013. The evolution, morphology, and development of fern leaves. In: BARTLETT, M. E. (Ed.) Frontiers in Plant Science 4: Article 4, pp. 1–16. VERMEULEN, J. & TSUKAYA, H. 2011. An assumedly rheophytic orchid: Bulbophyllum rheophyton n. sp., from Borneo. Pl. Syst. Evol. 293:71–73. WASER, N. M. & OLLERTON, J. (Eds) 2006. Plant-pollinator interactions: from specialization to generalization. University of Chicago Press, Chicago. YATABE-KAKUGAWA, Y., TSUTSUMI, C., HIRAYAMA, Y., TSUNEKI, S., MURAKAMI, N. & KATO, M. 2013. Transmission ratio distortion of molecular markers in a doubled haploid population originated from a natural hybrid between Osmunda japonica and O. lancea. J. Pl. Res. 126: 469–482. YORIFUJI, E., ISHIKAWA, N., OKADA, H. & TSUKAYA, H. 2015. Arundina graminifolia var. revoluta (Arethuseae, Orchidaceae) has fern-type rheophyte characteristics in the leaves. J. Pl. Res. 128: 239–247

APPENDIX Rheophytic ferns and suggested rheophytes. Family or subfamily names are followed by numbers of taxa.

Aspleniaceae (9) --- Asplenium delicatulum C.Presl, Asplenium dichotomum Hook. [facultative], Asplenium subaquatile Ces., Asplenium triculum A.R.Sm.; Hymenasplenium murakami-hatanakae Nakaike, Hymenasplenium obliquissimum (Hayata) Sugim. & Sa. Kurata, Hymenasplenium obtusifolium (L.) L.Regalado & Prada, Hymenasplenium riparium (Liebm.) L.Regalado & Prada [facultative], Hymenasplenium triquetrum (N. Murak. & R.C. Moran) L.Regalado & Prada Athyriaceae (7) --- Athyrium haleakalae K.R.Wood & W.L.Wagner; Deparia biserialis (Baker) M.Kato, Deparia confluens (Kunze) M.Kato, Deparia lancea (Thunb.) Fraser-Jenk. var. rheophila (M.Kato, Darnaedi & K.Iwats.) M.Kato; Diplazium aequibasale (Baker) C.Chr., Diplazium esculentum (Retz.) Sw. [facultative], Diplazium wahauense M.Kato, Darnaedi & K.Iwats. Blechnaceae (6) --- Austroblechnum difforme (Copel.) Gaspe & V.A.O.Dittrich; Cranfillia fluviatilis (R.Br.) Gaspe & V.A.O.Dittrich; Oceaniopteris francii (Rosenst.) Gaspe & Salino, Oceaniopteris gibba (Lab.) Gaspe & Salino; Parablechnum glaziovii (Christ) Gaspe & Salino, Parablechnum rheophyticum (R.C.Moran) Gaspe & Salino Cyatheaceae (1) --- Alsophila esmeraldensis R.C.Moran Dennstaedtiaceae (1) --- Microlepia rheophila K.Iwats. & M.Kato Dipteridaceae (1) --- Dipteris lobbiana (Hook.) T.Moore Dryopteridaceae subfamily Dryopteroideae (3) --- Ctenitis vilis (Kunze) Ching [facultative]; Dryopteris rheophila Mitsuta ex Darnaedi, M.Kato & K.Iwats.; Polystichum yaeyamense (Makino) Makino Dryopteridaceae subfamily Elaphoglossoideae (17) --- Bolbitis fluviatilis (Hook.) Ching in C.Chr., Bolbitis heteroclita (C.Presl) Ching in C.Chr. [facultative], 178 FERN GAZ. 20(5):169-179. 2017

(Bory) Alston, Bolbitis interlineata (Copel.) Ching in C.Chr. [facultative], Bolbitis interpreta (C.Presl) Hennipman [rheophyte?], Bolbitis presliana (Fée) Ching in C.Chr., Bolbitis quoyana (Gaud.) Ching in C.Chr., Bolbitis repanda Schott, Bolbitis rivularis (Brack.) Ching in C.Chr., Bolbitis salicina (Hook.) Ching in C.Chr., Bolbitis ×singaporensis Holttum [rheophyte?], Bolbitis sinuata (C.Presl) Hennipman [facultative], Bolbitis taylorii (F.M.Bailey) Ching in C.Chr.; Elaphoglossum dumrongii Tagawa & K.Iwats., Elaphoglossum resiniferum Holttum, Elaphoglossum rheophilum M.Kato, Darnaedi & K.Iwats.; Mickelia furcata R.C.Moran, Labiak & Sundue Hymenophyllaceae (2) --- Cephalomanes javanicum C.Presl var. asplenioides (C.Presl) K.Iwats. and var. sumatranum (Alderw.) K.Iwats. [facultative] Isoëtaceae (2) --- Isoëtes hawaiiensis W.C.Taylor & W.H.Wagner [facultative], Isoëtes martii A.Br. ex Kuhn (13) --- Lindsaea decaryana (C.Chr.) K.U.Kramer [rheophyte?], Lindsaea ensifolia Sw. var. rheophila K.Iwats., Lindsaea integra Holttum, Lindsaea lucida Blume [facultative?], Lindsaea plicata Baker; chinensis (L.) J.Sm. var. rheophila (K.U.Kramer) Parris, Odontosoria gracilis (Tagawa) Ralf Knapp, Odontosoria minutula (Sa.Kurata) Ebihara; Osmolindsaea odorata (Roxb.) Lehtonen & Christenh. [facultative], Osmolindsaea japonica (Baker) Lehtonen & Christenh.; Tapeinidium pinnatum (Cav.) C.Chr. [facultative], Tapeinidium prionoides K.U.Kramer [facultative?]; Xyropteris stortii (Alderw.) K.U.Kramer Ophioglossaceae (2) --- Ophioglossum gramineum Willd. var. majus (Alderw.) Wieferring, Ophioglossum reticulatum L. var. dilatatum (Miq.) Wieferring [facultative] Osmundaceae (4) --- Osmunda (Plenasium) angustifolia Ching, Osmunda (Plenasium) bromeliifolia (C.Presl) Copel. [facultative?], Osmunda hybrida Tsutsumi et al. [rheophyte?], Osmunda lancea Thunb. Polypodiaceae (14) --- Calymmodon asiaticus Copel.; Leptochilus macrophyllus (Blume) Noot. var. fluviatilis (Lauterb.) Noot., Leptochilus minor Fée [facultative?], Leptochilus ×trifidus Alderw.; Loxogramme antrophyoides (Baker) C.Chr. var. rheophila M.Kato, Darnaedi & K.Iwats.; Microsorum krayanense M.Kato, Darnaedi & K.Iwats.; Microsorum paucijugum (Alderw.) K.Iwats. & M.Kato, Microsorum pteropus (Blume) Copel., Microsorum submarginale M.Kato, Darnaedi & K.Iwats.; Oreogrammitis universa (Baker) Parris [facultative]; Selliguea oodes (Kunze) Hovenkamp, Selliguea yakushimensis (Makino) Fraser-Jenk.; Xiphopterella alternidens (Ces.) Parris; Xiphopterella gracilis Parris. Pteridaceae subfamily Pteridoideae (2) --- Pteris ensiformis Burm.f. var. rheophila M.Kato, Darnaedi & K.Iwats.; Taenitis interrupta Hook. & Grev. Pteridaceae subfamily (3) --- vittarioides Baker [facultative?]; Austrogramme asplenioides (Holttum) Hennipman, Austrogramme decipiens (Mett.) Hennipman [facultative] Tectariaceae (10) --- Tectaria acutifolia (Hieron.) Maxon, Tectaria ×chaconiana A.Rajas, Tectaria fauriei Tagawa [facultative?], Tectaria jacobsii Holttum, Tectaria lobbii (Hook.) Copel., Tectaria menyanthidis (C.Presl) Copel., Tectaria microlepis Holttum, Tectaria rheophytica Holttum, Tectaria subdigitata (Baker) Copel., Tectaria trifida (Fée) Price Thelypteridaceae (44) --- Amauropelta aspidioides (Willd.) Pic.Serm., Amauropelta consanguinea (Fée) Salino & T.E.Almeida [facultative], Amauropelta fluminalis (A.R.Sm.) Salino & T.E.Almeida, Amauropelta ptarmica (Kunze ex Mett.) Pic.Serm., Amauropelta rheophyta (Proctor) Salino & T.E.Almeida, Amauropelta sancta (L.) KATO: DIVERSITY AND ADAPTATIONS OF RHEOPHYTIC FERNS 179

Pic.Serm., Amauropelta saxicola (Sw.) Salino & T.E.Almeida, Amauropelta soridepressa (Salino & V.A.O.Dittrich) Salino & T.E.Almeida, Amauropelta villana (L.D.Gómez) Salino & T.E.Almeida; Ampelopteris prolifera (Retz.) Copel. [facultative]; Christella subpubescens (Blume) Holttum [facultative]; Goniopteris cumingiana (Kunze) de Jonch. & S.Sen, Goniopteris francoana (E.Fourn.) A.Löve & D.Löve, Goniopteris riograndensis (Lindm.) Ching, Goniopteris skinneri (Hook.) Salino & T.E.Almeida; Meniscium angustifolium Willd., Meniscium arborescens Humb. & Bonpl. ex Willd. [facultative]; Meniscium chrysodioides Fée [facultative], Meniscium lanceum (A.R.Sm.) R.S. Fernandes & Salino [facultative], Meniscium nesioticum (Maxon & C.V.Morton) Pic.Serm. [facultative]; Menisorus pauciflorus (Hook.) Alston [facultative?]; Mesophlebion oligodictyon (Baker) Holttum; Plesioneuron prenticei (Carr.) Holttum; Pronephrium aquatiloides (Copel.) Holttum, Pronephrium firmulum (Baker) Holttum, Pronephrium heterophyllum (C.Presl) Holttum [rheophyte?], Pronephrium hosei (Baker) Holttum, Pronephrium menisciicarpon (Blume) Holttum, Pronephrium peltatum (Alderw.) Holttum var. persetiferum Holttum [rheophyte?], Pronephrium rubidium (Hook.) Holttum [rheophyte?], Pronephrium salicifolium (Wall. ex Hook.) Holttum; Pseudocyclosorus falcilobus (Hook.) Ching, Pseudocyclosorus esquirolii (Christ) Ching [facultative]; Sphaerostephanos acrostichoides (Desv.) Holttum (=Thelypteris riparia (Copel.) C.F.Reed), Sphaerostephanos aquatilis (Copel.) Holttum, Sphaerostephanos cataractorum (W.H.Wagner & Grether) Holttum, Sphaerostephanos hispidifolius (Alderw.) Holttum, Sphaerostephanos mutabilis (Brause) Holttum, Sphaerostephanos perglanduliferus (Alderw.) Holttum, Sphaerostephanos warburgii (Kuhn & Christ) B.C.Stone; Steiropteris hottensis (C.Chr.) Salino & T.E.Almeida, Steiropteris wrightii (Mett. ex D.C.Eaton) Pic.Serm.; Trigonospora calcarata (Blume) Holttum in B.K.Nayar & S.Kaur, Trigonospora ciliata (Benth.) Holttum 180 FERN GAZ. 20(5). 2017


Masahiro Kato was born in Japan in 1946. He graduated from Kyoto University and received Dr. Sci. in 1976 with a thesis entitled “Morphological and systematic studies of Athyriaceae.” After being employed as research assistant in the Department of Botany, Kyoto University, he moved to the University of Tokyo in 1982 and became professor in 1997. Then he moved to the National Museum of Nature and Science as director of the Department of Botany and director of Tsukuba Botanical Garden. Now he is professor emeritus of the University of Tokyo and researcher emeritus of the National Museum of Nature and Science. He has undertaken a number of fieldwork projects over tropical Asia. His research interest has focused on the fern flora of tropical Asia, classification and systematics of pteridophytes, such as Athyriaceae, Davalliaceae, Ophioglossaceae and Osmundaceae. He has also investigated the evolution of epiphytic Davalliaceae, and explored rheophytes in Borneo and adjacent areas. In extended research of rheophytes, he has studied the flora, morphology and evolution of the torrenticolous rheophytes Podostemaceae (rosid angiosperms). This research extended into the genetic background of their unique morphology. Additionally, he researched the evolution of primitive angiosperms and single-leaf plants (Monophyllaea, Gesneriaceae), and the phylogenetic relationships of bryophytes and pteridophytes. FERN GAZ. 20(5):181-196. 2017 181



1 Am Quambusch 25, 58135 Hagen, Germany; [email protected] 2 Department of Biology, University of Konstanz, Universitätsstraße 10, 78457 Konstanz, Germany; [email protected]

Key Words: Equisetum sylvaticum, hybrids, macromorphology, micromorphology, SEM, Senja, Norway, distribution

ABSTRACT Equisetum ×mildeanum (E. pratense × E. sylvaticum) and E. ×lofotense (E. arvense × E. sylvaticum) have been discovered on the island of Senja (Troms, Norway). Their appearance in the field and macromorphology are extensively compared and imaged. Additionally micromorphological features of the shoot and stomata of both rare hybrid taxa and the involved parental species have been examined by SEM technology. Distribution maps illustrate the known occurrence for the European Nordic countries. A third taxon, E. ×bowmanii (E. sylvaticum × E. telmateia), is included in the comparative study to include all known E. sylvaticum hybrids in a single SEM investigation for the first time.

INTRODUCTION Wood Horsetail, Equisetum sylvaticum L., belongs to the smooth horsetails (subgenus Equisetum) within the family of Equisetaceae (in comparison to the rough textured members of subgenus Hippochaete frequently called “scouring rushes”) and is a delicate plant occurring over a wide range of wet woodland communities in the nemoral to boreo- arctic zone of and North America (Hultén, 1950; Hultén & Fries, 1986; Jalas & Suominen, 1972). Of the 11 hybrids that have been described within the subgenus (see overview in Lubienski, 2010; Page & Gureyeva, 2009; 2013) three have E. sylvaticum as one parent: E. ×mildeanum Rothm. (E. pratense Ehrh. × E. sylvaticum), E. ×bowmanii C.N. Page (E. sylvaticum × E. telmateia Ehrh.), and E. ×lofotense Lubienski (E. arvense L. × E. sylvaticum). E. ×mildeanum was originally described from the Baltic Isle of Rügen of North Eastern Germany by Rothmaler (1944) and has been known to occur there since the 1950s (Lubienski, 2013; Lubienski & Dörken, 2015). More recently the hybrid has been recorded from (Page, 1988; Acock, 2015), Norway (Lubienski, 2003, 2009), and Sweden (Lubienski & Dörken, 2015). Unfortunately it is often confused with the parents, and its real distribution in the British Isles may be overestimated (see Acock, 2015). E. ×bowmanii is recorded from a few localities within the New Forest area of Southern England (Page, 1988). Additionally a second site was reported from Scotland more recently (Acock, 2015), but this likely to be an error and confused with E. sylvaticum (H. McHaffie & P. Acock, pers. comm.). Consequently the New Forest sites remain exclusive for this unique plant. 182 FERN GAZ. 20(5):181-196. 2017

In 2009 E. ×lofotense, a similarly remarkable hybrid incorporating E. sylvaticum, was detected at a single site in the far north of Norway on the Lofoten island of Austvågøya (Lubienski, 2010). During a trip to the island of Senja (Troms, Norway) four colonies of E. sylvaticum hybrids were found. Two were identified as E. ×mildeanum and seem to fit into a series of E. ×mildeanum sites lying on the islands of North Western Norway, whereas the latter two represent the second record for E. ×lofotense. At first glance the two hybrid taxa are quite similar in habit and certain macromorphological features. Their adjacent sites allowed for study and direct comparison of their macromorphology in situ. Additional micromorphological investigation using SEM focused on the silica spikes on the ridges of the shoots and the structure of the stomata. As part of the morphological comparison, the hybrids were compared with E. sylvaticum and other taxa to confirm the identity of the second parents.

MATERIAL AND METHODS Material Plant material was collected on the the island of Senja (Troms province) that is located northeast of the Lofoten and Vesterålen islands. It is situated in the transition between the Northern and Middle boreal vegetation zone (Sjörs et al., 2004). The islands and coastal mainland of Northwestern Norway display an oceanic climate with high precipitation values (Sjörs et al., 2004).There are two colonies of each hybrid alongside a single fjord (Mefjorden) in the northern part of the island (Figure 1).

Figure 1. Mefjorden in the northern part of the island of Senja (Troms, Norway) showing the four sites of the Equisetum sylvaticum hybrids (E. ×mildeanum 1 & 2, E. ×lofotense 3 & 4) (based on Google Earth ©, Google Inc., 26.12.2016).

Equisetum ×mildeanum (E. pratense × E. sylvaticum)

1. Mefjorden near Svarthola, Berg kommune, Senja, Troms fylke N 69°27’53.3’’ / E 17°34’29.2’’, c. 9 m a.s.l.

Growing over a length of approximately 90 metres along the wet western roadside ditch beneath the northeastern slope of Litlehesten mountain, south of Bratthesten tunnel (Figure 2a, b). LUBIENSKI & DÖRKEN: TWO HYBRIDS OF EQUISETUM SYLVATICUM 183

Figure 2. a, b. Equisetum ×mildeanum at its site near Svarthola (25.07.2016, M. Lubienski).

2. Mefjorden east of Mefjordvær, Berg kommune, Senja, Troms fylke N 69°30’54.4’’ / E 17°28’08.2’’, c. 16 m a.s.l.

Occupying both ditches and shoulders of the road east of the town of Mefjordvær and west of Mykjeneset beneath the northern slope of Kyle mountain. The colony stretches over a length of approximately 100 metres (Figure 3a, b).

Figure 3. a, b. Equisetum ×mildeanum at its site near Mefjordvær (30.07.2016, M. Lubienski).

Equisetum ×lofotense (E. arvense × E. sylvaticum)

3. Mefjorden near Svarthola, Berg kommune, Senja, Troms fylke N 69°27’42.2’’ / E 17°34’51.1’’, c. 5-13 m a.s.l.

Growing along a small creek that rises between the northeastern slopes of Store Hesten and Litlehesten mountain, crosses the road south of Bratthesten tunnel and flows into Mefjorden just a few metres beneath. The hybrid occupies the wet slope and the roadside ditch west of the road over an area of approximately 40 × 40 metres. The eastern side of the road descends to the sea and is colonized by E. ×lofotense over an area of approximately 40 × 5 metres (Figure 4a, b). 184 FERN GAZ. 20(5):181-196. 2017

Figure 4. a, b. Equisetum ×lofotense at its site near Svarthola (25.07.2016, M. Lubienski).

4. Mefjorden north of Kvalvika, Berg kommune, Senja, Troms fylke N 69°28’54.8’’ / E 17°33’15.9’’, c. 5-13 m a.s.l.

The site is situated by the same road two kilometres to the north, north of Bratthesten tunnel. The hybrid grows over an area 10 × 30 metres on the wet slope between Burstind and Bringtinden mountain and in the western roadside ditch; it is also found on the other (eastern) side of the road, where it accompanies a small rivulet to the sea (Figure 5 a, b).

Figure 5. a, b. Equisetum ×lofotense at its site near Kvalvika (25.07.2016, M. Lubienski).

Methods Freshly collected shoots were fixed in FAA (100 ml FAA = 90 ml 70% ethanol + 5 ml acetic acid 100% + 5 ml formaldehyde solution 37%) before being stored in 70% ethanol. For SEM with AURIGA ZEISS TM the fixed material was dehydrated in FDA (formaldehyde-dimethyl-acetal) and critical-point dried (CPD 030, BALZERS). CP- dried material was sputtered with gold-palladium (thickness 5 nm) with a BALTEC SputterCoater SCD 030.

RESULTS Comparison of morphology and field observations of Equisetum ×mildeanum and E. ×lofotense on Senja In the field both hybrids appear similar to E. sylvaticum at first glance and look like odd LUBIENSKI & DÖRKEN: TWO HYBRIDS OF EQUISETUM SYLVATICUM 185

Figure 6. SEM images of the micromorphology of the shoot ridges and stomata of Equisetum arvense (a-b), E. sylvaticum (c-d), E. pratense (e-f), and E. telmateia (g-h). Arrows and letters indicate ridges (r), furrows (f), mamillae (m) and pilulae (p). 186 FERN GAZ. 20(5):181-196. 2017 E. pratense (10–) 30 – 40 (-50) cm semi-dimorphic branches simple (sometimes with irregular secondary branches) often slightly bilaterally compressed not spreading away from the internode above slightly shorter than to as long as the adjacent nodal sheath pale green with short brown scarious appendices commissure obscure ridge-groove less distinct green-chlorophyllous, spherical, -bearing mildeanum × E. (10–) 30 – 40 (–50) cm branches with irregular secondary branches (sometimes simple) often distinctly bilaterally compressed not spreading away from the internode above non-chlorophyllous, irregularly shaped, without semi-dimorphic as long to twice long as the adjacent nodal sheath (never shorter) pale green with distinct light-brown scarious appendices commissure obscure ridge-groove less distinct and their parent species. E. sylvaticum  mildeanum (10–) 30 – 50 (–80) cm semi-dimorphic branches with regular secondary branches bilaterally compressed only at exposed sites spreading away from the internode above twice as long the adjacent nodal sheath (very rarely as long as) commissure obscure ridge-groove less distinct light-to reddish-brown and scarious throughout green-chlorophyllous, spherical, elater-bearing E.  lofotense , lofotense × E. E. branches with irregular secondary branches not or only slightly spreading away from the internode above non-chlorophyllous, irregularly shaped, without elaters (10–) 30 – 50 (–90) cm semi-dimorphic often distinctly bilaterally compressed twice as long the adjacent nodal sheath (rarely as long as) green with very short light- to reddish-brown scarious appendices commissure distinct, of a pale green to whitish colour ridge-groove often distinct arvense E. (10–) 30 – 50 (–80) cm branches simple (sometimes with irregular secondary branches) not bilaterally compressed, variable spreading away from the internode above (sometimes not spreading) twice (or more) as long the adjacent nodal sheath (rarely as long as) green-chlorophyllous, spherical, elater-bearing commissure distinct, of a pale green to whitish colour ridge-groove distinct Dimorphic green throughout branch st Macro- and micromorphological characters of Table 1. Table Sterile shoot – nodal sheath Fertile shoot – phenology Sterile shoot – shape Sterile shoot – branches (length ratio of 1 internode to nodal sheath) Size Sterile shoot – branching pattern Sterile shoot – branch teeth Sterile shoot – branches (ochreole) Spores LUBIENSKI & DÖRKEN: TWO HYBRIDS OF EQUISETUM SYLVATICUM 187 shorter than the sheath short, with a small dark centre and a broad, white, scarious margin not adhering together densely covered with short globose pilulae density of pilulae thinning towards the margins surface and pilulae not covered with wax crystals ring of fused pilulae absent pilulae at the stomatal pore stalked with head or globose ridges sharpened, hardly broadened silica mamillae 1-3, aligned, forming a single row furrows very small and acute shorter than or as long the sheath oblong, with a brown to reddish-brown centre and a distinct light-brown scarious margin adhering together only at the lowermost sheaths ridges sharpened to slightly flattened and broadened silica mamillae 2-4, aligned, forming a single sometimes row, individually side by furrows very small and acute, slightly broadened densely covered with short globose pilulae density of pilulae not thinning towards the margins surface and pilulae covered with wax crystals ring of fused pilulae absent to indistinct pilulae at the stomatal pore globose, not stalked slightly longer than or as long as the sheath long, with a reddish- brown centre and a very broad light-brown scarious margin adhering together throughout the shoot ridges broadened with two distinct margins silica mamillae thorn- like, forming two rows of alongside the margins the ridge furrows broad and rounded sparsely covered with short globose pilulae pilulae only scattered, often indistinct to nearly absent surface and pilulae densely covered with wax crystals bordered by a ring of fused pilulae pilulae at the stomatal pore absent to indistinct globose slightly shorter than or as long as the sheath long, with a dark-brown to reddish-brown centre and a distinct white scarious margin adhering together only at the lowermost sheaths ridges rounded to slightly broadened silica mamillae indistinct and scattered furrows broad and rounded sparsely covered with short globose pilulae pilulae only scattered, often indistinct to nearly absent surface and pilulae covered with wax crystals bordered by a ring of fused pilulae pilulae at the stomatal pore absent to indistinct globose covered with short globose pilulae density of pilulae not thinning towards the margins surface and pilulae covered with wax crystals or wax cristals absent bordered by a ring of fused pilulae pilulae at the stomatal pore globose (sometimes nearly absent) ridges rounded silica mamillae indistinct shorter than the sheath short, dark-brown with a very small white scarious margin not or sometimes adhering together and scattered furrows broad and rounded Sterile shoot – micromorphology of stomata (visible as two subsidiary cells) Sterile shoot – nodal sheath (teeth) Sterile shoot – micromorphology of internodes 188 FERN GAZ. 20(5):181-196. 2017 variants of that species. However detailed differences from the parents in macro- and micromorphological aspects have been listed earlier: Page (1988; 1997), Lubienski (2003; 2009; 2013), Lubienski & Dörken (2015) (for E. ×mildeanum) and Lubienski (2010; 2013) (for E. ×lofotense). Additionally both hybrids show irregular meiotic cell division resulting in aborted spores (for images see Lubienski, 2009; 2010; Lubienski & Dörken, 2015). In both taxa a remarkable discrepancy between their macro- and micromorphological characters is expressed. Traits of the macromorphological dominant parent E. sylvaticum are much more obscure in the micromorphology of the main shoot of both hybrids (especially regarding the highly distinctive ridge architecture, Figures 6, 7). Macro- and micromorphological features of both hybrids and their parental species

Figure 7. SEM images of the micromorphology of the shoot ridges and stomata of Equisetum ×lofotense (a-b), E. ×mildeanum (c-d), and E. ×bowmanii (e-f). Arrows and letters indicate ridges (r), furrows (f), mamillae (m) and pilulae (p). LUBIENSKI & DÖRKEN: TWO HYBRIDS OF EQUISETUM SYLVATICUM 189 are listed in Table 1. The characteristic and highly distinct flattened and two-edged ridges bordered by two rows of silica spikes of E. sylvaticum are not found in E. ×mildeanum and E. ×lofotense. Both show a micromorphology more similar to their second parent, E. pratense and E. arvense respectively (Lubienski, 2010; Lubienski & Dörken, 2015). Interestingly this recessive behaviour of E. sylvaticum micromorphology is expressed to a similar degree in the third known E. sylvaticum hybrid, E. ×bowmanii, also a hybrid clearly indicating Wood Horsetail as one parent in its macromorphological appearance. It therefore is added in Figures 6 and 7, so that the micromorphological characters of all three known E. sylvaticum hybrids and their parental species can be compared directly. Because of the rarity of E. ×lofotense, in comparison with E. ×mildeanum, some further field notes of the hybrid are given, thus supplementing the data given in Lubienski (2010; 2013). Plants on Senja were detected within the mixed stands with E. sylvaticum, because they displayed a characteristic asymmetric overhanging growth habit especially in the exposed roadside ditches and slopes (Figure 8a, b). In this character the hybrid is similar to E. ×mildeanum (Figure 8c, d). Nevertheless in all dimensions it is a more robust and taller plant, reaching heights up to 90 cm (the tallest shoot found on Senja was 78 cm long, Figure 9a), which is never found in E. ×mildeanum (max. height approx. 40 cm, Figure 9b). Additionally both E. sylvaticum hybrids show distinctly the influence of their second parent in their nodal sheath and branch characters (Figure 10) and can be differentiated by these. An additional task in the field is to distinguish E. ×lofotense from its E. sylvaticum parent with which it frequently co-occurs. Differences between both are seen in the branches, the branch teeth, the sheath teeth, the shoot surface, and the fertile shoots in general. The distinct branching pattern of all E. sylvaticum hybrids (Lubienski, 2010) seems to be a very stable and reliable character. The side branches are long, spreading, and secondarily branched only in their proximal part, so that a distal unbranched distinctly overhanging part results, giving the whole shoot a somehow irregular habit (Figure 11, 14b; see also image in Lubienski 2010). This differs from the very symmetric and storied branching pattern of E. sylvaticum resulting in brush-like side branch clusters (Figure 14a). In comparison with E. sylvaticum the teeth of the side branches of E. ×lofotense are not, or to a much lesser degree, spreading from the internode above (Figure 12). The sheath teeth are less reddish-brown, shorter or as long as the sheath itself (longer than the sheath in E. sylvaticum), only irregular adhering together, becoming darker brown, shorter and more free in the upper parts of the shoot, i.e. E. arvense-like. The main shoot surface is not as rough as in E. sylvaticum, a direct consequence of the micromorphology, where silica spikes are lacking on the less prominent ridges (Figure 7a). This difference is also clearly seen at the surface of the nodal sheath, which shows shallow ridges with a smooth surface like E. arvense, in contrast to prominent ridges with rough silica projections in E. sylvaticum (Figure 13). The fertile shoots of E. ×lofotense have a very striking appearance at the Senja sites. These are very sturdy in general habit and bigger in all dimensions than the sterile shoots. Although this is a typical feature of the fertile shoots of all semidimorphic species and hybrids (e.g. E. sylvaticum, E. pratense, E. ×mildeanum, E. ×bowmanii), the fertile E. ×lofotense shoots from Senja are remarkable in their sturdy -like habit. When viewed from above these shoots appear star-like and distinctly different from the fertile shoots of E. sylvaticum with which they grow intermixed. This difference results from 190 FERN GAZ. 20(5):181-196. 2017

Figure 8. Equisetum ×lofotense (a & b) and E. ×mildeanum (c & d), showing characteristic overhanging growth habit.

Figure. 9. Shoots of Equisetum ×lofotense (a) and E. ×mildeanum (b), showing differences in size (bar = approx. 10 cm). LUBIENSKI & DÖRKEN: TWO HYBRIDS OF EQUISETUM SYLVATICUM 191

Figure 10. Sterile shoots of Equisetum ×lofotense (a) and E. ×mildeanum (b), showing influence of the second parent species E. arvense in E. ×lofotense (a) (oblong nodal sheaths and sheath teeth, low number of teeth and side branches) and E. pratense in E. ×mildeanum (b) (short nodal sheaths and sheath teeth, high number of densely spaced teeth and side branches).

Figure 11. Sterile and fertile shoots of Equisetum ×lofotense, showing irregular branching pattern.

Figure 12. Sterile shoots of Equisetum sylvaticum (a) and E. ×lofotense (b), showing differences in branch teeth, spreading (a) and not spreading (b) from the internode above. 192 FERN GAZ. 20(5):181-196. 2017

Figure 13. Nodal sheaths of sterile shoots of Equisetum sylvaticum (a) and E. ×lofotense (b), showing differences in sheath surface and length and sheath teeth colour and length.

Figure 14. Fertile shoots of Equisetum sylvaticum (a) and E. ×lofotense (b), showing differences in branching pattern.

Figure 15 Equisetum ×lofotense, nodal sheaths, sterile (a) and fertile (b) shoot, showing different influence of the parent species E. arvense and E. sylvaticum in the macromorphological appearance of the hybrid. LUBIENSKI & DÖRKEN: TWO HYBRIDS OF EQUISETUM SYLVATICUM 193 the fact that the 1st order side branches of the fertile shoots of E. ×lofotense are in general longer than in E. sylvaticum, while having 2nd order side branches shorter than in E. sylvaticum (Figure 14). Concerning the sterile and fertile shoots of E. ×lofotense, a different influence of the parent species is also expressed in the macromorphological appearance of the hybrid, making the sterile shoots more E. arvense-like and the fertile shoots more E. sylvaticum- like (Figure 15).

Distribution of Equisetum ×mildeanum and E. ×lofotense Figure 16a shows the known distribution of E. ×mildeanum for whole Norden, and it can be presumed that other populations may be found within this area. Figure 16b shows the distribution of E. ×lofotense, at present an endemic of northwestern Norway. Further field work in this area should explore whether it is actually more frequent than currently known. The vigour of the hybrid populations on Austvågøya and Senja makes this suggestion likely.

DISCUSSION All four sites of E. ×mildeanum and E. ×lofotense are very similar from a geomorphological and ecological point of view, being situated within a single fjord in the north of Senja over a distance of approximately 7.25 kilometres. The habitat is exclusively a wet, north- to east-facing mountain slope characterised by huge boulders and sparse woodland, interrupted at its base by a road only a few metres above and near to the sea. All sites and the whole area in general harbour extensive stands of the parent species E. sylvaticum, E. arvense and E. pratense. Horsetail hybrid colonies are often found in disturbed sites such as roadside ditches or railway embankments. This can be caused either by vegetative spreading alongside

Figure 16. Distribution of Equisetum ×mildeanum (a) and E. ×lofotense (b) for Norden (arrows indicate two localities, the semi-filled dot indicates the historic occurrence of E. ×mildeanum at its type locality on the Isle of Rügen, Germany 194 FERN GAZ. 20(5):181-196. 2017 the traffic system by rhizome or shoot cuttings or through an initial hybridization event due to ecological conditions that favour cross fertilization between Equisetum gametophytes. Wet roadside ditches like those on Senja display perfect conditions for germination of the chlorophyllous and short-lived Equisetum spores and the establishment of gametophytes. These are open raw soils without higher plant or competition, especially after roads are periodically cleared and straightened (Hauke, 1967; Page, 1967; Duckett & Duckett, 1974; Mesler & Lu, 1977; Duckett & Duckett, 1980; Duckett, 1985). Such pioneer habitats fit with the rather narrow ecological requirements of Equisetum gametophytes and for this reason can harbour mixed gametophyte populations, that increase the probability of crossing events between different species (Hauke, 1978; Page & Barker, 1985; Husby, 2013). Intergametophytic fertilization and therefore hybridization in Equisetum is additionally favoured by the reproduction biology of the genus in general. Equisetum gametophytes are initially unisexual, potentially becoming bisexual under changing growing conditions (e. g. accumulation of metabolites) (Duckett, 1972; Duckett & Duckett, 1980). However such an accumulation rarely takes place under natural growing conditions (Duckett & Duckett, 1980) and therefore bisexuality is obviously rare among wild gametophyte populations (Duckett & Duckett, 1974, 1980; Duckett, 1979). Consequently, in Equisetum, intergametophytic fertilization is highly effective in nature, thus outbreeding processes are facilitated. This effect is further emphasized where gametophytes of different species grow together, which in addition favours hybridization events (Duckett & Duckett, 1974; Duckett, 1979). It seems likely that E. ×mildeanum and E. ×lofotense both originated in the roadside ditches of Mefjorden and succeeded in colonising the adjacent slopes. Each of the four colonies may have started from an individual crossing event or there may have been just two events with subsequent vegetative spread. As the two sites of E. ×lofotense are only two kilometres apart, a single de novo hybridization event between the parent species to form this rare hybrid seems to be most likely, followed by vegetative spread as a result of human disturbance. Nevertheless, the fact that all four colonies stretch over at least several hundred square metres means they are unlikely to be of very recent origin.

ACKNOWLEDGEMENTS We would like to thank Torbjørn Alm (Tromsø, Norway) for help with the map of Norden and Michael Laumann and Lauretta Nejedli (Electron Microscopy Center, Department of Biology, University of Konstanz, Germany) for technical support (SEM). Patrick Acock (St. Mary Cray, Great Britain) and Heather McHaffie (Edinburgh, Great Britain) provided valuable advice concerning E. ×bowmanii in the British Isles.

REFERENCES ACOCK, P. 2015. Equisetum L. In: STACE, C. A., PRESTON, C. D. & PEARMAN, D. A. (Eds) Hybrid Flora of the British Isles, pp. 7-13, Botanical Society of Britain and Ireland, Bristol. DUCKETT, J. G. 1972. Sexual behaviour of the genus Equisetum subgenus Hippochaete. Bot. J. Linn. Soc. 65: 87-108. DUCKETT, J. G. 1979. An experimental study of the reproductive biology and hybridization in the European and North American species of Equisetum. Bot. J. Linn. Soc. 79: 205-229. LUBIENSKI & DÖRKEN: TWO HYBRIDS OF EQUISETUM SYLVATICUM 195

DUCKETT, J. G. 1985. Wild gametophytes of Equisetum sylvaticum. Amer. Fern J. 75: 120-127. DUCKETT, J. G. & DUCKETT, A. R. 1974. The ecology of Equisetum gametophytes. Amer. J. Bot. 61 Suppl.: 36. DUCKETT, J. G. & DUCKETT, A. R. 1980. Reproductive biology and population dynamics of wild gametophytes of Equisetum. Bot. J. Linn. Soc. 80: 1-40. HAUKE, R. L. 1967. Sexuality in a wild population of Equisetum arvense gametophytes. Amer. Fern J. 57: 59-66. HAUKE, R. L. 1978. A taxonomic monograph of Equisetum subgenus Equisetum. Nova Hedwigia 30: 385-455. HULTÉN, E. 1950. Atlas över växternas utbredning i Norden. Fanerogamer och ormbunksväxter. Generalstabens Litografiska Anstalts Förlag, Stockholm. HULTÉN, E. & FRIES, M. 1986. Atlas of North European Vascular Plants. North of the tropic of cancer. I. Introduction. Taxonomic Index to the maps 1-996. Maps 1-996. Koeltz Scientific Books, Königsstein. HUSBY, C. 2013. Biology and functional ecology of Equisetum with emphasis on the Giant Horsetails. Bot. Rev. 79: 147-177. JALAS, J & SUOMINEN, J. (Eds) 1972. Atlas Florae Europaeae. 1. Pteridophyta (Psilotaceae to Azollaceae). The Committee for Mapping the Flora of Europe and Societas Biological Fennica Vanamo. Helsinki. LUBIENSKI, M. 2003. Equisetum ×mildeanum Rothm. (E. pratense Ehrh. × E. sylvaticum L.), en snellehybrid ny for Norge. Blyttia 61: 171-178. LUBIENSKI, M. 2009. Nye funn av Equisetum ×mildeanum (E. pratense × E. sylvaticum) i Norge. Blyttia 67: 270-275. LUBIENSKI, M. 2010. A new hybrid horsetail Equisetum ×lofotense (E. arvense × E. sylvaticum, Equisetaceae) from Norway. Nord. J. Bot. 28: 530-540. LUBIENSKI, M. 2013. Hybriden der Gattung Equisetum (Equisetaceae, Equisetopsida, Monilophyta) in Europa. Ber. Inst. Landschafts- Pflanzenökologie Univ. Hohenheim, Beih. 22: 91-124. LUBIENSKI, M. & DÖRKEN, V. M. 2015. Ängsfräken × skogsfräken funnen i Sverige – samt en översikt över de nordiska fräkenhybriderna. [Equisetum ×mildeanum Rothm. found in Sweden.] Svensk Bot. Tidskr. 109: 228-239. MESLER, M. R. & LU, K. L. 1977. Large gametophytes of Equisetum hyemale in northern California. Amer. Fern J. 67: 97-98. PAGE, C. N. 1967. Sporelings of Equisetum arvense in the wild. Br. Fern Gaz. 9: 335- 338. PAGE, C. N. 1988. Two hybrids of Equisetum sylvaticum L. new to the British flora. Watsonia 17: 273-277. PAGE, C. N. 1997. The ferns of Britain and Ireland. 2nd ed. Cambridge University Press, Cambridge. PAGE, C. N. & BARKER, M. 1985. Ecology and geography of hybridization in British and Irish horsetails. Proc. Roy. Soc. Edinb. 86b: 265-272. PAGE, C. N. & GUREYEVA, I. I. 2009. A new horsetail hybrid from Western . Animadversiones Systematicae ex Herbario Kryloviano Universitatis Tomskensis 101: 15-21. PAGE, C. N. & GUREYEVA, I. I. 2013. Equisetum × sergijevskianum, a hybrid horsetail from Siberia. Fern Gaz. 19: 181-190. ROTHMALER, W. 1944. Pteridophyten-Studien I. Feddes Repert. 54: 55-82. 196 FERN GAZ. 20(5):181-196. 2017

SJÖRS, H., JONSELL, B. & ELVEN, R. 2004. Features of Nordic environment and vegetation. In: Jonsell, B. (Ed) Flora Nordica. General Volume, pp. 37–100, The Bergius Foundation, The Royal Swedish Academy of Sciences, Stockholm. FERN GAZ. 20(5):197-201. 2017 197



1Universidad de la Amazonia, Programa de Biología, Calle 17, diagonal 17 con carrera 3F, Barrio el Porvenir, Florencia-Caquetá, Colombia; 2Current address: Instituto de Ciências Biológicas, Departamento de Botânica, Universidade Federal de Minas Gerais, Caixa Postal 486, 30123-970, Belo Horizonte, Minas Gerais, Brazil; 3Herbario Nacional de Bolivia, Instituto de Ecología, Universidad Mayor de San Andrés, Cota cota, calle 27, Campus Universitario, La Paz, Bolivia; 4Semillero de Investigación en Biogeografía Evolutiva (SIBE), Universidad de la Amazonia, Programa de Biología, Florencia-Caquetá, Colombia; 5Universidad de la Amazonia, Programa de Ingenieria Agroecológica, Florencia-Caquetá, Colombia. *Email: [email protected]

Key words: Andean-Amazonian foothills; Caquetá; Huila; ferns; Florencia; distribution range; epipetric habit.

ABSTRACT Serpocaulon obscurinervium is considered as a rare and threatened species, with only four exemplars registered including the type. It was recorded from Colombia and Ecuador, but most commonly reported for central southern Colombia (central west of Chocó and south of Nariño departments). Here, we expand its distribution by reporting new records for this species on both sides of the Eastern Cordillera of the Colombian Andes, specifically from the high Andean forest of Florencia, in the Caquetá department and Suaza in the Huila department. Furthermore, this is the first record of the species as an epipetric plant. Pictures of the plant and an updated distribution map are provided.

INTRODUCTION Serpocaulon A. R. Sm. (Polypodiaceae) is a neotropical fern genus, which contains 52 taxa (Sanín 2015). These species occur mainly in humid forests (Smith et al. 2006). Colombia harbours 27 species that are distributed in the inter Andean valleys, the Chocó Region, the Sierra Nevada de Santa Marta, Oriental valley and Amazon, but most species occur in the humid forests of the Andes and the Chocó Region (Sanín in press). Serpocaulon obscurinervium D. Sanín (Figure 1) is an Andean species that was described in 2014 from herbarium specimens deposited in collections of Colombia (CHOCO, COL, CUVC and PSO), France (P) and United States (MO and UC) (Sanín 2014). The first specimen was collected in Ecuador by R. Spruce in 1909. In 1973, other specimens were collected in Nariño at 2500m by O. Hagemann & L. Leist. The most recent collections were recorded in 1988 in the Andean zone of the Department of Chocó by P. Silverstone–Sopkin, and in La Planada Reserve, Nariño Department, by H. Herrera 198 FERN GAZ. 20(5):197-201. 2017

(Sanín 2014). Because the distribution of the species within Colombia was restricted to high montane forests outside of any protected area, its conservation status was considered as Endangered (EN) following the IUCN criteria (Sanín 2014). Since its first description, the species had never been found again or reported alive in nature. During the ongoing botanic exploration of the Caquetá and Huila departments, Serpocaulon obscurinervium was recorded growing in sites on both sides of the Eastern Cordillera. The specimens were found along the roadside of Puente Cerro Negro in El Portico, at the western slope in a forest at 2190m and in Gabinete military station at 2050m on the border of Caquetá and Huila Departments on the Eastern slope of the Eastern Andean Cordillera. In Huila Department, it was also found on the road to Neiva, in the municipality of Suaza at 2190m (Figure 2). Two to five vouchers were collected per site and were deposited at the following herbaria: CAUP, COAH, COL, HUA, FAUC & PSO. The exemplars from Caquetá (Puente Cerro Negro and Gabinete) are the first records of Serpocaulon obscurinervium as an epipetric fern. In addition, it is the first time that the species has been collected in the Caquetá and Huila departments (Murillo-P. et al. 2016), the easternmost distribution of the species with its nearest known population at

Figure 1. Serpocaulon obscurinervium. A: Habit of the plant, scale bar (5 cm). B: Rhizome, scale bar (1 cm). C: Rhizome bundles, scale bar (3.5 cm). D: Petiole bundles, scale bar (2 cm). E: Fiddlehead, scale bar (1 cm). F: Lamina base, scale bar (3 cm). G: Lamina apex, scale bar (3 cm). H: Immature sori, scale bar (8 cm). I: Mature sori, scale bar (8 cm). J: Pinna veins, scale bar (8 cm). All from Sanín & Peña 6371 (COL, HUA). Photos by J.L. Peña. SANÍN ET AL: SERPOCAULON OBSCURINERVIUM 199

250 km distance on the road of La Victoria−Monopamba, Dept. Nariño, in central south Colombia. Serpocaulon obscurinervium is closely related to S. eleutherophlebium (Fée) A. R. Sm., a species that is distributed from Costa Rica to Ecuador and Venezuela (Moran 1995, Smith et al. 2006), and in Colombia is widely recorded in high Andean forest and

Figure 2. Distribution of Serpocaulon obscurinervium. The black dots are previous registers of the species: 1) Cerro del Torrá, Valle del Cauca, Colombia; 2) La Planada Reserve, Nariño, Colombia; 3) road La Victoria−Monopamba, Nariño, Colombia; 4) Tungurahua, Mt. Abitagua, Ecuador. The asterisks are the new registers on the Oriental Andean Cordillera of Colombia: 5) El Portico and 6) El Gabinete from Florencia, Caquetá department and 7) From Suaza, Huila department. 200 FERN GAZ. 20(5):197-201. 2017 subpáramos at 2430-3950m, epiphytic, terrestrial and rupicolus (Sanín in press).The identification of these two taxa has been confused. Both species have similar long-creeping rhizomes, scales with patent apices, concolorous and pinnatisect laminae. However, S. obscurinervium differs from S. eleutherophlebium in the scales characters, being linear-lanceolate (versus subulate), dark orange (versus dark brown), larger sized 5.2-(6.3)-8.5 × 1.6-(1.7)-2.3 mm (versus 3.5-(5.1)-7.1 × 0.5-(1.1)-1.7 mm), and with longer petioles 24-(2.6)-28 cm (versus 3.4-(12)-25.5 cm), more areoles along the middle segment 19-(20)-28 rows (versus 6-(12)-21 rows), inconspicuous fertile veinlets (versus conspicuous fertile veinlets), and the presence of 2-4 celled, dispersed, catenate, terete and dark brown trichomes on the blades and rachises (versus absent trichomes) (for a complete comparison see Sanín 2014). Even though three more populations of Serpocaulon obscurinervium were discovered in the Caquetá and Huila departments, it is still categorized as Endangered [EN B1ab(i, ii, iii) + B2ab(i, ii, iii)] following the IUCN criteria. Here we present more support for this, and include Criterion D to its IUCN categorization: [EN B1ab(i, ii, iii) + B2ab(i, ii, iii) + D], the latter given that the population size of S. obscurinervium is estimated to be fewer than 250 mature individuals. The sparse and vicariant distribution of the species on the Andean Cordillera of Ecuador and Colombia (Sanín 2014), and the reduction of high montane forest vegetation caused by human settlements and agricultural expansion (Murcia et al. 2010), impose a permanent and increasing danger to the populations of this species, especially as all the records of this species are outwith any protected area. In addition, from five to 10 individuals were recorded in these new localities, including both mature and juvenile individuals (pers. obs.).

MATERIAL EXAMINED Serpocaulon obscurinervium—Caquetá: Florencia, Via Neiva, vereda El Condor, a mano derecha de la vía, a 100m del Puente Cerro Negro [1°45'19.62" N , 75°44'59.98" W], 2050m, 27 Jul 2015, Sanín & Peña 6371 (CAUP, PSO, COL, HUA); Florencia, Via Guadalupe, El Gabinete, a mano izquierda de la vía, a 100m de la estación militar [75º40’58.4” O, 1º52’44” N], 2700–2800m, 20 Feb 2016, Sanín, García & Camargo- García 6396 (COAH, COL, FAUC, HUA); Huila: Suaza, via Neiva, vereda Campo Hermoso, costado izquierdo de la via, km 40 [1°46'11.71" N, 75°46'37.52" O], 2190m, 1 Apr 2016, Peña 542 (COL) Chocó: San José del Palmar, Cerro Torrá, Filo de Cumbre, [04º48’ N, 76º29’ W], 2700–2800m, 21 Aug 1988, Silverstone-Sopkin 4633 (Paratypes: CHOCO, CUVC, MO, UC n.v.). Nariño: Reserva Natural La Planada, sendero entre El Hondón y Los Horquetas, Bosque primario, 01º10’ N, 78º00’ W, 1820m, 31 Jan [no year given], Herrera 9302, 9193 (Paratypes: PSO, CUVC, UC n.v.); Carretera La Victoria−Monopamba, [01º10’ N, 78º00’ W], 2500m, 10 Jan 1973, Hagemann & Leist 1898 (holotype: PSO; isotype: COL).

ACKNOWLEDGEMENTS DS would like to thank the primatologist Javier Garcia for promoting the biological research in Caquetá department. DS is in debt to the Biology program, especially to Professor Alexander Velasquez-Valencia and the staff of the Natural History Museum of the University of Amazonia for providing infrastructure. Special thanks to Drs Mary Gibby and Klaus Melhtreter for comments that substantially improved the manuscript. The University of the Amazonia funded this research. SANÍN ET AL: SERPOCAULON OBSCURINERVIUM 201

REFERENCES IUCN, 2001. IUCN Red List Categories and Criteria: Version 3.1. [online]. Available from http://www.iucnredlist.org/info/catego- ries_criteria2001 [accessed 24 Oct. 2016] MORAN, R.C. 1995. Polypodiaceae. Pp. 333–366. in: G. DAVIDSE, M. SOUSA, AND S. KNAPP (eds). Flora Mesoamericana, Psilotaceaea a Salviniceae. México: Universidad Nacional Autónoma de México. MURCIA, G.U.G., HUERTAS, M.C., RODRÍGUEZ, J.M., & CASTELLANOS, H.O. 2010. Cambios multitemporales de los bosques y otras coberturas de la Amazonia colombiana, a escala 1:100.000, en el periodo 2002 al 2007. Instituto Amazónico de Investigaciones Científicas SINCHI. Bogotá, D. C. MURILLO-P., M.T., J. MURILLO-A. & A. LEÓN-P. 2016. Serpocaulon obscurinervium D. Sanín. In: BERNAL, R. GRADSTEIN, S.R. & CELIS, M. (Eds.). 2015. Catálogo de plantas y líquenes de Colombia. Instituto de Ciencias Naturales, Universidad Nacional de Colombia, Bogotá. http://catalogoplantascolombia.unal.edu.co SANÍN, D. 2011. Revisión taxonómica de Serpocaulon (Polypodiaceae), para la Cordillera Central y el Macizo Colombiano. MSc thesis, 96 pp., Universidad de Caldas, Manizales, Colombia. SANÍN, D. 2014. Serpocaulon obscurinervium (Polypodiaceae), a new fern species from Colombia and Ecuador. Plant Ecol. Evol. 147: 127–133. SANÍN, D. 2015. Serpocaulon tayronae (Polypodiaceae), a new species from the Sierra Nevada de Santa Marta, Colombia. Phytotaxa 213: 243–252. SMITH, A.R., KREIER, H.-P., HAUFLER, C.H., RANKER, T.A. & SCHNEIDER, H. 2006. Serpocaulon (Polypodiaceae), a new genus segregated from Polypodium. Taxon 55 (4): 919-930. 202 FERN GAZ. 20(5). 2017 FERN GAZ. 20(5):203-213. 2017 203



Roelingsbeek 1, 8033 BM Zwolle, The Netherlands ([email protected])

Key words: Polystichum lonchitis, Kuinderbos, monitoring, mortality

ABSTRACT Polystichum lonchitis was found in 1978 for the first time in The Netherlands on a dry ditch bank, on calcareous sand within a planted woodland (Kuinderbos). In subsequent years six other specimens were found at three other sites, in a woodland, on a scree in a chalk stone quarry and on a wall, and four of these plants became fertile. Mortality in five out of seven plants resulted from a small landslide, habitat destruction, browsing and frost damage. Long term monitoring of two plants showed the impact of rainfall and frost on vitality. No recruitment was observed in the spore shadow of adult plants.

INTRODUCTION Polystichum lonchitis (L.) Roth. is a montane-alpine species, with a Holarctic distribution. In North America it is a species found mostly in boreal and subalpine regions between 0 and 3200 m (Morin, 1993). In Europe the species is found between 900 and 2100 m and rarely at lower altitude up to 300 m, except for the coastal area of Norway (Mutter et al., 1998). It is also found in Greenland, Iceland and in isolated parts of Russia but is rare in Japan (Dostál & Reichstein, 1984). Within the UK it is more widespread in the central Scottish Highlands (c. 610 – 915 m). Occasionally it descends to lower altitudes in steep upland river valleys (c. 150 m) and lower levels near the west coast, with its cool summers (Page, 1982). In 1978, Polystichum lonchitis was recorded for the first time in the Netherlands during a survey of 120 km of dry ditches in the Kuinderbos. Two plants were found growing at the sides of two adjacent dry ditches on the bottom of the former Zuiderzee at ca. 1 m below sea level (Bremer, 1980). Since then this small population has been monitored annually for the first 15 years and less intensely in subsequent years. After 1979 the species was found at three other sites in the Netherlands, and monitored annually near Lelystad (Smit, 1989). This paper analyses these records to try to determine whether a montane-alpine plant species is able to build up a viable lowland population.

METHODS Two plants (plant 1 and 3, both in Flevoland, Table 1) were monitored for 24 years (Kuinderbos) and 27 years (Lelystad) with annual counting of the number of fronds and the maximum frond length and fertility at the end of the summer season or start of autumn, when frond formation had stopped and sori produced. Change in maximum frond length, change in number of fronds and the log transformed product of both parameters (always year t to year t + 1) were used as response variables to rainfall and the number of frost days. The average values in Flevoland were used for annual rainfall 204 FERN GAZ. 20(5):203-213. 2017 Ls Ls Fd Fd Br, Hd Hd Mo Fr Fr - Fr-Qu - - Fr,Cb Tree layer Tree Mo = cause of mortality: of cause = Mo NE E N SSW N SW SW Asp Carpinus betulinus. Carpinus 62 80 45 90 45 40 >30 Sl , Cb = = Cb , robur Quercus fine sand fine sand sandy clay cement loamy soil on chalk loamy soil on chalk fine sand Substrate . Nr = plant number, as mentioned in the paper. First = first year first = First paper. the in mentioned as number, plant = Nr lonchitis . Polystichum , Qu = = Qu , excelsior Fraxinus ditch side, woodland wall quarry quarry ditch side, woodland ditch side, woodland ditch side, woodland habitat B. van As B. van Bremer P. P. Bremer P. J. Egelsmeer J. Egelsmeer P. Bremer P. A. Smit Leg 0 F F ? 0 F F Fer 2011 2014 1984 1994 1998 Last 1978 1979 1998 1998 1986 2011 1994 First Some characteristics of the Dutch population of population Dutch the of characteristics Some `t Rooth Lelystad Kuinderbos Kuinderbos Kuinderbos Vlaardingen `t Rooth Location 1 2 3 4 5 6 7 of recording, Last = last year of recording, year of death. Fer = Fertility of plant: 0 = not fertile, F = = fertile Fr during layer: period of recording, Tree ? = aspect, not = Asp °, in slope = Sl known, Table 1. Table Ls = small scale land slide, Hd habitat destruction, Fd frost damage, Br browsing . nr BREMER & SMIT: POLYSTICHUM LONCHITIS IN THE NETHERLANDS 205

Table 2. Correlation matrix with doses and effect parameters with correlation (r) and significance (p) shown (and significant levels p < 0.05 marked with asterix *). ▲ n = change in number of fronds between year t and t + 1, ▲ Fm = change in maximum frond length between year t and t + 1

Plant 1, Kuinderbos

▲ n ▲ Fm ▲ (nxFr) log ▲ (nxFr)

Rainfall year t r 0.1 0.01 0.77 -0.01

p 0.67 0.96 0.75 0.96

Rainfall April year t+1 r 0.33 0.2 0.37 0.17

p 0.17 0.41 0.12 0.5

Rainfall May-July year t+1 r 0.62 0.17 0.42 0.61

p 0.01* 0.49 0.07 0.01*

Rainfall May-July year t r -0.14 0.19 0.14 0.14

p 0.95 0.43 0.58 0.58

Number of frost days r -0.25 -0.4 -0.32 -0.23

p 0.3 0.09 0.18 0.34

Plant 3, Lelystad

▲ n ▲ Fm ▲(nxFr) log ▲ (nxFr)

Rainfall year t r -0.43 0.02 -0.36 -0.28

p 0.03* 0.93 0.07 0.17

Rainfall April year t+1 r 0.2 0.08 0.16 0.21

p 0.32 0.7 0.44 0.31

Rainfall May-July year t+1 r 0.09 0.42 0.22 0.27

p 0.66 0.03* 0.29 0.17

Rainfall May-July year t r -0.38 -0.11 -0.32 -0.39

p 0.06 0.58 0.11 0.05*

Number of frost days r -0.12 -0.45 -0.28 -0.28

p 0.56 0.02* 0.17 0.16 206 FERN GAZ. 20(5):203-213. 2017 in year t, rainfall in April in year t + 1, rainfall in May-July in year t and t + 1, as we expected drought in or early summer to play a role. The number of frost days per winter (maximum day temperature below 0°C) was used as indicator for the low temperature impact in winters regional data used (www.knmi.nl). These were the same parameters as used by Bremer & Jongejans (2010) in their study on the demography of Asplenium scolopendrium L. SPSS 10.1 was used to calculate correlations. Per location data about habitat, substrate, inclination, aspect and composition of the tree layer and herb layer were collected in order to classify the sites and vegetation.

RESULTS Habitat and vegetation Over a period of 39 years seven plants were found at the former sea floor in Flevoland, in the city of Vlaardingen and in a chalk quarry in the southern part of the Netherlands (Figure 1). The Kuinderbos plants grew in dry ditch sides within plantations of Fraxinus excelsior L., accompanied by of the Fissidentietum taxifolii bryophyte community (Bremer, 1999) and fern species such as Asplenium scolopendrium and Polystichum aculeatum (L.) Roth. Near Lelystad the plant grew under a canopy of Fraxinus excelsior L., with Oxyrhynchium hians (Hedw.) Loeske, striatum (Hedw.) Schimp. and urbanum L. The non-native Potentilla indica (Andrews) Wolf had invaded the site. This vegetation is related to the Fraxino-Ulmetum, a woodland community confined to clayey soils in the Netherlands. Both `t Rooth plants were found growing among flint stones deposited at a chalk quarry. The vegetation at this site was characterized by mass growth of Asplenium trichomanes L., Gymnocarpium robertianum (Hoffm.) Newman and Asplenium scolopendrium. In the bryophyte layer Encalypta streptocarpa Hedw. was a prominent species. Even Asplenium ruta-muraria L. was growing among flint stones, one of the few examples where it shows terrestrial growth in the Netherlands. The vegetation belongs to the Asplenietum ruto-murario-trichomanes, a community strongly linked to wall habitats (Segal, 1969) (OV39, British National Vegetation Classification, Rodwell, 1992). The Vlaardingen site was by a wall, which was destroyed two years after the discovery of Polystichum lonchitis. The vegetation belonged to the Filici-Saginetum, a wall community with Dryopteris filix-mas (L.) Schott and Asplenium scolopendrium. All sites have a high pH in common and are related to gradients. There was no difference in aspect comparing N-NE with S-SW (χ2 = 0.14 ns) (Table 1).

Distribution and trend The number of plants varied from one to two plants per location. The small population in the Netherland showed a positive trend until 2010 with decline in recent years (Figure 2). Most plants were found as adults; the Vlaardingen plant was a juvenile/subadult plant, rather small in size, as is often the case with wall dwelling fern species. Five plants died because of landslip, habitat destruction, frost damage and browsing or because of a combination of these factors (Table 1). At Vlaardingen, prior to wall destruction, the plant was conserved and planted in a garden but was subsequently lost through theft within two years. At `t Rooth quarry one of the sites was destroyed in favour of pond digging because of a threatened toad species. The other plant died as a result of browsing in combination with frost damage (Bremer & Egelsmeer, 2015). In the Kuinderbos, one plant slipped to the bottom of the dry ditch after a small scale land slip (initiated by heavy rain fall) and drowned as the normally dry ditches contain water (0.1 – 0.3 m depth) in BREMER & SMIT: POLYSTICHUM LONCHITIS IN THE NETHERLANDS 207 winter time. Four out of seven plants became adults, producing spores nearly every year. No recruitment took place in the spore shadow.

The impact of severe winters and rainfall The first plant found in the Netherlands (Kuinderbos, plant number 1) has been monitored since 1979. The maximum frond length (Lm) increased exponentially between 1979 and 1985. During the severe winter of 1984 - 1985 it was not harmed at all; number of fronds and maximum frond length increased, despite 22 frost days during that winter. The winter 1985 - 1986 was also long and cold, with the same number of frost days as in the

Figure 1. Distribution of Polystichum lonchitis in the Netherlands with number of plants per location (small dot = 1 plant, larger dot = 2 plants). The Kuinderbos is at 52°46` N 5° 49`E. 208 FERN GAZ. 20(5):203-213. 2017

Figure 2. The number of plants during half a century in The Netherlands since 1978.

Figure 3. The change in maximum frond length between year t and year t + 1 related to the number of frost days per winter between year t and year t + 1 in the Lelystad plant. (plant number 3). BREMER & SMIT: POLYSTICHUM LONCHITIS IN THE NETHERLANDS 209 preceding winter. The plant produced fewer and smaller fronds and the plant was sterile for one year. In subsequent years the plants recovered. Over the whole period the vitality parameters in plant 1 did not correlate significantly with the number of frost days (Table 2). The Lelystad plant however showed a significant negative correlation between change in frond length and the number of frost days per winter (n = 26, r2 = 0.18 p = 0.03) (Figure 3). When analysing rainfall and growth a positive correlation was found in Kuinderbos plant 1 between the change in number of fronds and the May-July rainfall in the year t + 1 (n = 19, r2 = 0.38 p = 0.01) (Figure 4) and in plant 3 (Lelystad, Figure 5) between the change in frond length and May-July rainfall in year t + 1 (n = 26, r2 = 0.18, p = 0.03). The log transformed product of the change in maximum frond length x change in number of fronds showed a strong correlation in plant 1 (n = 19, r2 = 0.36, p = 0.01). The annual rainfall had a negative impact on plant 3 (n = 26, r2 = -0.16, p = 0.03).

DISCUSSION The site in `t Rooth is an artificial flint stone scree in a chalk quarry, which resembles the more natural habitat. In Scotland stable boulder scree slopes may contain a rich fern vegetation with Asplenium viride Huds., Cystopteris fragilis (L.) Bernh and Dryopters expansa (C.Presl) Fraser-Jenk.& Jermy (Page, 1982). On ledges it grows among low- growing flowering plants in the Dryas octopetala – Silene acaulis ledge community (Rodwell, 1992). Probably the dry ditches under a canopy can at best be compared with the gryke habitat - fissures in exposed limestone habitat that are usually 1 – 2 m deep (Lake et al., 2015). From the syntaxonomical point of view the woodland sites have

Figure 4. The increase of number fronds (year t and year t + 1) related to the rainfall in the period May – July in year t + 1 in the Kuinderbos plant (plant number 1). 210 FERN GAZ. 20(5):203-213. 2017

Figure 5. The Lelystad plant (plant number 3) accompanied by Potentilla indica in 2007, 21 years after the first record (photo P. Bremer)

Figure 6. The plant recorded in the Kuinderbos in 2015 (plant number 7), first discovered four years after the first record (photo P. Bremer). BREMER & SMIT: POLYSTICHUM LONCHITIS IN THE NETHERLANDS 211 much in common with, the Fraxinus excelsior-Acer campestre-Mercurialis perennis woodland and in particular the Geranium robertianum-subcommunity, where sometimes it is found under a miniaturised version of this woodland in the Limestone grykes in the Yorkshire Dales (Rodwell, 1998). The montane-alpine species was not expected to colonise a former sea floor. In neighbouring countries lowland sites for this species are not known (e.g. www.floraweb.de). In the year Polystichum lonchitis was recorded for the first time, Asplenium viride Huds. was also found in the Kuinderbos (Bremer, 1981), and some montane bryophytes had been found on dry ditch sides on the former seafloor [e.g. Distichium inclinatum (Hedw.) Bruch & Schimp. (Bremer & Ott, 1979) and fungi (e.g. Suillus tridentinus (Bres.) Singer]. The Kuinderbos is the only known site within the Netherlands with a small viable population of the montane Knautia dipsacifolia Kreutzer (Meijer & Bremer, 1986). All these species came by long distance dispersal by wind, with Knautia dipsacifolia being the exception (probably with humans as vector). Page (1982) states that the species occasionally descends to lower altitudes near the extreme west coasts of Scotland because of its cool summers. At open sites in the Netherlands the preferred N-E aspect was confirmed. In the Dutch woodlands it thrives with a SW preference, although in April, without the protection of a sprouted canopy, it can be warm in these ditches. A high temperature is probably compensated by evaporation of water, as dry ditches always contain water in this time of the year. There is a continuous flow of diaspores from mountainous areas spreading over the Netherlands, implicating that under optimal conditions plants can establish from spores. But the chance of establishment is dependent on the extent of an area which is suitable for colonisation, spore density, spore longevity (sporebank) and the ability of intragametophytic selfing (Klekowski 1979). In the Kuinderbos 300 ha of calcareous deposits on peat drained by more than 100 km of dry ditches provided a large area, with colonization by numerous rare fern species (Bremer 2007, de Groot 2012). De Groot (2012) showed in four species [Asplenium scolopendrium, A. trichomanes, Polystichum aculeatum, P. setiferum (Forssk.) Woynar] a very high selfing capacity in both diploid and polyploid species. Single-spore establishment following long-distance dispersal may have resulted in selection for selfing genotypes. There was no recruitment from the adult plants of diploid Polystichum lonchitis, at woodland sites. This might indicate that selfing is less likely in a second generation and that the species depends on intergametophytic crossing. There is a very low chance that spores from other genotypes are able to establish within the spore shadow of an adult plant because of the large distance to British and Central European populations. Polystichum lonchitis does have Arbuscular Mycorrhiza (Harley & Harley, 1987), but it is unknown whether fungi play a crucial role in establishment. With only two plants left, stochastic might occur. In the Netherlands Atlantic species such as Asplenium scolopendrium, Dryopteris borreri Newman, D. affinis (Lowe) Fraser-Jenk. and Polystichum setiferum are increasing in number and becoming more common. Global warming with more precipitation in summer and higher temperatures in winter plays a role. With its fluctuating small population Polystichum lonchitis remains a threatened species on the Dutch Red List (Sparrius et al., 2012). Landslip within dry ditches, frost damage, habitat destruction and browsing played a role in the mortality of five out of seven plants. One plant lived for ca. 45 years. Northridge writes that one of five Fermanagh plants (N. Ireland) was found as a mature plant in 1979 and was still in excellent condition 32 years later (www.habitas.org.uk/priority/species). Although plants in the UK seem to be long-lived, 212 FERN GAZ. 20(5):203-213. 2017 no demographic studies are known for this species. In the Netherlands frost damage played a role in vitality and survival. One would expect a montane-alpine species to more adapted to low temperatures, yet effects of severe winters were observed in four of seven plants, with plants becoming smaller, with lower number of fronds per plant, smaller fronds per plant or lower fertility. The same was observed in Asplenium scolopendrium, but this species has an Atlantic distribution, and is susceptible to frost. In this species snow cover can protect plants from heavy damage (Bremer & Jongejans, 2010). The more leathery fronds of Polystichum lonchitis (see Figure 6) seem to be more protective than those of Asplenium scolopendrium. However, our data suggest that snow cover might be crucial, a suggestion also made by Dostál & Reichstein (1984), indicating growth in snow-rich habitats. Mutter et al. (1998) did not find a role of extensive snow cover. For many subalpine species snow cover is a prerequisite for survival during winters (Körner & Larcher, 1988) as snow protects from low temperatures. Buried beneath a layer of snow plants are exposed to milder temperatures close to 0°C. Without snow cover desiccation plays a role or excessive natural radiation may result in frost damage. As old fronds might serve as nutrient storage organs frost damage impacts the new generation of fronds (Landi et al., 2014). Within the Netherlands long term monitoring of a complete plant population is exceptional. While in other groups, e.g. breeding birds, annual monitoring is commonplace, it is much more difficult in plants. Individuals in many species are more difficult to identify [e.g. Pteridium aquilinum (L.) Kuhn] and a complete survey of a large area time consuming. Plant species involved mostly have a restricted number of populations, as is the case in Woodsia ilvensis (L.) R.Br. (McHaffie, 2010) and more long term monitoring in ferns is needed (Sharpe & Mehlteter, 2010).

ACKNOWLEDGEMENTS We thank Ben van As, Olaf Opdenkamp and Jan Egelsmeer for their data and Klaus Mehltreter for reviewing the paper.

REFERENCES BREMER, P. 1980. The ferns of the Kuinderbos (The Netherlands), the establishment op 23 species in a planted forest. Acta Botanica Neerlandica 29(5/6): 351 – 357. BREMER, P. 1981. Polystichum lonchitis (L.) Roth. en Asplenium viride Huds. nieuw voor Nederland. Gorteria 10(7): 113 - 120. BREMER, P. 1999. Mosgemeenschappen op greppelkanten in de Flevolandse bossen. Stratiotes 19: 56 - 65. BREMER, P. 2007. The colonisation of a former sea floor by ferns. PhD thesis, Wageningen University. BREMER, P. &. OTT, E.C.J. 1990. The establishment and distribution of bryophytes in the woods of the IJsselmeerpolders, The Nether lands. Lind bergia 16:3 - 18. BREMER, P. & JONGEJANS, E. 2010. Frost and Forest Stand Effects on the Population Dynamics of Asplenium scolopendrium. Population Ecology 52: 211 - 222. BREMER, P. & EGELMEERS, J. 2016. De Lansvaren in Nederland. Varenvaria 29(1): 8 - 13. GROOT, A. DE. 2012. The fate of a colonizer: successful but lonely? The establishment of inter- and intraspecific diversity in ferns by means of long-distance dispersal. PhD thesis, Utrecht University. DOSTáL, J. & REICHSTEIN, T. 1984. Polystichum. In: G. Hegi. Illustrierte Flora von BREMER & SMIT: POLYSTICHUM LONCHITIS IN THE NETHERLANDS 213

Mitteleuropa. Band I. Teil 1: 169 - 187. HARLEY, J.L & HARLEY, E.O. 1987. A check-list of mycorrhiza in the British flora. New Phytologist 105: 1 - 102. KLEKOWSKI, E.J., 1979. The genetics and reproductive biology of ferns. In: A.F. Dyer (red.). The experimental biology of ferns. Pp. 133 – 170. London, Academic Press. KÖRNER, C & W. LARCHER, 1988. Plant life in cold climates. In: Plants and Temperature. Eds. S. Long & F.I. Woodward. Cambridge: pp. 25 – 57. LAKE, S., D. LILEY, R.STILL & SWASH, A., 2015. Britain`s Habitats. A guide to the Wildlife Habitats of Britain and Ireland. Princeton University Press. LANDI, M., ZOCCOLA, A., BACARO, G & ANGIOLINE, C., 2014. Phenology of Dryopteris affinis ssp. affinis and Polystichum aculeatum: modelling relationships to the climatic variables in a Mediterranean area. Plant Species Biology 29(2): 129 – 137. MCHAFFIE, H., 2010. Recent monitoring of wild populations of Woodsia ilvensis. Pteridologist 5(3): 191 - 192. MEIJER, K. & BREMER, P. 1986. Knautia dipsacifolia Kreutzer nieuw voor Nederland. Gorteria 13: 74 - 77. MORIN, N.R. 1993. Flora of North America. North of Mexico. Volume 2. Pteridophytes and Gymnosperms. North America Editorial Committee. Oxford University Press. MUTTER, H., H.J.B. BIRKSAND & ODLAND, A. 1998. The comparative ecology of Polystichum aculeatum, P. braunii and P. lonchitis in Hordaland, western Norway. Nordic Journal of Botany 18(3): 267 - 288. PAGE, C.N., 1982. The Ferns of Britain and Ireland. Cambridge University Press RODWELL. J.S. 1992. British Plant Communities Volume 3 - Grasslands and montane communities. Cambridge University Press. RODWELL, J.S. 1998. British Plant Communities. Volume 1. Woodlands and scrub. Cambridge University Press. SEGAL, S. 1969. Ecological notes on wall vegetation. PhD thesis, Universiteit of Amsterdam. SHARPE , J.M. & K. MEHLTRETER, K. 2010. Ecological insight form fern population dynamics. In: K. MEHLTRETER, L.R. WALKER & J.M. SHARPE. Fern Ecology: 61 – 110. Cambridge University Press. SMIT, A. 1989. De naaldvarens (Polystichum-soorten) van het Overijssels Hout bij Lelystad. Gorteria 15: 90 – 94. SPARRIUS, L., B.ODE & R.BERINGEN, 2012. Basisrapport voor de Rode Lijst vaatplanten 2012. FLORON. http://www.floron.nl/publicaties/rode-lijst-2012 214 FERN GAZ. 20(5). 2017


An Annotated Checklist of Indian Pteridophytes Part – 1 (Lycopodiaceae to Thelypteridaceae. Fraser-Jenkins, C.R., Gandhi, K.N., Kholia, B.S. & Benniamin, A. 2017. pp 562. Bishen Singh Mahendra Pal Singh, Dehra Dun, India.

This new checklist of Indian pteridophytes is a welcome addition to our knowledge of the diversity of Asian ferns and lycophytes, and adds to the recent publications on pteridophytes from China (Wu et al. 2013) and Nepal (Fraser-Jenkins et al. 2015). This volume covers Indian Lycopodiaceae, Selaginellaceae, Isoetaceae, Equisetaceae and 19 fern families (Psilotaceae to Thelypteridaceae). In addition to the detailed checklist that forms the bulk of the volume, there is a short introduction that sets the work in context and a summary of the numbers of families, genera and species that are to be included in both Parts 1 and 2. The checklist itself is followed by a series of 37 colour images, that range from some significant herbarium specimens to many field photographs of living plants, either in close-up or as habitat shots, and all of these providing a beautiful visual introduction to the vast range of diversity among Indian pteridophytes. In generating the information for this detailed checklist, the authors have combined an extensive literature review with detailed studies of herbarium specimens and distributions from both public and private herbaria from India and elsewhere, and sound field knowledge. Critical comparison has been made with records from adjacent areas, and this has led to the inclusion of 34 families, 130 genera, 1107 species and 50 additional subspecies for the whole of the checklist, of which 23 families, 59 genera, 572 species and 32 additional subspecies are covered in Part 1. In addition, for each of the genera, the authors provide lists of excluded taxa; this is very valuable in clarifying the presence/absence of taxa for which past records have proved to be erroneous, often the result of confusion of nomenclature. The new checklist has a traditional taxonomic framework; the authors have considered recent taxonomic approaches built on phylogenetic analysis of DNA sequence data but have given greater weight to morphological classification. One of the strengths of this checklist is in the detailed citations for families, genera and species, including citation of types and synonyms, and information on distribution within and outwith India. For many of the species there are extensive notes to clarify previous reports, to point out mis-identified material or instances where there has been mis-application of names, sometimes in attempted lectotypification. This detailed clarification of the names used (and those excluded) in the checklist is invaluable for other researchers, and comparison can be made with, for example, fern taxa from elsewhere in Asia. In a taxonomic work of this detail it is not surprising to find that some 55 nomenclatural changes have been made, for example, Mickelopteris Fraser-Jenk., gen. nov. in the family Pteridaceae, and these changes can be identified readily as they are listed in bold in the taxonomically arranged index of accepted and excluded taxa at the end of Part 1. The authors are to be congratulated on a very welcome addition to current knowledge of the rich diversity of pteridophytes in Asia. FERN GAZ. 20(5). 2017 215

REFERENCES Fraser-Jenkins C.R., Kandel D.R. & Pariyar S. 2015. Ferns and Fern-allies of Nepal, Volume 1. pp 508. Kathmandu, Nepal. Wu, Z., Raven, P.H. & Hong, D. 2013. Flora of China, vols. 2-3, Lycopodiaceae through Polypodiaceae. Missouri Botanical Garden Press.

M. Gibby 216 FERN GAZ. 20(5). 2017


PAPERS should not usually exceed 20 printed pages and are generally expected to be considerably shorter. As well as reports of original research, review articles and short notes, e.g. new records, are encouraged. The senior author should supply an email address to facilitate correspondence. REVIEWS should cover recent developments in any aspect of research on ferns and lycopods. This can include unpublished research by the author, and/or the possible direction of future research. They should be aimed at the general reader who may not be an expert in the chosen topic. Proposals for a review should be discussed first with the review editors. MANUSCRIPTS should be submitted in English (British) in electronic format in 10-point Times New Roman font and double spaced. Electronic versions of text and tables should be compatible with WORD, with figures as pdf or jpg files, and sent as email attachments or CDroms. All manuscripts will be refereed. THE TITLE should reflect the content of the paper and be in BOLD CAPITALS (11-point) and centrally aligned. Generic and specific names should be in italics and any title containing a generic or specific name should be followed by the family name in brackets e.g. TRICHOMANES SPECIOSUM (HYMENOPHYLLACEAE) IN SOUTHERN SPAIN AUTHOR ABBREVIATIONS should follow Pichi Sermolli’s (1996) Authors of scientific names in Pteridophyta, Royal Botanic Gardens, Kew. MAIN HEADINGS: should be in BOLD CAPITALS (10-point) and centrally aligned. SUBSIDIARY HEADINGS: should be in bold, the first letter of each word in capitals, the rest in lower case and left-aligned. AUTHORS’ NAMES AND FULL ADDRESSES (including email address): follow the title and are centrally aligned. KEY WORDS: up to ten. ABSTRACT: should reflect the content of the paper. FIGURES: there is no distinction between photographs and line drawings in numbering. All should be presented in a form ready for reproduction, colour, TIF format and 300 dpi (please contact editor with queries), with a scale bar where appropriate. Lettering or numbers (Arabic) should be in the bottom left using uppercase Times Roman. Figure captions should be on a separate sheet. TABLES: can be printed in either portrait or landscape format. Authors should consider this when preparing tables. Authors should ensure that tables fit the printed page size in a legible form. PAGESIZE: The Gazette has a useable page size of 123 × 204 mm. This can be used in portrait or landscape mode but any supplied artwork should not be larger than this. MEASUREMENTS: follow the metric system. CHECKLISTS: follow the format of Baksh-Comeau, Fern Gaz. 16(1, 2): 11- 122. REFERENCES: follow the style of a recent issue of The Fern Gazette, e.g.:- HOOKER, W.J. 1864. Species Filicum, 5. Dulau & Co., London. MORTON, C.V. 1947. The American species of Hymenophyllum, section Sphaeroconium. Contr. U.S. Natl. Herb. 29(3): 139-201. STEVENSON, D.W. & LOCONTE, H. 1996. Ordinal and familial relationships of pteridophyte genera. In: CAMUS, J.M., GIBBY, M. & JOHNS, R.J. (Eds) Pteridology in perspective, pp. 435-467. Royal Botanic Gardens, Kew. JOURNAL ABBREVIATIONS: should follow Botanico Periodicum Huntianum & Supplements. Alterations from the original text at proof stage will be charged for unless they are minor points of detail. A pdf will be provided free to the senior author. FERN GAZ. 20(6):217-242. 2017 217

REVIEW -DEVonIan oRIgInS of fERnS anD lycophytES - What WE knoW, What WE nEED to fInD out

P.G. GENsEl Department of Biology, University of North Carolina Chapel Hill, NC 27599-3280, UsA Email: [email protected]

Key words: silurian-Devonian, lycophytes, basal , ferns evolution, phylogenetic relationships.

aBStRact This represents a synopsis of current knowledge of the siluro-Devonian fossil record concerning evolution of lycophytes and ferns. This is the time period when several taxa or lineages at different grades of organisation existed that may be informative about the origins of these groups or structures typical of these groups. Considerable new data, including earlier first appearances of lineages and plant structures, new data about siluro-Devonian lycopsids or basal euphyllophytes, and new whole plant reconstructions of small to tree-size plants in both lineages, have been published in recent years. It is not possible to be completely comprehensive, but the taxa discussed are either central to established ideas, or provide new information in relation to phylogenetic relationships and evolutionary trends. It remains difficult to trace the phylogenetic relationships of early plants relative to extant lineages. New data are reviewed which may be important in reassessing homology of characters and/or hypotheses of such relationships or in determining which taxa to exclude. Including fossils in estimates of relationships of these major lineages of plants will provide a more accurate and comprehensive understanding of the past history of seedless vascular plants.

IntRoDuctIon A consensus classification of extant seedless vascular plants by PPG1 (2016), reflecting molecular and some morphological phylogenies (smith et al., 2006b; Kenrick & Crane, 1997; Pryer et al., 2001; 2009; schneider et al., 2009; and others) recognises (with three families and collectively referred to as lycophytes) and a grade “euphyllophytes” which consists of two clades - seed plants and Polypodiopsida (Figure 1). The latter includes four subclasses, , , Marattiidae and Polypodiidae- all referred to as ferns. lycopodiopsida appears to represent a monophyletic group defined by both molecular and morphological characters that diverged at least in the mid-silurian if not earlier (Kenrick & Crane, 1997; Nickrent et al., 2000; Renzaglia et al. 2000; Pryer et al., 2001; 2004; Qiu et al., 2007; li et al., 2014) but see Hao and Xue (2013) for a different interpretation. Relationships of several putative stem lycophytes such as some Cooksonia pertoni, Aberlemnia (formerly Cooksonia) caledonica, Renalia and several others, some within more derived extinct clades where critical characters are missing, are poorly resolved (Kenrick & Crane, 1997; Gensel & Berry, 2001; Hao & Xue, 2013). Additional data and more whole plant reconstructions of several Upper silurian or lower Devonian polysporangiophytes are needed to address these issues. 218 FERN GAZ. 20(6):217-242. 2017

The term “fern” or “fern-like” now encompasses the majority of extant and extinct non-lycophytic seed-free vascular plants and several extinct “stem” euphyllophytes (trimerophytes), those with “peripheral permanent protoxylems” such as cladoxylopsids, iridopterids, and in some cases, rhacophytes and stauropterids (Kenrick & Crane, 1997; Rothwell & stockey, 2008; Rothwell & Nixon, 2006; Berry & stein 2000; Berry & Wang 2006). This results in a broad view of what represents a “fern”. This is tied up in considerations of evolution of the so-called megaphyllous leaf via modification of branch systems. However it is clear such leaves evolved many times and possibly from different parts of lateral branch systems. Furthermore, using criteria such as presence/absence of a leaf gap and simple vs branched venation to distinguish microphyll vs megaphyll is not clear-cut. For a more complete discussion of these issues, see Tomescu (2009) and later sections of this paper. The term megaphyll is intentionally not used in this paper. Fossils were intentionally excluded from the PPG1 classification, because “the phylogenetic affinities of most extinct plants are rather unclear” (p.565). While a practical starting point for names, it, along with molecular- derived phylogenies of only extant forms, implies misleading relationships by ignoring some of the major discoveries in the fossil record of the past several decades. For example, stating that ferns are the closest living relative to seed plants is correct when only living plants are considered, but ignores the existence of extinct plants with unique combinations of characters representing one or more now-extinct lineages, notably , but also others, indicating a more distant relationship between seed plants and ferns. While molecular-based phylogenies may suggest a close relationship among the extant subclasses, incorporation of fossil and morphological data (Figure 2) suggest they are not monophyletic (Rothwell & Nixon, 2006). The fossil record, while still incomplete, presently suggests each Polypodiopsida subclass (PPG1 2016) differs in time of first appearance and possibly in origin. First appearances of undoubted extinct members of each are as follows: equisetophytes -the late Devonian, filicaleans (extinct Polypodiidae) - Early Carboniferous, Marattiidae -late Carboniferous, and Ophioglossidae -Cenozoic. Thus time gaps exist between the later appearing taxa/lineages and putative early representatives of those clades. Finding morphologically based synapomorphies to unite extant and extinct putative progenitor

figure 1. A simplified phylogeny of ferns and lycophytes, showing only the major lineages, modified from Figure 1 of the Pteridophyte Phylogeny Group 1 (PPG1) study. GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 219 or sister lineages is difficult. Characters that have been commonly employed among the Devonian age euphyllophytes are 1) branching systems, from which leaves are believed to have evolved and part or all of which are often interpreted as “leaf-equivalents”, 2) stelar anatomy and 3) protoxylem location. sometimes interpretations are retrospective, with extant leaf morphologies influencing ideas of what constitutes a leaf-equivalent. Not everyone agrees on which part or parts of a branching system constitutes a leaf- equivalent in each lineage, and lastly, for some, all leaves are considered homologous (ignoring the fossil record) rather than evaluating the fossil plants within the context of their time and environment (Berry & stein, 2000) and taking a more “bottom-up outlook” (Rothwell & stockey, 2008) of extinct plants relative to extant ones. Recently acquired fossil data and whole- plant reconstructions, coupled with new developmental information or tests of certain character distributions, may improve assessment of homology of structures and character state transformations and ultimately phylogenetic relationships (Tomescu, 2009; Corvez et al., 2012 and authors cited therein). Additional work on understanding variation in anatomy also is needed. As our understanding of certain plant types improve, the fossil record may also indicate which possible progenitor candidates to preclude.

What DoES thE foSSIl REcoRD cuRREntly tEll uS?

figure 2. From Rothwell and Nixon, 2006 showing a tree based on a combination of fossil and extant plant morphological and molecular characters. Inclusion of fossils causes changes in postulated relationships of so-called “fern-like plants” relative to filicalean (=Polypodiidae) ferns. Terminology is that used by Rothwell and Nixon (2006). lignophytes refer to progymnosperms and seed plants. OP= ophioglossid ferns and psilotophytes. Palaeozoic leptosporangiate ferns include taxa such as Metaclepsydropsis, Ankyropteris, Psalixochlaena, Botryopteris, and others. 220 FERN GAZ. 20(6):217-242. 2017

figure 3. A stratigraphic chart showing major events in plant evolution or first appearances of certain plant groups or plant structures, spanning to Tournaisian. GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 221

gEnERalItIES 1) Everything happened earlier (Figure 3): Dispersed cryptospores and trilete spores provide evidence for Kingdom Plantae in the early Middle Ordovician to late Middle Ordovician respectively (Rubenstein et al., 2014; steemans et al., 2009; 2010; Wellman et al., 2008; 2015; Kenrick et al., 2012; Edwards et al., 2014, Edwards & Kenrick, 2015, and references cited therein). Cryptospore types and diversity increase until the earliest Devonian, trilete spores diversify from silurian on. Earliest forms occur in Gondwanan regions, suggesting radiation of cryptospore producers and basal polysporangiates from Gondwanan microcontinents towards laurussia (Wellman et al., 2015; steemans et al., 2010). The absence of megafossils during this time span may partly reflect the lack of known terrestrial sediments in the Ordovician and silurian (Kenrick et al., 2012; Wellman et al., 2015). some tiny Upper silurian to lower Devonian plants produce cryptospores, termed cryptophytes (Edwards et al., 2014), and others produce trilete spores, some possibly representing precursors of a bryophyte or polysporangiophyte grade (Edwards et al., 2014). Unfortunately, only some dispersed spores can be tied to parent plants, which limits establishing evolutionary/phylogenetic links between cryptophyte, bryophyte or polysporangiate lineages, and thus to either lycophytes or fern-like precursors (Edwards et al., 2014).

2) Innovations: The silurian to Middle Devonian vascular plant diversification demonstrates a greater increase in plant diversity, size and complexity than previously known. silurian to Early Carboniferous taxa exhibit a trend from considerable morphological/anatomical plasticity to more predictable body plans or architecture in younger, more derived forms (Bateman et al., 1998). Recent research suggests a transition from apparently rhizomatous forms to upright trees by the Middle to late Devonian among lycopsids and euphyllophytes (Figure 3), tree size ranging from 3 to at least 8m tall and of varying trunk diameters (4 to ~20 cm wide). some formed new vegetation associations, most notably early forests. In euphyllophytes, no laminate leaves and no, or limited, secondary tissues exist (thus having alternative means of mechanical support) (stein et al., 2007; 2012; Giesen & Berry, 2013; Berry & Marshall, 2015). Archaeopteridalean forests of large trees with abundant secondary tissues dominated some regions in the late Devonian. Evidence of early evolution of leaves and roots occurs from late silurian on, as discussed in greater detail later. Another major innovation, aligned xylem meeting criteria for secondary xylem and interpreted as an early type of vascular cambium, is demonstrated by the Pragian Amoricaphyton (Gerrienne et al., 2011; strullu-Derrien et al., 2013; 2014; Gerrienne & Gensel, 2015) and plants from eastern Canada (Gerrienne et al., 2011; Hoffman & Tomescu, 2013; Gensel in prep.). It is unknown if these possessed uni- or bifacial cambia. That, and lack of fertile remains, renders affinities and potential relevance as to how vascular cambia evolved or to fern and lycophyte vs. lignophyte evolution unclear. Uni- and bifacial vascular cambia originated more than once, as they are known in several extinct lineages from Middle Devonian on, including lycopsids (uni), progymnosperms (bi), early seed plants (bi), and possibly cladoxyls (unclear if secondary xylem or primary, aligned metaxylem). is established by the Middle Devonian in lycopsids and some euphyllophytes (the incipiently heterosporous lower Devonian Chaleuria; the Upper Devonian archaeopterids and some lower Carboniferous stauropterids). The Middle Devonian putative ovule precursor, Runcaria (Gerrienne et al., 2004) and numerous pre- 222 FERN GAZ. 20(6):217-242. 2017 ovules of late Devonian (Famennian) age indicate an initial radiation of early seed plants at nearly the same time as heterosporous euphyllophytes occurred (Prestianni & Gerrienne, 2010), but prior to appearance of all fern clades except Equisitidae.

lycophytE EVolutIon The presence of late Wenlock Cooksonia sp. from Avalonia (Edwards & Feehan, 1980), and the Upper silurian (ludlow) occurrences of several , putative stem lycophytes, and the lycopsid Baragwanathia from Bathurst Island, Canada and/or Australia (Kotyk et al., 2004; Rickards, 2000) indicate several grades and clades of rhizomatous, homosporous lycophytes at that time and an early origin for lycophytes. Zosterophylls, stem lycopsids, pre-lycopsids and lycopsids occur widely in the Early Devonian. strobilate probable zosterophylls such as Distichophytum and plants similar to Bathurstia first appear in the late silurian and similar forms are diverse in the Early Devonian. Zosterophyllopsids are leafless but exhibit similar anatomy and sporangial morphology and dehiscence as lycopsids. Kaulangiophyton, lacking vascularized leaves, Asteroxylon with transfusion tissue in “leaves” (Hueber, 1992), and fertile Drepanophycus spp. all lack association of sporangia with leaves, and sometimes are considered pre-lycopsids (Gensel & Andrews, 1984; Gensel & Berry, 2001). leaves of differing morphology occur in the siluro-Devonian Baragwanathia, in the pre-lycopsid Drepanophycus, the putative lycopsid Zhenglia (Hao et al., 2006) and in some Pragian taxa of uncertain affinity from China such as Adoketophyton (li & Edwards, 1992; Hao et al., 2003). Mid-Upper Devonian and younger lycopsids exhibit mostly laminate univeined lycophylls, some of which are much-divided (Protolepidodendrales) or have laciniate to fimbriate margins (e.g. Longostachys, Wuxia). Ideas about how lycophylls may have evolved vary but are unresolved, ranging from change via progressive vascularization of enations, sterilization of sporangia (Crane & Kenrick, 1997), or by reduction of branch systems (reviewed in Tomescu, 2009). Growth habit among these basal groups (stem taxa, zosterophylls, pre-lycophytes, early lycopsids) is either rhizomatous or tufted (Gensel et al., 2001; Raven & Edwards, 2001; Gensel & Berry, 2001; Hao et al., 2010). Rooting structures appear to have arisen more than once, ranging from downward trending axes (some Zosterophyllum spp. of earliest to early Devonian age), to part of a bifurcation of a rhizome (H or K branching), with one half being stem-like and bearing enations or leaves, the other portion lacking those structures and appearing root-like (Bathurstia, Drepanophycus - Gensel et al., 2001). An un-named lycopsid from the lower Devonian of differs in that the initial K-branch produces a “root-bearing axis”, from which dichotomizing root-like structures depart (Matsunaga & Tomescu, 2016). These authors suggest rooting structures in lycophytes are de novo structures rather than modified stems. Hetherington and Dolan (2017) note commonalities among lycopsid root morphology as well as disparities, recognizing multiple types of structures that bear the branched roots. None of the early forms are anatomically preserved, thus features that distinguish roots from stems (root cap, root hairs, etc) in younger plants are lacking (Raven & Edwards, 2001; Gensel et al., 2001; Kenrick & strullu-Derrien, 2014). The order Protolepidodendrales, plants characterized by much divided or hastate leaves, epiphyllous oval to possibly round sporangia, and faintly to deeply lobed protosteles, is established by the Emsian (Leclercqia spp.) and taxa are much more diverse and widespread in the Middle and early late Devonian (Gensel & Berry, 2001). GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 223

Most specimens may represent more distal or aerial branches and presumably were rhizomatous or in one case, climbing (L. uncinata Xu et al., 2011). Oval to elongate sporangia attach to the upper surface of identical to vegetative leaves. Leclercqia complexa from the Middle Devonian Gilboa, NY locality is known to be ligulate; L. complexa, L. cf. complexa and L. uncinata yield spores showing them to be homosporous (Grierson & Bonamo, 1979; Richardson et al., 1993; Gensel & Kasper, 2005; Gensel & Albright, 2006; Wellman et al., 2009; Xu et al., 2011). spores and ligules are unknown for other genera. Rooting structures, which might aid in better understanding of relationships, are unknown in all taxa. These plants are often considered to be putative precursors to the ligulate heterosporous clades selaginellales and , but better understanding of all plant parts would allow a more precise assessment of their relationship to other extinct and extant clades. As noted below, additional putatively transitional forms exist, although many are incompletely preserved. A major change in growth habit and reproduction occurred in the late Mid Devonian, as shown by the bipolar/pseudobipolar, approximately half metre tall tree-like taxa Longostachys and Chamaedendron (Cai & Chen, 1996; schweitzer & li, 1996), both of uncertain affinity, in China. In situ (and some isolated) lycopsid stems from the upper Middle/lower Upper Devonian of svalbard, consisting of approximately 1m tall, incomplete trunk pieces, and identified as Protolepidodendropsis pulchra, are interpreted as a paleotropical forest (present in a low paleolatitude) by Berry and Marshall (2015). Sublepidodendron, especially S. songziense Wang et al. 2003, from the late Devonian of China, was at least a metre tall. A smaller, very slender, upright and unbranched late Devonian lycopsid, Clevelandodendron ohioensis (Chitaly & Pigg, 1996), exhibits a lobed/branched base and a bisporangiate reminiscent of some lepidodendrids. Wider stemmed lycopsids such as Cyclostigma, Lepidosigillaria, and the unidentified tree lycopsid from Gilboa, NY (Haughton, 1859; Chaloner, 1968; Kräusel & Weyland, 1949; stein et al., 2012) also occur in late Mid to early late Devonian in laurussia. These taxa not only differ in size (length, width), but also in branching (presence/absence, type), leaf base patterns, leaves, rooting and reproductive structures. They also clearly demonstrate the existence of upright lycopsids of varying heights, diameters, and branching architecture by Middle to late Devonian time, coexisting with the presumably rhizomatous protolepidodendrids and presaging growth habits and reproductive strategies present among the abundant Carboniferous and younger lycopsid lineages. Various isolated anatomically preserved stem remains, when viewed in comparison to anatomy in more extensively preserved plants, also show differences in some features, not yet well understood. secondary tissues occur in lycopsids from the Middle Devonian. Tracheid wall pitting among different taxa varies from one or more of the following pitting types: scalariform, pitted, circular bordered pitted, scalariform with fibrils connecting the bars or pitlet sheets. Much remains to be learned about its possible taxonomic implications. Different types of stem bases with rooting structures occur among these taxa. Longostachys and Chamaedendron stems (and possibly Clevelandodendron) terminate in a series of downward trending, bifurcating root-like structures, but details are poorly understood. The two major rooting structures of arborescent isoetalean lycopsids dominant in the Carboniferous occur by the Middle Devonian. The earliest records of cormose lycopsid bases may be those recorded by Xu and Wang (2016) from the late Middle-late Devonian Hujiersite Fm, China and the bases of the more extensively preserved Protolepidodendropsis pulchra, of similar age from svalbard (Berry & 224 FERN GAZ. 20(6):217-242. 2017

Marshall, 2015). late Devonian Leptophloem and Otzinachsonia also exhibit cormose bases. A more branched, stigmarian type rooting system (Wang et al., 2003) occurs in Sublepidodendron spp from China. laurussian forms are less easy to interpret. Pigg (2001) suggests that cormose and stigmarian forms represent variations of a single rooting type, perhaps being influenced by ecological factors. some of these Devonian lycopsid stems or branches bear sporangia aggregated into strobili, ranging from mono- to bisporangiate (Longostachys, Chamaedendron, Sublepidodendron, Cyclostigma), while others apparently have alternating fertile and vegetative zones (Monilostrobus Wang & Berry, 2003; Wuxia Berry et al., 2003), as also occurs in younger tree lycopsids. The Upper Devonian Wuxia is interesting in that megasporangiate “cones/fertile zones” occur just below branching points of axes but presumed microsporangiate ones are terminal (Berry et al., 2003). Hoxtolgaya robusta Xu et al. (2012), Middle Devonian of China, is interpreted as arborescent based on 9cm wide stems of unknown length, and exhibits sporangia yielding spores of one size, interpreted as homospory, but preservaton is too incomplete to determine with certainty. In strobili or fertile zones, sporophylls are either identical to vegetative leaves, or modified with a broad fimbriate to spiny base and distal lamina, as in the Middle to Upper Devonian small tree-like lycopsids such as Longostachys (Cai & Chen, 1996), Chamaedendron (schweitzer & li, 1996) or Wuxia (and others) (Berry et al., 2003). Berry et al. (2003) note that variation in fertile regions/structures and in stem anatomical features occurs earlier and more broadly in China than laurussia, suggesting that perhaps China may have been an important area of early evolutionary innovation in clade(s) from which the major lineages of arborescent, heterosporous lycopsids of the Carboniferous and later arose. More whole plant reconstructions of several of these lycopsids are needed, and particularly, clarification of the organisation of basal regions, presence/absence of branching, anatomical features, and type of reproductive structures among individual plants, in order to better understand their relationships to both earlier and later occurring lineages to resolve relationships of these several plants and other taxa all considered as part of the isoetalean clade. The record of herbaceous, homosporous lycopsids (e.g. lycopodiaceae) is far less well known in the Devonian and Carboniferous. Lycopodites oosensis from the late Devonian of Germany (Kräusel & Weyland, 1937) may represent one, but should be restudied. The lower Carboniferous herbaceous form Hestia possesses too few characters to assess affinities (Bateman et al., 2007) but might represent an eligulate form. Younger fossils have been assigned to the genus Lycopodites but it is difficult to determine the affinities of these leafy stems; one has been found to represent conifer twigs, others exhibit a mix of lycopodiacean and sellaginellacean characters but are usually allied to selaginellaceae (skog & Hill, 1992; Thomas, 1992). Rowe (1988) identified a rhizomatous plant from the late Early Carboniferous Drybrook sandstone as an early selaginallalean, Selaginellites resimus, but little is known about early evolution of selaginellales. several questions concerning early lycophyte evolution remain or are only partially answered by recent data. The paraphyly of some Zosterophyllum species and stem lycophytes remains unresolved despite new data. Much remains to be clarified about overall construction and relationships of the numerous Middle-late Devonian protolepidodendraleans and small tree lycopsids, as discussed above, including how many times have structures such as strobili, ligules, rooting structures, and leaf abscission arisen and in what combinations? How do they relate to later-occurring lineages? And GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 225 again, questions remain about the early history of herbaceous, eligulate, homosporous lycopodiaceae and ligulate, heterosporous selaginellaceae through much of the Devonian and Carboniferous and even early Mesozoic.

EuphyllophytE EVolutIon Extinct Devonian plants included in the Euphyllophyta mostly exhibit much branched aerial axes, some terminated by mostly fusiform sporangia. Anatomy consists of various protosteles (haplo-, actino-, dissected). Pertinent to fern evolution are stem euphyllophytes, such as Psilophyton and Pertica, the predominantly Mid-Upper Devonian (=Pseudosporochnales [or Cladoxylales] + Iridopteridales), the enigmatic, but putative iridopterid Rhacophyton, and possibly stauropteridaleans. The latter groups have been considered by many authors to be derived from a trimerophyte ancestor, and possible fern-like or fern precursors. Reasons vary; some derive from cladistic studies, others from older comparative studies. While lacking laminate leaves, either the whole or part of their lateral branch systems (lBs) are interpreted as showing possible stage(s) in the evolution of a leaf, as mentioned earlier (Kenrick & Crane, 1997; Gensel et al., 2001; Boyce & Knoll, 2002; sanders et al., 2007; Tomescu, 2008; Galtier, 2010; Corvez et al., 2012). These occurred simultaneously but at different rates in different lineages (Galtier, 2010), and are not homologous within ferns or between “ferns” and seed plants. secondly, some of the above taxa exhibit peripherally located protoxylem in various types of actino- or dissected protosteles (where anatomy is known) that differs from that present in aneurophytalean progymnosperms and early seed plants. The latter, and some basal euphyllophytes, exhibit radiate protoxylem (Beck & stein, 1993), in which actinosteles or variants thereof have a central protoxylem, which appears to extend out each arm of the stele to produce lateral traces. These are considered mesarch but this depends on whether the protoxylem at the tips of arms persist, a feature presently not well understood. Anatomy of this type is typical of aneurophytalean progymnosperms, possibly some of the larger (Pertica– grade) trimerophytes, stenokolealeans, and other plants of uncertain affinity. However, it remains unclear if these two protoxylem patterns represent a good indicator of phylogenetic relationships. For example, scheckler et al. (2006), in a re-study of Langoxylon stockmans, a taxon of uncertain affinity, point out that Langoxylon stems possess a mixture of these two characters. Pseudosporochnus hueberi lacks a central protoxylem, but exhibits peripheral protoxylems plus several separate protoxylems along the midplane of an arm (stein & Hueber, 1989), thus not all early euphyllophytes can be so easily categorized. The term “peripheral loop”, initially referring to an anatomical feature of Carboniferous zygopteridalean petioles, has been used to describe protoxylem features in many Devonian plants. As originally defined, a peripheral loop consists of a central rod of parenchyma surrounded by protoxylem (leclercq, 1970) located near the margin of an elongated . similar structures are termed the same in stems of Devonian taxa. several studies show that most of the latter represent protoxylem lacunae, resulting from break-down of thinner walled protoxylem during development or represent a different organisation of protoxylem with associated parenchyma (scheckler, 1974; scheckler et al., 2006; stein, 1982). This suggests that the presence of a so-called peripheral loop should be carefully evaluated in any Devonian plant, and is not phylogenetically useful. Furthermore, care should be taken in use of the term “clepsydroid” and in any phylogenetic implications that might follow, because strap- 226 FERN GAZ. 20(6):217-242. 2017 shaped strands in main axes or lateral branch traces in Devonian plants expand at their ends in preparation for producing the next higher order traces, thus appearing similar to the dumbbell-shaped petiole trace so termed among some early ferns. The earliest putative laminate leaf may date from the Pragian Eophyllophyton (Hao, 1988; Hao & Beck, 1993), considered basal and sister to other euphyllophytes by Hao and Xue (2013) but considered of uncertain affinity by this author. While laminate leaves are absent in most other coeval taxa, they occur in the Early to late Devonian Platyphyllum (=Flabellifolium?) and the late Devonian Ellesmeris (Hill et al., 1997), enigmatic Shougangia bella (Wang DM et al., 2015), archaeopteridaleans and early seed plants, appearing earlier in time in lignophytes than in filicales (Galtier, 2010). Clearly, resolution of what constitutes homologous structures in lBss among these different much- branched plants, and more documentation of the first appearance of laminae, would address remaining questions concerning number of times and modes of leaf evolution in these groups and non-homology of so-called megaphylls in these groups.

claDoXylS, IRIDoptERIDS anD RhacophytES- callED “fERn- lIkE” But? starting in the Mid-Eifelian and extending through the late Devonian, several taxa referable to Cladoxylopsids s.l. (e.g. including iridopterids, Berry & stein, 2000) and rhacophytes are part of a more complex vegetation, some of the former representing small to large trees recently described as major components of in situ Middle to early Upper Devonian forests in the UsA and Germany (stein et al., 2007; 2012). Concomitantly, major revision of taxon concepts for Pseudosporochnus (Berry & Fairon- Demaret, 1997; 2002), Calamophyton (now includes Duisbergia, Cladoxylon, and most species of Hyenia - Fairon-Demaret & Berry, 2000; Berry, 2005; Giesen & Berry, 2013) and Eospermatopteris/ (stein et al., 2007; 2012) has resulted in critical new information about the architecture of these leafless trees. pseudosporochnaleans Pseudosporochnus trees (Figure 4) produce rows of branches that abscise at their base; the branches are digitately divided and produce higher-order, divided laterals that are three-dimensional to subopposite and alternate, final divisions being vegetative and non- laminate or fertile (Berry & Fairon-Demaret, 2002). Calamophyton is a smaller tree, with a swollen base, stems enlarging near apex and bearing vertical rows of lateral branch systems that then divide digitately (Figure 5), and produce higher order helically arranged dichotomizing vegetative or fertile non-laminate units. Older branch systems break off above their base, leaving the Duisbergia type morphology (Giesen & Berry, 2013). Eospermatopteris is larger, with a swollen base, a wide stem, and crown of digitately divided branch systems of the Wattieza type (stein et al., 2007; 2012), abscising at their base. Terminal fusiform sporangia are known for all of these, being either recurved (Calamophyton, Wattieza) or erect (Pseudosporochnus). These apparently represent the earliest tree architectural types among euphyllophytes, being similar to some palms, and tree ferns (stein et al., 2007) in stem and root structure. Where anatomy is known in the pseudosporochnaleans, it varies from being a multistranded, anastomosing stele, producing similar but smaller multi-stranded steles in lateral branches, as in Calamophyton or Pseudosporochnus (Figure 6), to being comprised of individual radially oriented strands near the periphery of the stem adjacent GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 227

figure 4. A recent reconstruction of the cladoxylopsid Pseudosporochnus, showing its crown of relatively large branch systems, each of which is closely dichotomous so as to appear digitately divided. Reproduced from Berry and Fairon-Demaret, 2002, Figure 9, Int. J. Plant sci. 163(5) 699-713. © 2002 by The University of Chicago. 228 FERN GAZ. 20(6):217-242. 2017

figure 5. Calamophyton primaevum. Drawing of a stem showing two features- the remnant bases of branches along the stem and parts of the attached, digitately arranged branches at the crown. B. suggested anatomy of Calamophyton (Duisburgia) based on Mustafa (1978) showing primary xylem in black, surrounded by aligned, possibly secondary xylem (lines). Redrawn from Giesen and Berry, 2013; stem is from Figure 5a (in part), anatomy redrawn from Figure 17d. GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 229 to a large pith in main trunk (Figure 5) as in the presumed base of Calamophyton stem (Giesen & Berry, 2013) and Eospermatopteris/Wattieza (Boyer & Matten, 1996). Opinion differs as to whether some of these plants exhibit a limited amount of aligned metaxylem or secondary tissues. Coeval progymnosperms possess a eustele and bifacial vascular cambium. Habit, branching, aspects of rooting and internal construction of the large pseudosporochnalean cladoxylopsids might mean they already had diverged to the extent that they have little relationship with extant polypodiid or equisetid lineages, but instead represent a distinct lineage that flourished globally, then went extinct without any survivors (see Rothwell & stewart, 1993).

Iridopteridaleans Numerous Middle Devonian plants from laurussia, , and China comprise another lineage of Middle to Upper Devonian euphyllophytes, the Iridopteridales (stein, 1982; Fu et al., 2011) based on anatomy, morphology, or a combination of these preservation forms (Figure 7). These plants as presently known are smaller than cladoxylopsids, consisting of stems with a deeply ribbed mesarch actinostele comprised of two to three centrally united arms, each arm dividing one or more times to form six to twenty arms/ribs depending on the taxon. A single protoxylem region exists near the end of each arm (Figure 7 A-C). Branching pattern is essentially whorled, each protoxylem pole emitting a trace that supplies either a lateral branch or a smaller dichotomous appendage, the two types occurring in the same whorl (Figure 7D). sometimes branching occurs in only part of a whorl, with successive partial whorls offset (stein, 1982). sporangia terminate ultimate dichotomizing appendages borne from second order branches- some upright and some recurved (Berry &stein, 2000, Compsocradus). Other, often less extensively preserved, taxa attributed to the Cladoxylopsida are not treated here. Relationship to Polypodiidae is unclear; depending

figure 6. Anatomy of a lateral branch system of Pseudosporochnus, which is characteristic of many cladoxylopsids; the vascular bundles are arrayed in a manner interpreted as representing the beginning of a dichotomy. Redrawn from stein and Hueber, 1989. 230 FERN GAZ. 20(6):217-242. 2017 on what combination of characters are selected, they may be closer to Equisetidae; if others, of uncertain affinity (stein et al., 1984; Berry & stein, 2000).

Rhacophyton and putative relatives Rhacophyton (several spp) exhibits some characters that presage a fern type of organisation, but also possesses secondary tissues and some morphology reminiscent of progymnosperms or iridopteridaleans. somewhat rhizomatous to upright axes (?stems) bearing adventitious roots in R. ceratangium (Cornet et al., 1976) produce single or paired much divided lateral branch systems which overall appear three dimensional, with more two-dimensional second order axes. The secondary axes depart oppositely, and range from dichotomous to slightly pseudomonopodial. Fertile lateral branch systems are more complex, each pair with one vegetative and one fertile unit. The vegetative segments are elongate and bear divided higher order non-laminate ultimates. The fertile units are much divided and recurved, forming rounded masses of dividing axes terminating in fusiform sporangia with elongate tips. Anatomy of less extensively preserved, more upright appearing stems of R. zygopteroides was described by leclercq (1951) from poorly preserved specimens, and interpreted as an actinostele with unequally sized arms, although these sections have deterioriated so this cannot be confirmed. similarly arranged lateral branches produce clepsydroid shaped steles. Both were described as surrounded by secondary xylem. In R. ceratangium, both main axis and lateral branches are considered to have produced an elongated clepsydroid-shaped primary xylem, surrounded by secondary xylem with rays (Cornet et al., 1976; Dittrich et al., 1983). Traces to higher order axes also are known. R. condrusorum schultka (1978) is similar. Ellesmeris Hill et al. (1997) exhibits a simpler, but similar overall architecture, although the ultimate units are laminate, and fertile structures unknown. Anatomy is not intact, but the authors suggest main axis and lateral branch produce an elongate haplostele, with protoxylems near the tips of the arms. Knowing if all orders of branching possessed the same type of stele, or alternatively that stelar configuration changed from radial to

figure 7. line drawings of anatomy and stem portion of taxa included in Iridopteridalean cladoxylopsids. A. amphikoma; B. Iridopteris eriensis; C. Arachnoxylon kopfii; D. Portion of stem and branching structure of Anapaulia moodyi; whorls of branches alternate with dichotomizing structures similar to ultimate branchlets; each lateral branch bears second and ultimate order branches. A-C redrawn from stein, 1982. D redrawn from Berry and Edwards, 1996. GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 231 bilateral from main axis to lateral branch is of some significance in considering relationships and what to interpret as stem vs. leaf in these plants. For the present they usually are considered a separate lineage, Rhacophytaceae, although some authors previously placed them as a basal member of the zygopteridaleans and others consider them more allied to progymnosperms. Berry and Wang (2006) suggest, intriguingly, that Rhacophyton shares some characteristics of iridopteridaleans.

StEm EuphyllophytES anD RElEVancE to EVolutIon of latER foRmS Trimerophyte grade euphyllophytes exhibit either dichotomous, anisotomous or strongly pseudomonopodial branching of varying degrees of predictable cladotaxy. some of these terminate in fusiform sporangia, ranging from loosely (Psilophyton-grade plants) to densely (Pertica) clustered. Anatomy is known from parts of aerial systems of some Psilophyton species, while anatomy is unknown in published Pertica species. However, Gothanophyton and some undescribed plants at the Pertica-grade provide data about possible anatomical variation in these entities, showing some differentiation between ribbed main axes and lateral branches, and possibly indicating evolutionary trajectories different from Pertica, although affinities of most of these are poorly understood.

Psilophyton-grade basal euphyllophytes Psilophyton Dawson (Banks et al.) Doran (1980), initially based on dichotomous to slightly pseudomonopodially branched axes, where anatomically preserved, exhibits a haplostele with centrally located protoxylem (centrarch) with little or no differentiation between main axis and lateral branch traces (Figure 8A). It thus appears the least specialized of stem euphyllophytes, but that may be deceptive. At the same time, plants of this type may be more closely related to polypodiids, or lineages leading to polypodiids and equisetids, than many later-appearing taxa. Over 14 species of Psilophyton are now recognized, some based on morphology, one from anatomy only (P. coniculum) and others with both, although anatomy so far is not extensively known in most species. Based on current knowledge, preservation ranges from nearly complete plants (P. crenulatum) to more fragmentary remains. Phylogenetic analyses involving more than one species of Psilophyton indicate the genus is not monophyletic (Kenrick & Crane, 1997; Hao & Xue, 2013) and comparative evaluation of known species further suggests the possibility that these species might represent more than one taxon. Psilophyton crenulatum Doran (1980), an apparently rhizomatous plant with upright branch systems, exhibits dense regions of mostly vegetative branching, some of which appear immature (apices present) while more distally, spacing of vegetative and fertile lateral branches varies. Psilophyton dawsonii, based mainly on anatomy of a 2.5 cm long specimen (Banks et al., 1975), possesses regions of closely spaced vegetative lateral branches. The more distally located fertile lateral branches exhibit a trace suggested to differ in shape from vegetative ones (Banks et al., 1975). some short dichotomously divided lateral appendages are considered possible aphlebiae or roots. These features are not known for any other Psilophyton species, including P. coniculum (Trant & Gensel 1985), known only from anatomy (no sporangia attached). In this species, more closely spaced branching occurs mainly in lateral branches. Comparison of the many other Psilophyton species indicates that relative distribution of vegetative vs fertile regions is unknown, partly because the genus is best recognized 232 FERN GAZ. 20(6):217-242. 2017 morphologically from fertile specimens. In many instances the number of specimens available or mode of preservation limits information. Where numerous specimens are available, study shows branching is variously spaced. Frequently, lateral branches are incomplete and finding completely vegetative branch systems is difficult. It was previously questioned whether certain previously described species of Psilophyton conform sufficiently to the concept of the genus. P. kräuselii, from the Middle Devonian of Bohemia (Obrhel, 1959) is too poorly preserved to determine affinity, being based on fragmentary fertile and sterile remains (Hao & Xue, 2013; this paper). Hao and Xue (2008) also question the generic identity of P. szaferi, P. striatum (Wang & Berry, 2001) and P. dapsile (Kasper et al. 1974) because they are either too poorly known or lack twisted sporangia. Examination of P. dapsile sporangia in the author’s collection suggest that while small, they do show some torsion. Psilophyton szaferi, described by Zdebska (1986) from the lower Devonian of Poland, consists of a portion of a lateral branch system with upright sporangia borne in loose clusters more similar to those in Pertica and should be restudied. Psilophyton microspinosum from the Early Devonian of Maine (Kasper et al., 1974) exhibits a more rigid pattern of lateral branches and erect, untwisted sporangia, suggesting its affinity should be re-evaluated. The few morphologically preserved Pragian Armoricaphyton and the Emsian unnamed plant from Canada (Gerrienne et al., 2011; strullu-Derrien et al., 2014; Gerrienne & Gensel, 2015) with an early type of secondary tissue are similar in stem

figure 8. Diagrammatic drawings of small basal euphyllophytes; predominant mode of branching shown above, anatomy below. Axes approx. 2-5 mm. A. a generalized drawing of branch system of Psilopyton. Anatomy a centrarch haplostele, producing circular (or possibly square, if fertile) lateral branches. B. Unnamed plant from Emsian, New Brunswick (Gensel in prep). Ribbed, twice branched axes known. some stems with elongate haplostele producing lateral trace, others indicate same type of haplostele surrounded by secondary xylem. C. Armoricaphyton chateaupannense, Pragian, France (Gerrienne et al., 2011; Gerrienne and Gensel, 2016). Ribbed, twice branched axes. Primary xylem a round haplostele, surrounded by secondary xylem. Basal part of lateral branch with secondary xylem, more distal region without. Dark circles or lines in center of steles indicate protoxylem, white area primary xylem, lines = secondary xylem. GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 233 diameter and branching, to Psilophyton (Figure 8 B, C). No fertile remains are known. All of these taxa, along with Pertica type trimerophytes showing preserved anatomy, exhibit P-type tracheid wall patterning sensu Kenrick and Crane, 1997 (Gensel, unpublished data). This character may be less useful at lower taxonomic levels, as the combination of characters evident in these plants suggest broader disparity among these early simpler plants than previously realised. Morphologically preserved Middle Devonian plants such as Planatophyton (Gerrienne et al., 2014), Pauthecophyton (Xue et al., 2012), and Tsaia (Wang & Berry, 2001) are similar to Psilophyton in size and some aspects of branching but differ either in specifics of branching, sporangial orientation and/or dehiscence mode. These may represent distal regions of much larger plants or, alternatively, fragments of plants similar to Psilophyton in size. Additional study of these plants may reveal features useful in determining if they represent one, or several, lower to Middle Devonian genera and/or lineages and clarify their postulated phylogenetic relationships. larger basal euphyllophytes similar to Pertica Pertica and similar-appearing plants (Figure 9A-C) are taller and more robust in width, with a dominant main axis and variously arranged lateral branches. Pertica spp. exhibit mostly isotomously divided lateral branches with terminally borne, closely spaced, erect fusiform sporangia. Anatomy is unknown in published Pertica species. A new genus, initially considered Pertica-like but definitely an unrelated taxon (Figure 9A), has a dominant main axis, helically arranged lateral branches that are single or paired, and, in contrast to Pertica, dichotomously- divided ultimate units located along their length, some sterile and some terminating in fusiform sporangia. Main axis anatomy is an actinostele (usually) with a single central protoxylem and a protoxylem near the end of each arm (Gensel, 1984). The lateral branch trace(s) are ovoid. There is no evidence of secondary xylem. An undescribed plant from the lower Devonian of New Brunswick is similar to Pertica spp. in morphology (Figure 9B), with a dominant main axis, helical lateral branches, and dichotomous laterals terminating in tight clusters of fusiform sporangia (Gensel, in prep.). Anatomy is unusual, consisting of a “winged” stele in which four major arms emanate from an elongate central region, but with one arm dividing further (Figure 9B). At least two types of lateral branch traces are formed, differing from main axis stelar configuration, but preservation is incomplete. Do other perticas have similar anatomy or might there be variation? Gothanophyton Remy et Hass (Figure 9 C), with a 1cm wide main axis and single or paired lateral branches, exhibits a “winged” stele, consisting of an elongated central region with two major arms extending from either end (Remy & Hass, 1986a). One or more of the arms divide again, so that 4-7 armed steles are known. Protoxylems are located at either end of the central region near departure of major arms and near the end of each arm (Figure 9C). shape and/or lobing of lateral branch traces differ from the main axis configuration. Morphology is poorly preserved, but the authors suggest lateral branches departed singly, in pairs or in threes. Much more needs to be learned about this complex lower Devonian plant. In all of these plant types, protoxylems emanate from one or more of the main axis arms to supply lateral branches and only primary xylem is present. Architecture of these plants is morphologically and anatomically plastic, although with some common patterns, but evidence is building that these taxa exhibit enough differences to not be closely 234 FERN GAZ. 20(6):217-242. 2017

figure 9. Diagrammatic drawings of larger basal euphyllophytes, similar in size to Pertica (main axes approx. 1 cm) - note that none show evidence of secondary xylem. A. Plant described by Gensel (1984), with large main axis, single or paired lateral branches, dichotomously divided ultimates arranged along lateral branches. Anatomy a three-lobed stele with central protoxylem and protoxylem at end of each arm. First order lateral trace (or pair of traces) oval, second order trace circular. (Gensel, in prep) B. A Pertica-like plant with large main axis, helically arranged predominantly dichotomously divided lateral branch, terminating in bunched sporangia (not shown). Anatomy consists of slightly elongate central region from which four arms emanate, one of these divides again to produce five arms total. Protoxylem located at each end of central area, and ends of arms. First order traces are elongate to v-shaped or oval and in pairs as shown. Gensel (in prep). C. Gothanophyton zimmermanni Remy and Hass. large main axis with single or paired lateral branches (poorly preserved), bearing at least second order ones. Main axis stele consists of an elongate central region from which 4-7 arms emanate. Protoxylem located at each end of central region, along and at ends of arms. lateral traces change shape after departure to become 4-lobed. Protoxylem in black. Clear area in stele is primary xylem. C. redrawn from Remy and Hass, 1986. GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 235 related. They are similar in showing a change in shape between main axis and first-order lateral branches which conforms more closely to that seen in many ferns or extinct fern- like plants than anything known in progymnosperms, despite apparently comparing more closely to the radiate protoxylem group. Basal regions or rooting structures for Psilophyton and the taller euphyllophytes mentioned above are unknown. The fossils appear to represent aerial shoots extending from a rhizome, but it also is possible that some represent the main part of a non- rhizomatous plant that may have had a basal rooting system. Fairly wide, branched but detached rooting structures running perpendicular to bedding surfaces occur in sediments near some of the larger taxa in northern New Brunswick, while one long, downward extending axis was discovered by Elick (1998) in Gaspé which may represent a rooting structure.

Basal euphyllophytes incertae sedis several upper lower Devonian plants (late Emsian) have been excluded from phylogenetic studies, largely because their anatomy is unknown and branching pattern and other features are unique. These include Oocampsa catheta, interpreted as a trimerophyte- intermediate (Andrews et al., 1975), Chaleuria cirrosa (Andrews et al., 1974) and an undescribed plant with similar branching architecture from northern Maine (Gensel, in prep). They all exhibit a robust, fairly long, greater than 1 cm wide dominant main axis with densely spiraled lateral branches. In Oocampsa, higher order branches are several times irregularly dichotomous, bearing clusters of ovoid sporangia. Chaleuria and the new plant from Maine exhibit isotomous second-order branches, some terminating in fusiform to ovoid sporangia. Basal regions and thus growth habit(s) are unknown. One might expect either a multilobed or cladoxyl- like stelar configuration based on the major branching patterns. Oocampsa differs from the other two in higher order branching and sporangial morphology, while the other two represent one or more as yet unrecognized lineages, or may be early members of one of the later- occurring lineages such as cladoxyls or iridopterids. stenokolealeans are still poorly known, but recent studies indicate a possible affinity to early seed plants (Moment et al., 2016).

SummaRy significant new data now exist concerning the time of appearance of plants and morphology and anatomy of some taxa. Exceptionally preserved plant remains have indicated an earlier advent of plants, plant structures and habit, including transitions to upright, even arboreal types by the Middle Devonian. limited evidence indicates a wider array of growth strategies among these. Although the euphyllophyte and lycophyte lineages separate very early in time, with each demonstrating acquisition of major plant structures, gaps in preservation preclude establishing evolutionary trends or clear phylogenetic relationships within each lineage, especially early vs later Devonian plant types, and between Devonian and Carboniferous or younger lineages that appear more directly related to extant ferns or lycophytes (similar situation for seed plants). As whole plant reconstructions are made and new approaches employed, with new discoveries or re-investigation of earlier material, assessment of character homology will be more possible. several putative candidate lineages leading to equisetids or polypodiids remain, but it is clear that some, such as cladoxyls, are already highly specialized in development 236 FERN GAZ. 20(6):217-242. 2017 and construction by the Middle Devonian. Were more basal taxa similar, as suggested by Lorophyton Fairon-Demaret et li (1993), Foozia minuta Gerrienne (1992) or perhaps as yet unrecognized taxa? Basal euphyllophytes at the Psilophyton, Pertica, and Gothanophyton grade are increasingly interesting in that it is among plants of this type that early differentiation of anatomical configurations of main axis- lateral branch is established. Within this group one might possibly be able to discern early divergence of the fern sensu lato vs seed plant lineages. Further examination of anatomy and organisation of lateral branch architecture, incorporating signals of developmental differences, may aid in recognizing early stages in evolution of leaves- these clearly will be different in various lineages. similarly, the questions of transitions between predominantly rhizomatous and upright habit, and between types of rooting and of organisation of reproductive structures in both major lineages may become clearer with future discoveries or new approaches.

REfEREncES ANDREWs, H.N., GENsEl, P.G., & FORBEs, W.H. 1974. An apparently heterosporous plant from the Middle Devonian of New Brunswick. Palaeontol. 17: 387-408. ANDREWs, H.N., GENsEl, P.G. & KAsPER, A.E. 1975. A new fossil plant of probable intermediate affinities (Trimerophyte-Progymnosperm). Can. J. Bot. 53: 11719-1728. BANKs, H.P., lEClERCQ s., & HUEBER, F.M. 1975. Anatomy and morphology of Psilophyton dawsoni, sp. n. from the late lower Devonian of (Gaspé) and Ontario, Canada. Paleontographica Americana 8 (48): 77-127. BATEMAN, R.M., CRANE, P.R., DIMICHElE, W.A., KENRICK, P., ROWE, N.P., sPECK, T., sTEIN, W.E. 1998. Early Evolution of land plants: Phylogeny, physiology, and ecology of the primary terrestrial radiation. Ann. Rev. Ecol. syst. 29: 263-92. BATEMAN, R. M., KENRICK, P. & ROTHWEll, G.W. 2007. Do eligulate herbaceous lycopsids occur in Carboniferous strata? Hestia eremosa gen. et sp. nov. from the of Oxroad Bay, East lothian, scotland. Rev. Palaeobot. Palynol. 144 (3): 323-335. BECK, C.B. & sTEIN, W.E. 1993. Crossia virginiana gen. et sp. nov., a new member of the stenokoleales from the Middle Devonian of southwestern . Palaeontographica B229: 115-134. BERRY, C.M. 2005. ‘Hyenia’ vogtii Hoeg from the Middle Devonian of spitsbergen- its morphology and systematic position. Rev. Palaeobot. Palynol. 135(1-2), pp. 109- 116. BERRY, C. M. & FAIRON-DEMARET, M. 1997. Reinvestigation of the cladoxylopsid Pseudosporochnus nodosus leclercq et Banks from the Middle Devonian of Goe, Belgium. Int. J. Plant sci. 158(3), pp. 350-372 BERRY, C. M. & sTEIN, W.E. 2000. A new iridopteridalean from the Devonian of Venezuela. Int. J. Plant sci. 161(5), pp. 807-827. BERRY, C. M. & FAIRON-DEMARET, M. 2002. The architecture of Pseudosporochnus nodosus leclercq et Banks: a Middle Devonian cladoxylopsid from Belgium. Int. J. Plant sci. 163(5), pp. 699-713. BERRY, C. M. & WANG, Y. 2006. Eocladoxylon (Protopteridium) minutum (Halle) Koidzumi from the Middle Devonian of Yunnan, China: an early Rhacophyton-like GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 237

plant? Int. J. Plant sci. 167(3), pp. 551-566. BERRY, C. M. & MARsHAll, J.E.A. 2015. lycopsid forests in the early late Devonian paleoequatorial zone of svalbard. Geology 43(12): 1043-1046. BERRY, C. M., WANG, Y. & CHONGYANG, C. 2003. A lycopsid with novel reproductive structures from the Upper Devonian of Jiangsu, China Int. J. Plant sci. 164(2), pp. 263-273. BOYCE, C.K. & KNOll, A.H. 2002. Evolution of developmental potential and the multiple independent origins of leaves in Paleozoic vascular plants. Paleobiology 28(1): 70-100. BOYER, J. s. & MATTEN, l.C. 1996. Anatomy of Eospermatopteris eriana from the Upper Middle Devonian (Givetian) of New York. Int. Org. Palaeobot. 5, 13 (1996). CAI C-Y & CHEN, l-Z. 1996. On a Chinese Givetian lycopod, Longostachys latisporophyllus Zhu, Hu and Feng, emend.: its morphology, anatomy and reconstruction. Palaeontogr. Abt B. 1996; 238: 1–43. CHAlONER, W. G. 1968. The cone of Cyclostigma kiltorkense Haughton, from the Upper Devonian or Ireland. J. linn. soc. london (Botany) 61: 25-36. CHITAlY, s. & PIGG, K.B. 1996. Clevelandodendron ohioensis, Gen. et sp. Nov., A slender upright lycopsid from the late Devonian Cleveland shale of Ohio. Am. J. Bot. 83(6): 781-789. CORNET, B., PHIllIPs, T.l. & ANDREWs, H.N.1976. The morphology and variation in Rhacophyton ceratangium from the Upper Devonian and its bearing on frond evolution. Palaeotographica B 158: 105-129. CORVEZ, A., BARIEl, B. & DUBUIsssON, J-Y. 2012. Diversity and evolution of the megaphyll in Euphyllophytes: phylogenetic hypotheses and the problem of foliar organ definition. Comptes Rend. Palevol. 11: 403-418. CRANE, P.R. AND KENRICK, P. 1997. Diverted development of reproductive organs: a source of morphological innovation in land plants. Plant syst. & Evol. 206 (1997): 161-174. DITTRICH, H.s., MATTEN, l.C. & PHIllIPs, T.l. 1983. Anatomy of Rhacophyton ceratangium from the Upper Devonian (Famennian) of . Rev. Palaeobot. Palynol. 40: 127-147. DORAN, J.B. 1980. A new species of Psilophyton from the lower Devonian of northern New Brunswick, Canada. Can. J. Bot. 58: 2241-2262. EDWARDs, D. & FEEHAN, J. 1980. Records of Cooksonia-type sporangia from the late Wenlock strata of Ireland. Nature 287: 41-42. EDWARDs, D. & KENRICK, P. 2015. The early evolution of land plants, from fossils to genomics: a commentary on lang (1937) ‘On plant remains from the Downtonian of England and Wales’. Phil. Trans. R. soc. B 370: 20140343. EDWARDs, D., MORRIs, J. l., RICHARDsON, J.B. & KENRICK, P. 2014. Cryptospores and cryptophytes reveal hidden diversity in early land floras. New Phytol. 202: 50-78. ElICK, J. M., DRIEsE, s.G. & MORA, C.I. 1998. Very large plant and root traces from Early to Middle Devonian: Implications for early terrestrial ecosystems and atmospheric p (CO2) estimations. Geology 26: 143-146. FAIRON-DEMARET, M. & lI, C-s. 1993. Lorophyton goense gen. et sp. nov. from the lower Givetian of Belgium and a discussion of the Middle Devonian Cladoxylopsida. Rev. Palaeobot. Palynol. 77(1-2): 1-22. FAIRON-DEMARET, M. & BERRY, C.M. 2000. A reconsideration of Hyenia elegans 238 FERN GAZ. 20(6):217-242. 2017

Krausel et Weyland and Hyenia ‘complexa’ leclercq: Two Middle Devonian Cladoxylopsida from western Europe. Int. J. Pl. sci. 161(3): 473-494. FU, Q., WANG, Y., BERRY, C.M. & XU, H-H.. 2011. Complex branching patterns in a newly recognized species of Compsocradus Berry et stein (Iridopteridales) from the Middle Devonian of North Xinjiang, China. Int. J. Pl. sci. 172(5): 707-724. GAlTIER, J. 2010. The origins and early evolution of the megaphyllous leaf. Int. J. Plant sci. 171(6):641-661. GENsEl, P.G. & ANDREWs, H.N. 1984. Plant life in the Devonian. New York: Praeger Press, 379 pp. GENsEl, P.G. & KAsPER, A.E. 2005. A new species of the Devonian lycopod genus, Leclercqia, from New Brunswick, Canada. Rev. Palaeobot. Palynol. 136, 105–123. GENsEl, P.G. & AlBRIGHT, V.M. 2006. Leclercqia complexa from the Early Devonian (Emsian) of northern New Brunswick, Canada. Rev. Palaeobot. Palynol. 142: 103-121. GENsEl, P.G. & BERRY, C.M. 2001. Early lycophyte evolution. Am. Fern J. 91: 74-98. GENsEl P.G., KOTYK, M.E. & BAsINGER, J.F. 2001. Morphology of above- and below-ground structures in Early Devonian (Pragian-Emsian) plants In GENsEl P.G. & D. EDWARDs, eds, Plants Invade the land: Evolutionary and Environmental Perspectives. Columbia University Press, New York, pp. 83–102. GERRIENNE, P. 1992. The Emsian plants from Fooz-Wépion (Belgium). III. Foozia minuta gen. et spec. nov., a new taxon with probable cladoxylalean affinities. Rev. Palaeobot. Palynol. 74: 139-157. GERRIENNE, P.M. & GENsEl, P.G. 2016. New data about anatomy, branching, and inferred growth patterns in the Early Devonian plant Armoricaphyton chateaupannense, Montjean-sur-loire, France. Rev. Palaeobot. Palynol. 224: 38-53. GERRIENNE, P., MEYER-BERTHAUD, B., FAIRON-DEMARET, M., sTREEl, M. & sTEEMANs, P. 2004. Runcaria, a Middle Devonian seed plant precursor. science 306: 856-858. GERRIENNE, P., MEYER-BERTHAUD, B., YANG, N., sTEEMANs, P., & li, C-s. 2014. Planatophyton gen. nov., a late Early or Middle Devonian euphyllophyte from Xinjiang, North-west China. Rev. Palaeobot. Palynol. 208:55-64. GERRIENNE, P., GENsEl, P.G., sTRUllU-DERRIEN, C. lARDEUX, H., sTEEMANs, P., & PREsTIANNI, C. 2011. A simple type of wood in two Early Devonian plants. 2011. science 333(6044):837. GIEsEN, P. & BERRY, C.M. 2013. Reconstruction and growth of the early tree Calamophyton (Pseudosporochnales, Cladoxylopsida) based on exceptionally complete specimens from lindlar, Germany (Mid-Devonian): Organic connection of Calamophyton branches and Duisbergia trunks. Int. J. Plant sci. 174 (4): 665-686. GRIERsON, J.D. & BONAMO, P.M. 1979. Leclercqia complexa: Earliest ligulate lycopod (Middle Devonian). Am. J. Bot. 474-476. HAO, s-G. 1988. A new lower Devonian genus from Yunnan, with notes on the origin of leaves (in Chinese). Acta Bot. sin. 30: 441-448. HAO, s G. & BECK, C. B. 1993. Further observations on Eophyllophyton bellum from the lower Devonian (siegenian of Yunnan, China. Palaeontogr. B 230: 27-41 HAO, s-G, & XUE, J. 2013. The Early Devonian Posongchong flora of Yunnan- a contribution to an understanding of the evolution and early diversification of vascular plants. science Press, 366 pp. HAO s, XUE, J., GUO, D, & WANG, D-M.. (2010) Earliest rooting system and GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 239

root:shoot ratio from a new Zosterophyllum plant. New Phytol 185: 217–225 HAO, s., WANG, D., WANG, Q. & XUE, J. 2006. A New lycopsid, Zhenglia radiata gen. et sp. nov., from the lower Devonian Posongchong Formation of southeastern Yunnan, China, and Its Evolutionary significance. Acta Geol. sin. 80(1): 11-19. HAUGHTON, s. 1859. On Cyclostigma, a new genus of fossil plants from the Old Red sandstone of Kiltorcan Co Kilkenny. J. Roy. soc. Dublin 2:407-420. HETHERINGTON, A.J. & DOlAN, l. 2017. The evolution of lycopsid rooting structures: conservatism and disparity. New Phytologist 215 (2): 538-544. HIll, s.A., sCHECKlER, s.E. & BAsINGER, J.F. 1997. Ellesmeris sphenopteroides, gen. et sp. nov., a new zygopterid fern from the Upper Devonian () of Ellesmere, N.W.T., Arctic Canada. Am. J. Bot. 84: 85-93. HOFFMAN, l.A. & TOMEsCU, A.M. F. 2013. An early origin of : Franhueberia gerriennei gen. et sp. nov. from the lower Devonian of Gaspé (Quebec, Canada). Amer. J. Bot. 100, 754-763. HUEBER, F.M. 1983. A new species of Baragwanathia from the sextant Formation (Emsian) Northern Ontario, Canada. Bot. J. linn. soc. 86:57-79. HUEBER, F.M. 1992. Thoughts on the early lycopsids and zosterophylls. Ann. Mo. Bot. Gdn. 79:474-499. JOHNsON, T. 1913. On Bothrodendron (Cyclostigma) kiltorkense Haughton, sp. Roy. soc. Dublin, sci. Proceed. 13 (n.s.): 500-528. KAsPER, A.E, ANDREWs, H.N. & FORBEs, W.H. 1974. New fertile species of Psilophyton from the Devonian of Maine. Am. J. Bot. 61: 339-359. KENRICK P. 2013. The origin of roots. In: Eshel A, Beeckamn T, eds. Plant roots: the hidden half. london, UK: Taylor & Francis, 1–14. KENRICK P. & CRANE, P. R. 1997. The origin and early diversification of land plants: A cladistic study. Washington: smithsonian. KENRICK P. & sTRUllU-DERRIEN, C. 2014. The Origin and early evolution of roots. Plant Phys. 166: 570-580. KENRICK, P., WEllMAN, C.H., sCHNEIDER, H. & EDGECOMBE, G.D. 2012. A timeline for terrestrialization: consequences for the carbon cycle in the Palaeozoic. Phil. Trans. Roy. soc. B 367: 519-536. KOTYK, M. E., BAsINGER, J.F., GENsEl, P. G. & DE FREITAs, T. A. 2004. Morphologically complex plant macrofossils from the late silurian of Arctic Canada. Am. J. Bot. 89: 1004-1013. KRÄUsEl, R. & WEYlAND, H. 1937. Pflanzenreste aus dem Devon. X. Zwei Pflanzenfunde im Oberdevon der Eifel. senckenbergiana 19: 338-355. KRÄUsEl, R. & WEYlAND, H. 1949. Gilboaphyton und die Protolepidophytales. senckenbergiana 30: 129-152. lEClERCQ, s. 1951. Étude morphologique et anatomique d’une Fougère du Dévonien superieur. Ann. soc. Geol. Belg..Mem. Vol. XIV (3): 39 pp. lEClERCQ, s. 1970. Classe des Cladoxylopsida Pichi-sermolli, 1959. In: Boureau, E> (Ed.). Traité de Paléobotanique. IV. Fasc. I. Filicophyta. Masson, Paris, pp. 119-165. lI, F-W., VIllARREAl, J.C., KEllY, s., ROTHFEls, C.J., MElKONIAN, M., FRANGEDAKIs, E., RUHsAM, M., sIGEl, E.M., DER, J.P., PITTERMANN, J., BURGE, D.O., POKORNY, l., lARssON, A., CHEN, T., WEsTsTRAND, s., THOMAs, P., CARPENTER, E., ZHANG, Y., TIAN, Z., CHEN, l., YAN, Z., ZHU, Y., sUN, X., WANG, J., sTEVENsON, D.W., CRANDAll-sTOTlER, B.J., 240 FERN GAZ. 20(6):217-242. 2017

sHAW, A. J., DEYHOlOs, M.K. sOlTIs, D.E. ,GRAHAM, s.W., WINDHAM, M.D., lANGDAlE, J.A., WONG, G.K-s. MATHEWs, s. & PRYER, K.M. 2014. Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns. PNAs, 111:6672-6677 MATsUNAGA, K.K.s. & TOMEsCU, A.M.F. 2016. Root evolution at the base of the lycophyte clade: insights from an Early Devonian lycophyte. Ann. Bot 117(4):585-598. MOMENT, N., GERRIENNE, P. & PREsTIANNI, C. 2015. Brabantophyton, a new genus of stenokolealean affinities from the Middle to earliest Upper Devonian locality from Belgium. Rev. Palaeobot. Palynol. 227: 77-96. MUsTAFA, H. 1978. Beiträge zur Devonflora III. Argum. Palaeobot. 5: 91-132. NICKRENT, D. l., PARKINsON, C. l., PAlMER, J. D. & DUFF, R. J. 2000. Multigene phylogeny of land plants with special reference to bryophytes and the earliest land plants. Mol. Biol. Evol. 17:1885–1895. OBRHEl, J. 1959. Neue pflanzenfunde in den sbrsko-schichten (Mitteldevon). Vestn. Ustred. Ustavu Geol. 34: 384-388. PPG1. 2016. A community-derived classification for extant lycophytes and ferns. J. syst. Evol. 54 (6): 563–603. PREsTIANNI, C. & GERRIENNE, P.M. 2010. Early seed plant radiation: an ecological hypothesis pp. 71-80 in Vecoli, M., Clement, G. & Meyer-Berthaud, B. (eds). The Terrestrialization Process: Modelling Complex Interactions at the Biosphere-Geosphere Interface. Geol. soc. lond. spec. Pub., 339. PRYER, K.M., sMITH, A.R., & ROTHFEls, C. 2009. Polypodiopsida (14 January 2009) [online]. In: Tree of life Web Project: http://tolweb.org/ Polypodiopsida/20615/2009.01.14 [accessed 7 October 2016]. PRYER K.M., sCHNEIDER H., sMITH A.R., CRANFIll R., WOlF P.G., HUNT J.s. 2001. Horsetails and ferns are a monophyletic group and the closest living relatives to seed plants. Nature 409: 618–621. PRYER, K.M., sCHUETTPElZ, E., WOlF, P.G., sCHNEIDER H., sMITH A.R., & CRANFIll, R. 2004. Phylogeny and evolution of ferns (monilophytes) with a focus on the early leptosporangiate divergences. Amer. J. Bot. 91: 1582–1598 QIU Y-l, lI, l., WANG, B., CHEN, Z., DOMBROVsKA, O., lEE, J., KENT, l., lI, R., JOBsON, R.W., HENDRY, T.A., TAYlOR, D.W. 2007. A nonflowering land plant phylogeny inferred from nucleotide sequences of seven chloroplast, mitochondrial, and nuclear genes. Int. J. Plant sci. 168: 691–708. RAVEN J.A. & EDWARDs, D. (2001) Roots: evolutionary origins and biogeochemical significance. J. Exp. Bot. 52: 381–401 REMY, W. & HAss, H. 1986. Gothanophyton zimmermanni nov.gen., nov. spec., eine Pflanze mit komplexem stelar-Korper aus dem Emsian. Arg. Palaeobotanica 7 (9): 9-69. RENZAGlIA, K. s., DUFF, R. J. T, NICKRENT, D. l. & GARBARY, D. J. 2000. Vegetative and reproductive innovations of early land plants: implications for a unified phylogeny. Phil. Trans. R. soc. lond. B 355:769–793 RICHARDsON, J.B., BONAMO, P. M. & McGREGOR, D.C. 1993. The spores of Leclercqia and the dispersed spore morphon Acinosporites lindlarensis Riegel: a case of gradualistic evolution. Bull. Nat. Hist. Mus., lond. (Geol.) 49, 121–155. RICKARDs, R. B. 2000. The age of the earliest club mosses: the silurian Baragwanathia flora in Victoria, Australia. Geol. Mag. 137(2): 207-209. GENsEl: SILURIAN-DEVONIAN ORIGINS OF FERNS AND LYCOPHYTES 241

ROTHWEll, G.W. & NIXON, K. C. 2006. How does inclusion of fossil data change our conclusions about the phylogenetic history of euphyllophytes? Int. J. Plant sci. 167(3): 737-749. ROTHWEll, G. W. & sTOCKEY, R. A. 2008. Phylogeny and evolution of ferns: a paleontological perspective. Pp. 332-364, Ranker, T.A. & Haufler, C. H. (eds) Biology and Evolution of Ferns and lycophytes. Cambridge Uiversity Press. ROWE, N. 1988. A herbaceous lycophyte from the lower Carboniferous Drybrook sandstone of the Forest of Dean, Gloucestershire. Palaeontol. 31 (1): 69-83. RUBINsTEIN, C.V.; GERRIENNE, P. DE lA PUENTE, G.s., AsTINI, R.A. & sTEEMANs, P. (2010). Early Middle Ordovician evidence for land plants in Argentina (eastern Gondwana). New Phytologist. 188 (2): 365–369 sANDERs, H., ROTHWEll, G.W. & WYATT, s. 2007. Paleontological context for the developmental mechanisms of evolution. Int. J. Plant sci. 168: 719-728. sCHECKlER, s.E., 1974. systematic characters of Devonian ferns. An. Mo. Bot. Gard. 61:462-473. sCHECKlER, s.E., sKOG, J.E. & BANKs, H.P.. 2006 Langoxylon asterochlaenoideum stockmans: Anatomy and relationships of a fern-like plant from the Middle Devonian of Belgium. Rev. Palaeobot Palynol. 142: 193-217. sCHNEIDER H, sMITH, A. R. & PRYER, K. M. 2009. Is morphology really at odds with molecules in estimating fern phylogeny? sys. Bot. 34: 455–475. sCHUlTKA, s. 1978. Beiträge zur Anatomie von Rhacophyton condrusorum Crepin. Arg. Palaeobot. 5: 11-22. sCHWEITZER H-J & lI, C-s. 1996 Chamaeodendron nov. gen., eine multisporangiate lycopsid aus dem Frasnium sudchinas. Palaeontogr B 238:45–69. sKOG, J. & HIll, C. 1992. The Mesozoic herbaceous lycopsids. Ann. Mo. Bot. Gard. 79: 648-675. sMITH A.R., PRYER, K.M., sCHUETTPElZ, E. KORAll, P., sCHNEIDER, H. & WOlF. P.G. 2006b. A classification for extant ferns. Taxon 55: 705–731. sTEEMANs, P., WEllMAN, C.H. & GERRIENNE, P. 2010. "Paleogeographic and paleoclimatic considerations based on Ordovician to lochkovian vegetation". Geological Society, London, Special Publications. 339 (1): 49– 58. doi:10.1144/sP339.5 sTEEMANs, P., lE HÉRIssÉ, A., MElVIN, J., MIllER, M.A., PARIs, F., VERNIERs, J., WEllMAN, C.H.. 2009. Origin and radiation of the earliest vascular plants. science 324: 353. sTEIN, W. 1982. Iridopteris eriensis from the Middle Devonian of North America, with systematics of apparently related taxa. Bot. Gaz. 143(3): 401-416. sTEIN, W. & HUEBER, F. M. 1989. The anatomy of Pseudosporochnus: P. hueberi from the Devonian of New York. Rev. Palaeobot. Palynol. 60: 311-359. sTEIN, W., Wight, D. C. & Beck, C. B. 1984. Possible alternatives for the origin of sphenopsida. syst. Bot. 9: 102-118. sTEIN, W., MANNOlINI, F., HERNICK, l V-A., lANDING, E., & BERRY, C.M.. 2007. Giant cladoxylopsid trees resolve the enigma of the Earth’s earliest forest stumps at Gilboa. Nature 446: 904-907. sTEIN, W., BERRY, C.M., HERNICK, l V-A. & MANNOlINI, F. 2012. surprisingly complex community discovered in the mid-Devonian fossil forest at Gilboa. Nature 483(7387), pp. 78-81. sTRUllU-DERRIEN, C., KENRICK, P., TAFFOREAU, P., COCHARD, H., JEAN- 242 FERN GAZ. 20(6):217-242. 2017

lOUIs BONNEMAIN, J-l, lE HÉRIssÉ, A., lARDEUX, H. & BADEl, E. 2014. The earliest wood and its hydraulic properties documented in c. 407-million-year- old fossils using synchrotron microtomography. Bot. J. linn. soc. 175: 423-437. THOMAs, B. 1992. Paleozoic herbaceous lycopsids and the beginnings of extant Lycopodium sens. lat. and Selaginella sens. lat. Ann. Mo. Bot. Gdn 79: 623-631. TOMEsCU, A.M.F. 2009. Megaphylls, microphylls and the evolution of leaf development. Trends Plant sci.14:5-12. TRANT, C. & GENsEl, P.G. 1985. Branching in Psilophyton: a new species from the lower Devonian of New Brunswick, Canada. Am. J. Bot. 72(8): 1256-1273. WANG, DM, XU, H-H., XUE, J-Z., WANG,Q., lIU, l. 2015. leaf evolution in early- diverging ferns: insights from a new fern-like plant from the late Devonian of China. Ann. Bot. 115: 1133-1148. WANG, QI, HAO, s-G., WANG, D-M., WANG, Y. & DENK, T. 2003. A late Devonian arborescent lycopsid Sublepidodendron songziense Chen emend. (sublepidodendraceae Krausel et Weyland 1949) from China, with a revision of the genus Sublepidodendron (Nathorst) Hirmer 1927. Rev. Palaeobot. Palynol. 127: 269- 305. WANG, Y. & BERRY, C.M. 2001. A new plant from the Xichong Formation (Middle Devonian) south China. Rev. Palaeobot. Palynol. 116:63-85. WANG, Y. & BERRY, C.M. 2003. A novel lycopsid from the Upper Devonian of Jiangsu, China. Palaeontology 46(6): 1297-1311. WEllMAN, C.H., sTEEMANs, P., MIllER, M.A. 2008. Trilete spores from the Ordovician of saudi Arabia: earliest evidence for vascular plants and their immediate predecessors (“protracheophytes”). Terra Nostra 2, 304. WEllMAN, C.H., sTEEMANs, P., MIllER, M.A. 2015. spore assemblages from Upper Ordovician and lowermost silurian sediments recovered from the Qusaiba-1 shallow core hole, Qasim region, central saudi Arabia. Rev. Palaeobot. Palynol. 212: 111-126. XU, H.H. & WANG, Y. 2016. The earliest cormose rhizomorph of putative lycopsid affinity from the Middle Devonian of West Junggar, Xinjiang, China. Rev. Palaeobot. Palynol. 226: 54-57. XU, H-H, BERRY, C.M., WANG, Y., MARsHAll, J.E.A. 2011. A new species of Leclercqia Banks, Bonamo et Grierson (lycopsida) from the Middle Devonian of North Xinjiang, China, with a possible climbing habit. Int. J. Plant sci. 172 (6): 836- 846. XUE, J.Z., HAO, s-G, ZHU, X., WANG, D-M. 2012. A new basal euphyllophyte, Pauthecophyton gen. nov., from the lower Devonian (Pragian) of Yunnan, China. Rev. Palaeobot. Palynol. 183(1): 9-20. ZDEBsKA, D. 1986. Psilophyton szaferi sp. nov., from the lower Devonian of the Holy cross Mountains, Poland. Acta soc. Bot. Poland 55: 315-324. FERN GAZ. 20(6). 2017 243

a ShoRt BIogRaphy of thE authoR Patricia G. Gensel is a professor in the Biology Department, University of North Carolina at Chapel HiIl, having started there in 1975. she received her B.A. at Hope College, Holland, Michigan, then she worked as a research assistant in the Geology Department, King's College, Univ. of london, England, for Dr. John Richardson in the area of Devonian palynology. Patricia returned to the U.s.A. and embarked on graduate studies of fossil plants and their spores with Professor Henry Andrews at the University of Connecticut, storrs, CT., where she earned a M.s. and then Ph.D. After a few years of post-doctoral work, again with H. N. Andrews, she joined the faculty of (initially) the Botany Department, and later, Biology Department, at UNC. Patricia continued work on both Devonian (from U.s. and Canada) and Early Carboniferous plants (from Us), studying both megafossils and where possible, in situ spores. she also had students who worked on fossils from North Carolina and prepared a field guide to Plants of the Deep River Basin, NC. she co- authored the book Plant Life in the Devonian with H.N. Andrews (1984), and co-edited a book of contributed chapters entitled Plants Invade the Land with Dianne Edwards (2001). 244 FERN GAZ. 20(6). 2017

she has authored or co-authored at least 68 peer-reviewed publications, the majority on Devonian plants, but also some about in situ spores, spore ultrastructure, and Early Carboniferous plants, several book chapters, reviews or field guides. Patricia has organized many symposia, mostly about early land plant evolution, trained several Ms students and a few PhD students. Her major interests center on producing a better understanding of the morphology, structure, evolutionary relationships or patterns of evolutionary change, and where possible ecology, of early land plants, mainly of Devonian and Early Carboniferous age. she continues her interest in palynology, especially in situ spores or spore ultrastructure, in terms of what they may contribute relative to systematics or phylogenies of early plants. Recent publications include a report of early wood from the very Early to Early Devonian of France and New Brunswick (Gerrienne et al., 2011, Gerrienne & Gensel, 2016, work in prep), a revision of sporangial morphology and attachment in the iconic Sawdonia ornata (Gensel & Berry, 2016) and paleoenvironmental interpretations of some of the major plant-bearing deposits in New Brunswick or early coal (Kennedy et al., 2012; Kennedy et al., 2013). Her on-going work focuses on elucidating anatomy and morphology of basal euphyllophytes (formerly trimerophytes), additional work on Sawdonia spp., and other Devonian or Carboniferous plants.

REFERENCEs GENsEl, P. & BERRY, C.M. 2016. sporangial Morphology of the Early Devonian Zosterophyll Sawdonia ornata from the type locality (Gaspé). Int. J. Pl. sci. 177: 618-632. GERRIENNE, P.M. & GENsEl, P.G. 2016. New data about anatomy, branching, and inferred growth patterns in the Early Devonian plant Armoricaphyton chateaupannense, Montjean-sur-loire, France. Rev. Palaeobot. Palynol. 224: 38-53. GERRIENNE, P., GENsEl, P.G., sTRUllU-DERRIEN, C. lARDEUX, H., sTEEMANs, P., & PREsTIANNI, C. 2011. A simple type of wood in two Early Devonian plants. science 333(6044):837. KENNEDY, K.l, GENsEl, P.G. & GIBlING, M.R., 2012. Paleoenvironmental inferences from the classic Early Devonian plant-bearing locality of the Campbellton Formation, New Brunswick, Canada. Palaios 27: 424–438. KENNEDY, K.l., GIBlING, M.R, EBlE, C.F., GAsTAlDO, R.A., GENsEl, P.G., WERNER-ZWANZIGER, U. & WIlsON, R.A. 2013. lower Devonian coaly shales of northern New Brunswick, Canada: plant accumulations in the early stages of terrestrial colonization. J. sediment. Res. 83:1202–1215 FERN GAZ. 20(6):245-254. 2017 245

a nEW REcoRD of BRoWn Rot DISEaSE In WatER fERn AZOLLA MICROPHYLLA (aZollacEaE): loSS of ImpoRtant BIo-RESouRcE

s. DEY, M. HORE, J. BIsWAs, M. BIsWAs, B.K. MANDAl, P. DAs & s. GUPTA

Pteridology and Palaeobotany section, Department of Botany, University of Kalyani, Kalyani- 741235, West Bengal, India e-mail id: [email protected]

Key words: cottony white mycelium; yellow pigmentation; macroconidia; microconidia; chlamydospores; pathogenic fungus; Fusarium thapsinum

aBStRact severely infected water fern, Azolla microphylla Kaulf., has been observed in natural habitats in different districts in the lower Gangetic plain of West Bengal, India. The infected plants have turned dark brown and been rotted entirely within 5-7 days, causing loss of natural resource as the plant species has immense commercial value. specific growing colonies with cottony white mycelium, yellow pigmentation on the cultured medium, macro- and micro- conidial features, and absence of chlamydospore indicate the causal pathogen to be Fusarium thapsinum Klittich., lesile, Nelson, Marases. The identification of the causal pathogen has been authenticated by standard pathogenicity test (Koch’s Postulates). Extensive literature survey reveals that this is a new record of brown rot disease in A. microphylla.

IntRoDuctIon Azolla lam. is a free-floating, fast growing water fern. The plant is widely distributed in tropical, subtropical, and warm temperate zones in different countries of the World including India (Wagner, 1997). Here Azolla spp. are naturally available, mostly on moist soils, freshwater ditches, ponds, lakes, sluggish rivers and marshy lands. Azolla is treated as a ‘green gold mine’, because of its high nutritive value and it is considered as ‘super plant’ due to its fast-growing capacity (Wagner, 1997). They can even grow in nitrogen deficit areas and can double the biomass in 3-5 days because of its unique symbiotic relationship with the nitrogen fixing endophytic blue-green cyanobacterium, Anabaena azollae sterberger (Wagner, 1997). High nutritive values (Buckingham et al., 1978; Alcantara & Querubin, 1985; Paoletti et al., 1987; lejeune et al., 2000; Alalade & lyayi, 2006) and huge productivity through easy cultivation methods (Dao & Tran, 1979; lumpkin & Plucknett, 1982; liu et al., 2008) are the key factors for utilizing Azolla as one of the most promising aquatic plants for livestock feed (Becerra et al., 1995; Alalade & lyayi, 2006; Cagauan & Pullin, 1991; shiomi & Kitoh, 2001; Fiogbe et al., 2004), and as an effective natural fertilizer to increase rice yield successfully (Peters, 1978; Tung & shen, 1985; Watanabe & liu, 1992; Yadav et al., 2014). The plant has been employed in experiments during space and planetary travel (liu et al., 2008; Carrapico, 2002; Katayama et al., 2008), and used for the production of non-polluting high energy biofuel (Peters et al., 1976; Newton, 1976; Das et al., 1994; Hall et al., 1995), especially biodiesel (salehzadeh et al., 2014). The plant can act as reducer of various greenhouse gases by ammonia volatilization 246 FERN GAZ. 20(6):245-254. 2017

(Watanabe & liu, 1992), by inhibiting methane production from rice field (Prasanna et al., 2002), and noticeably by sequestering atmospheric CO2 (Brinkhuis et al., 2006). In addition, weed control (Krock et al., 1991), mosquito repellence (Ansari & sharma ,1991), phyto-remediation from waste water (Jain et al., 1989; saxena, 1995; Costa et al., 1999; Antunes et al., 2001; Khosravi et al., 2005; Umali et al., 2006; Rai, 2008; Costa et al., 2009; Rai & Tripathi, 2009; Elmachliy et al., 2010; Rai, 2010a, b; sood et al., 2012) and soil (Umali et al., 2006; Cohen et al., 2002 and Mashkani & Ghazvini, 2009) are added approaches for utilizing this vital bio-resource. With view to the many valuable uses of Azolla, the present authors have attempted to explore the species under various abiotic stresses through experimental set-up by culture of collected specimens from natural habitats of the lower Gangetic plains of West Bengal, India. During field survey, it has been observed that among the available Azolla species, A. microphylla Kaulf. grows dominantly over others and it can resist direct sunlight in summer. However, the species has been found to be severely infected by a fungal pathogen at the end of winter season (January-March) in most of the collection sites, which disrupts normal growth of the plant and ultimately causes death within 5-7 days. From the available literature, it seems that pathogenic reports on Azolla spp. are rather meagre (sasi et al., 1982; Kannaiyan, 1985; Kannaiyan & Kumar, 2005; lee et al., 2011; Banihashemi, 2014). The present communication describes a new causal organism for A. microphylla, which may help in identifying suitable measure(s) to protect this important natural resource.

matERIal anD mEthoDS Fresh and infected samples of Azolla microphylla Kaulf. were collected from different sites of three districts, namely, North 24 Parganas (22.936 N, 88.392 E; 22.825 N, 88.786 E), south 24 Parganas (22.021 N, 88.615 E), and Nadia (22.983 N, 88.445 E) in the lower Gangetic plain of West Bengal. The morphological studies of infected and non-infected plants were made by hand lens and stereo zoom microscope (model lEICA s8APO). The anatomical details of infected plants and that of the isolated pathogen were thoroughly studied under light microscope (model leitz laborlux s). Macro and micro photographic documentations were made using Nikon D3200 DslR and EC3 scientific cameras. To identify the pathogen from morphological characteristics, first the isolations were made from the frond of the naturally infected A. microphylla plant using a sterile inoculating loop and plated onto 2.25% Potato Dextrose Agar (PDA). A total of five replicas of culture and three replicas of control plates were incubated at 25˚C temperature in complete darkness. After 2–3 days of inoculation, the fungal colony was observed to grow in each of the five replications of cultured plates but no changes were observed in control plates. The colony morphology, growth pattern, size and shape of macro- and micro- conidia were characterized thoroughly. To confirm the identity of the pathogen, the standard pathogenicity test (Koch’s Postulates) was performed stepwise. student’s t-test was done to compare the variations in two sets of data of pathogens detected from inoculated and re-inoculated pure cultured plates prepared through the pathogenicity test. The tests were performed by inoculating naturally-collected, healthy A. microphylla plants of a single genotype, because the disease was observed in A. microphylla only in natural habitats. A total of 45 experimental plants were surface sterilized through rinsing in 70% ethanol for 30 sec. followed by 1% DEY et al.: BROWN ROT DISEASE IN AZOLLA MICROPHYLLA 247 sodium hypochlorite solution for 1 min., and sterile distilled water for three times. Out of 45 surface sterilized plants, 30 healthy plants were wounded on the fronds by sterile needle. A small mycelial plug taken from a month-old colony of the pathogen was inserted into the wounds using a sterile toothpick. Both 30 inoculated and 15 non-inoculated plants were then placed onto nutrient medium, distributing five plants in each plate so that six replicas were made for inoculated plants and three for non-inoculated ones. The nutrient media was prepared using HIMEDIA PTO10 Murashige and skoog medium with 17.04 gm./lit. and 10 gm. of agar. The plates were kept in a growth chamber at 25˚±2˚C temperature and 1800 lux intensity of light in 16:8 hr day and night cycle.

RESultS anD DIScuSSIon severe infection was observed in A. microphylla during the months of January to March in the field (Figure. 1A, B), as well as in a tank placed in the experimental garden of the Botany Department, Kalyani University (Figure 1C). In a fresh individual plant (Figure 1D), at first the infection was observed in roots (Figure 1E), followed by dark brown-coloured spots on the leaf (Figure 1F). later, the infected areas sequentially enlarged (Figure 1G), and eventually the whole leaf turned into brown, showing bloch notch symptoms (Figure 1H). Finally, the plant rotted within 5-7 days (Figure 1I). Outwardly protruding, cottony, white mycelial growth covering the entire leaf surface was observed with disease severity (Figure 1J). The transverse section of infected leaves at the initial stage showed loosening of leaf tissues with distinct entrance of hyphae (Figure 1K). Anatomical section of leaf was not possible at the final stage of infection due to total disintegration of leaf tissues. The browning and rotting of leaves and roots are characteristic symptoms produced by the pathogen in A. microphylla. The dark brown coloration of the infected parts of the plant is because of the degradation of chlorophyllous pigments and probably production of phenolic compounds as resistance to pathogens (lattanzio et al., 2006). By comparing the symptoms of rot diseases (Agrios, 2005), the studied infection in present investigation has been designated as brown rot disease of A. microphylla. Each replica of cultured PDA media produced fungal colonies with very thick, abundant, cottony white mycelium, finally attaining a diameter of 48.71±3.0 mm (Figure 1l) after seven days of incubation. The growth pattern of the mycelium was radial. A characteristic yellow pigmentation was found to appear on the agar plate (Figure 1l) after 3-4 days of incubation. After 5-6 days, a slight purplish pigmentation was observed to ooze out and persisted for 1-2 days only. No sporodochia was observed in any of the cultured plates. Under the microscope, the fungal hyphae appeared hyaline and septate (Figure 1M). Conidiogenous cells were mostly monophialidic (Figure 1N) and rarely polyphialidic. Monophilalidic conidiogenous cells were long (range 10.53-33.94 µm), with an average length of 21.43±7.41 µm (Table 1). The microscopic examination of the fungi taken from the pure culture plates revealed hyphae with characteristic macro- and microconidia (Table 1). Microconidia were produced in relatively short chains (Figure 1O) or in a false head; conidiophores were branched or unbranched, bearing hyaline, monophialidic, conidiogenous cells. Three different forms of microconidia [oval (Figure 1P), fusiform (Figure 1Q), and napiform (Figure 1R)] were found, which were hyaline, thin-walled, and yellowish in colour. Fusiform shaped microconidia had a single septum. The average sizes of the oval, fusiform, and napiform shaped microconidia were 248 FERN GAZ. 20(6):245-254. 2017

figure. 1(a.-W.): Caption opposite page DEY et al.: BROWN ROT DISEASE IN AZOLLA MICROPHYLLA 249

10.73±3.31x2.48±0.33 µm, 13.27±1.98 x 4.85±0.48 µm, and 7.17±0.77 x 3.37±0.18 µm respectively (Table 1). The macroconidia were found abundantly in aerial parts of the hyphae of the infected leaf; 3-5 septate, but rarely 6; straight to falcate, thin walled, hyaline; slightly curved and tapering in a banana shape (Figure 1s). The average size of macroconidia is of 33.10±5.92 x 3.62±0.28 µm (Table 1). The apical cell of each macroconidia was conical, whereas the basal cell was relatively ill-developed and not the usual foot-shaped. The average length of the apical cell (6.47±0.77 µm) was longer than the penultimate cell (5.29±0.83 µm) (Table 1). It is significant to note that no chlamydospore was found in any one of the culture plates. The growth pattern of the cultured pathogen onto PDA media and the occurrence of distinctive banana-shaped macroconidia and specific types of microconidia indicate that the causal pathogen is Fusarium link (Moretti, 2009). Fusarium is a well-known plant pathogenic fungus, which is soil borne in nature and causes severe plant diseases around the world (Agrios, 2005). The Fusarium strain that produces diffusing yellow pigment was separated as a new species, F. thapsinum Klittich., lesile, Nelson, Marases (Teleomorph, Gibberella thapsina) (Klittich et al., 1997, summerell et al., 2003; leslie & summerell, 2006). The characteristic features of cottony white mycelium, yellow pigmentation onto pure culture PDA medium, absence of chlamydospores, shape and size of macro- and microconidia corroborate with the diagnostic features of the holotype BPI 737885 (Klittich et al., 1997) and hence identify the species epithet of the present causal pathogen as F. thapsinum. After seven days incubation in the pathogenicity tests, identical disease symptoms with brown-coloured lesions and protruding cottony white mycelium were observed only on the inoculated plants (Figure 1V) and the fungus was consistently re-isolated from lesions. The identity of the re-isolated pathogen was confirmed as F. thapsinum again by pure culturing onto PDA medium (Figure 1W) maintained at similar conditions mentioned before and subsequent microscopic examinations of the hyphae with

figure 1(a.-W.): Non-infected, and infected Azolla microphylla plant showing symptoms of brown rot disease, cultured colony of causal pathogen, and microscopic observation of different features of causal organism (bar= 0.1 mm except otherwise mentioned). A. & B. Naturally infected plant on pond (A) and in rice field (B); C. Infected plant grown in tank at experimental garden of Botany Department, Kalyani University; D. Non-infected plant; E. Infected root showing brown coloration; F. Brown coloured spots on leaf (arrow marked) at initial stage of infection; G. Gradual enlargement of infected area on leaf; H. leaf showing bloch notch symptom; I. Entirely rotten plant at final stage of infection; J. Cottony white mycelial growth on infected leaf; K. Transverse section of infected leaf showing hyphal infiltration; l. Growth pattern of pure cultured colony showing white mycelium and diffuse yellow pigmentation (dyp); M. septate fungal hyphae; N. Monophialidic conidiogenous cells; O. Occurrence of microconidia in short chain; P.-R. Microconidia of different shapes- P. oval, Q. fusiform, R. napiform; s. Banana-shaped macroconidia; T. Experimentally diseased plants in pathogenicity test; U. Control plants in pathogenicity test; V. Magnified view of experimentally diseased plants showing same disease symptoms; W. Growth pattern of pure cultured colony prepared from re-isolated pathogens from experimental disease plants showing white mycelium and diffuse yellow pigmentation (dyp). 250 FERN GAZ. 20(6):245-254. 2017 table 1: Characterization of macro- and micro- conidia grown in pure cultures prepared from the isolated inoculum of naturally diseased plant (NDP) and the re-isolated inoculum of experimentally diseased plant (EDP) of Azolla microphylla

measurement (in µm) of macro- and micro- conidia *t value at attributes NDP EDP 18 df Range Mean ± sD Range Mean ± sD macRoconIDIa

No. of 3 - 6 5.00± 0.79 3 - 6 5.00± 0.94 --- septation length of 4.82 – 7.58 6.47 ± 0.77 4.78 – 7.83 6.27 ± 1.14 0.4485 apical cell length of penultimate 3.89 – 6.51 5.29 ± 0.83 4.12 – 6.62 5.00 ± 0.78 0.7648 cell

Ratio of apical to penultimate 0.95 – 1.58 1.25 ± 0.22 0.98 – 1.65 1.26 ± 0.18 0.1004 cell size [length 19.43 – 39.23 (l) 33.10 ± 5.92 x 21.26 – 40.79 (l) 32.53 ± 4.66 x 0.2319 (l) (l) x width 3.06 – 3.98 (w) 3.62 ± 0.28 2.80 – 4.00 (w) 3.61 ± 0.35 0.0341 (w) (w)] length width 7.63 – 11.50 9.18 ± 1.70 5.84 – 11.39 9.09 ± 1.57 0.1253 ratio mIcRoconIDIa length of conidiogenous 10.53 – 33.94 21.43 ± 7.41 10.96 – 34.08 22.42 ± 6.87 0.3012 cell size of oval shaped 7.22 - 16.08 (l) 10.73 ±3.31 x 6.99 - 17.09 (l) 12.45 ±3.11 x 1.1672 (l) microconidia 2.05 – 2.92 (w) 2.48 ± 0.33 1.98 – 3.03 (w) 2.58 ± 0.35 0.6400 (w) [l x w] size of napiform 6.18 – 8.26 (l) 7.17 ± 0.77 x 6.10 – 8.63 (l) 7.47 ± 0.80 x 0.8199 (l) shaped 3.14 – 3.68 (w) 3.37 ± 0.18 3.23 – 3.71 (w) 3.42 ± 0.16 0.5843 (w) microconidia [l x w] size of fusiform 10.08 – 16.86 (l) 13.27 ± 1.98 x 9.77 – 16.23 (l) 12.86 ± 2.20 x 0.4284 (l) shaped 3.97 – 5.41 (w) 4.85 ± 0.48 4.06 – 5.41 (w) 4.76 ± 0.49 0.3894 (w) microconidia [l x w] * p>0.01 in all cases DEY et al.: BROWN ROT DISEASE IN AZOLLA MICROPHYLLA 251 characteristics macro and micro conidia (Table 1). The control plants, however, did not show any disease symptoms and no traces of F. thapsinum were recovered from them. Thorough literature survey reveals that previously reported diseases in Azolla are black rots in A. pinnata from India (Kannaiyan, 1985), Rhizoctonia blight in A. japonica from (lee et al., 2011), and A. filiculoides from Iran (Banihashemi, 2014). Kanniyan and Kumar (2005) identified a fungal complex as a causal organism for black rot disease in A. pinnata, with Rhizoctonia solani as the primary infecting organism.

concluSIon The present investigation reports a new record of brown rot disease in Azolla microphylla caused by the fungal pathogen Fusarium thapsinum. Because of the immense value of this plant, it will be essential to explore preventive measure(s) to protect this important bio-resource from destructive fungal attack.

acknoWlEDgmEntS The financial grant is aided by UGC-MRP (F. No. 41-489/2012 (sR) dated 16th July, 2012) and DsT-PURsE programme of University of Kalyani. The first author acknowledges the financial support received from University of Kalyani as a University Research scholar. The authors gratefully acknowledge the kind help provided by Prof. Animesh Kumar Datta of Botany Department, University of Kalyani. The authors gratefully acknowledge the valuable comments and suggestions given by anonymous reviewers which helped a lot to improve quality of the manuscript.

REfEREncES AGRIOs, G.N. 2005. Plant pathology. In: DREIBElBIs, D. (Ed) Plant disease caused by Fungi, pp 534-540. Elsevier academic press, UsA. AlAlADE, O.A. & lYAYI, E.A. 2006. Chemical composition and the feeding value of Azolla (Azolla pinnata) meal for egg-type chicks. Int. J. Poult. sci. 5(2):137-141. AlCANTARA, P.F. & QUERUBIN, l.J. 1985. Feeding value of Azolla meal for broilers. Philipp. J. Vet. Anim. sci. 11:1-8. ANsARI, M.A. & sHARMA, V.P. 1991. Role of Azolla in controlling mosquito breeding in Ghaziabad district villages (U P). Indian J. Malariol. 28(1):51-54. ANTUNEs, A.P.M., WATKINs, G.M. & DUNCAN, J.R. 2001. Batch studies on the removal of gold(III) from aqueous solution by Azolla filiculoides. Biotechnol. lett. 23:249-251. BANIHAsHEMI, Z. 2014. Rhizoctonia blight of Azolla in rice paddy fields in Northern Iran. Iran J. Plant Path. 50(4):189-190. BECERRA, M., PREsTON, T.R. & OGlE, B. 1995. Effect of replacing whole boiled soya beans with Azolla in the diets of growing ducks. livest. Res. Rural Devt. 7(3):1-10. BRINKHUIs, H., sCHOUTEN, s., COllINsON, M.E., slUIJs, A., DAMsTÃ, J.s., DICKENs, G.R., HUBER, M., CRONIN, T.M., ONODERA, J. & TAKAHAsHI, K. 2006. Episodic fresh surface waters in the Eocene Arctic Ocean. Nature 441:606-609 BUCKINGHAM, K.W., ElA, s.W., MORRIs, J.G. & GOlDMAN, C.R. 1978. Nutritive value of the nitrogen-fixing aquatic fern Azolla filiculoides. J. Agri. Food Chem. 26(5):1230-1234. CAGAUAN, A.G. & PUllIN, R.s.V. 1991. Azolla in aquaculture: Past present and 252 FERN GAZ. 20(6):245-254. 2017

future. In: MUIR, J., ROBERTs, R.J. (Eds) Recent Advances in Aquaculture, pp 104-130. Blackwell science, Oxford. CARRAPICO, F. 2002. The Azolla-Anabaena-Bacterial system as a natural microcosm. In: sPIE (Ed) Proceedings of sPIE Astrobiology Conference on Instruments Methods and Missions for Astrobiology, IV. pp 261-265. sPIE, san Diego. COHEN, M.F., WIllIAMs, J. & YAMAsAKI, H. 2002. Biodegradation of diesel fuel by an Azolla-derived bacterial consortium. J. Environ. sci. Health A Tox. Hazard. subst. Environ. Engg. A37(9):1593-1606. COsTA, M. l., sANTOs, M.C. & CARRAPICO, F. 1999. Biomass characterization of Azolla filiculoides grown in natural ecosystems and wastewater. Hydrobiologia 415:323–327. COsTA, M.l., sANTOs, M.C.R., CARRAPICO, F. & PEREIRA, A.l. 2009. Azolla-Anabaena’s behaviour in urban wastewater and artificial media – Influence of combined nitrogen. Water Res. 43(15):3743-3750. DAO, T.T. & TRAN, Q.T. 1979. Use of Azolla in rice production in Vietnam. In: IRRI (Ed) Nitrogen and Rice, pp 395-405. Int. Rice Res. Inst., Philippines. DAs, D., sIKDAR, K. & CHETTERJEE, A.K. 1994. Potential of Azolla pinnata as biogas generator and as a fish-feed. Indian J. Environm. Health 36(3):186-191. ElMACHlIY, s., CHEFETZ, B., TEl-OR, E., VIDAl, l., CANAls, A. & GEDANKEN, A. 2010. Removal of silver and lead ions from water wastes using Azolla filiculoides an which adsorbs and reduces the ions into the corresponding metallic nanoparticles under microwave radiation in 5 min. Water Air soil Pollut. 218:365-370. FIOGBE, E. D., MICHA, J.C. & VAN, H.C. 2004. Use of a natural aquatic fern, Azolla microphylla as a main component in food for the omnivorous-phytoplanktonophagous tilapia, Oreochromis niloticus l. J. Appl. Ichthyol. 20(6):517–520. HAll, D.O., MARKOV, s.A., WATANABE, Y., RAO, K.K. 1995. The potential applications of cyanobacterial photosynthesis for clean technologies. Photosyn. Res. 46(1):159-167. JAIN, s.K., VAsUDEVAN, P.K. & JHA, N.K. 1989. Removal of some heavy metals from polluted water by aquatic plants: studies on duckweed and water velvet. Biol. Wastes 28(2):115-126. KANNAIYAN, s. & KUMAR, K. 2005. Black rot disease interactions and nitrogen fixation in Azolla. In: Daya Publishing House (Ed) Azolla biofertilizer for sustainable rice production, pp 404-421. Daya Publishing House, Delhi. KANNIAIYAN, s. 1985. Use of Azolla in India. In: IRRI (Ed) Proceedings of the Workshop on Azolla Use, pp 109-118. Fuzhou Fujian, China. KATAYAMA, N., YAMAsHITA, M., KIsHIDA, Y., lIU, C.C., WATANABE, I., WADA, H. & sPACE AGRICUlTURE TAsK FORCE. 2008. Azolla as a component of the space diet during habitation on Mars. Acta Astronautica. 63(7-10):1093-1099. KHOsRAVI, M., RAKHsHAEE, R. & GANJI M.T. 2005. Pre-treatment processes of Azolla filiculoides to remove Pb(II), Cd(II), Ni(II) and ZN(II) from aqueous solution in the batch and fixed-bed reactors. J. Hazard. Mater 127(1-3):228-237. KlITTICH. C.J.R., lEslIE, J.F., NElsON, P.E. & MARAsAs, W.F.O. 1997. Fusarium thapsinum (Gibberella thapsina): A new species in section Liseola from sorghum. Mycologia 89(4):643-652. KROCK, T., AlKAMPER, J. & WATANABE, I. 1991. Azolla’s contribution to weed control in rice cultivation. Plant Res. Developm. 34:117-125. DEY et al.: BROWN ROT DISEASE IN AZOLLA MICROPHYLLA 253 lATTANZIO, V., lATTANZIO, V. M.T. & CARDINAlI, A. 2006. Phytochemistry: Advances in Research. In: IMPERATO F. (Ed) Role of phenolics in the resistance mechanisms of plants against fungal pathogens and , pp 23-67. IsBN: 81-308-0034-9. Research signpost, India lEE, J., CHA, J., NOH, G., HAN, K., BAE, D., KWON, Y., lIM, C, JEONG, s, KWON, J., PARK, C. & KWAK, Y. 2011. Rhizoctonia blight of Azolla japonica caused by Rhizoctonia solani. Res. Plant Dis. 17(3):405-409. lEJEUNE, A., PENGA, J., BOUlENGE, E., lARONDEllE, Y. & VAN, H.C. 2000. Carotene content of Azolla and its variations during drying and storage treatments. Anim. Feed sci. Tech. 84:295-301. lEslIE J.F. & sUMMEREll, B.A. 2006. species descriptions. In: The Fusarium laboratory manual, pp 264-265. Blackwell Publishing, Iowa. lIU, X., MIN, C., XIA-sHI, l. & CHUNGCHU, l. 2008. Research on some functions of Azolla in CElss system. Acta Astronaut. 63:1061-1066. lUMPKIN, T.A. & PlUCKNETT D.l. 1982. Azolla as a green manure: Use and management in crop production, Westview Press, Boulder Colorado. MAsHKANI, s.G. & GHAZVINI, P.T.M. 2009. Biotechnological potential of Azolla filiculoides for biosorption of Cs and sr: Application of micro-PIXE for measurement of Biosorption. Bioresource Technology 100(6):1915-1921. MORETTI, A.N. 2009. Taxonomy of Fusarium genus a continuous flight between lumpers and splitters. Proc. Nat. sci. Matica. srpska. Novi. sad. 117:7-13. NEWTON, J.W. 1976. Photoproduction of molecular hydrogen by a plant-algal symbiotic system. sci. 191(4227):559-561. PAOlETTI, C., BOCCI, F., lERCKER, G., CAPEllA, P. & MATERAssI, R. 1987. lipid composition of Azolla caroliniana biomass and its seasonal variation. Phytochem. 26(4):1045–1047. PETERs, G.A. 1978. Blue- and algal associations. Bioscience 28(9):580-585. PETERs, G.A., EVANs, W.R., TOIA, R.E. 1976 Azolla-Anabaena azollae relationship. Plant Physiol. 58:119-126. PRAsANNA, R., KUMAR, V., KUMAR, s., YADAV, A.K., TRIPATHI, U., sINGH, A.K., JAIN, M.C., GUPTA, P., sINGH, P.K. & sETHUNATHAN, N. 2002. Methane production in rice soil is inhibited by cyanobacteria. Microbiol. Res. 157(1):1-6. RAI, P.K. & TRIPATHI, B.D. 2009. Comparative assessment of Azolla pinnata and Vallisneria spiralis in Hg removal from G.B Pant sagar of singrauli Industrial region India. Environ. Monit. Assess. 148(1):75-84. RAI, P.K. 2008. Phytoremediation of Hg and Cd from industrial effluents using an aquatic free floating macrophyte Azolla pinnata. Int. J. Phytoremediation. 10(5):430- 439. RAI, P.K. 2010a. Microcosm investigation on phytoremediation of Cr using Azolla pinnata. Int. J. Phytoremediation 12(1):96-104. RAI, P.K. 2010b. Heavy metal pollution in lentic ecosystem of subtropical industrial region and its phytoremediation. Int. J. Phytoremediation 12(3):226-242. sAlEHZADEH, A., NAEEMI, A.s. & ARAsTEH, A. 2014. Biodiesel production from Azolla filiculoides (water fern). Trop. J. Pharm. Res. 13(6):957-960. sAsI, P.s., WIlsON, K.I. & sAsIDHARAN, N.K. 1982. Incidence of Rhizoctonia solani on Azolla pinnata. Curr. sci. 48(5):216. sAXENA, D.K. 1995. Purification efficiency of Lemna and Azolla in WIMCO effluents. Proc. Natl. Acad. sci. India sect. B Biol. sci. 65:61-65. 254 FERN GAZ. 20(6):245-254. 2017 sHIOMI, N., KITOH, s. 2001. Culture of Azolla in a pond nutrient composition and use as fish feed. soil sci. Plant Nutr. 47(1):27–34. sOOD, A., UNIYAl, P.l., PRAsANNA, R., AHlUWAlIA, A.s. 2012. Phytoremediation potential of aquatic macrophyte Azolla. Ambio. 41:122-137 sUMMEREll, B.A., sAllEH, B. & lEslIE J.F. 2003. A Utilitarian Approach to Fusarium Identification. Plant Dis. 87(2):117-128. TUNG, H.F. & sHEN, T.C. 1985. studies of the Azolla pinnata-Anabaena azollae symbiosis: Concurrent growth of Azolla with rice. Aquatic Bot. 22(2):145-152. UMAlI, l.J., DUNCAN, J.R. & BURGEss, J.E. 2006. Performance of dead Azolla filiculoides biomass in bioabsorption of Au from wastewater. Biotechnol. lett. 28(1):45-49. WAGNER, G.M. 1997. Azolla: A review of its biology and utilization. Bot. Rev. 63(1):1-26. WATANABE, I. & lIU, C.C. 1992. Improving nitrogen-fixing systems and integrating them in to sustainable rice farming. Plant soil 141:57-67. YADAV, R.K., ABRAHAM, G., sINGH, Y.V. & sINGH P.K. 2014. Advancements in the utilization of Azolla-Anabaena system in relation to sustainable agricultural practices. Proc. Indian Natn. Acad. sci. sect. B Biol. sci. 80(2):301-316. FERN GAZ. 20(6):255-266. 2017 255


D.J. TENNANT low Missise Farm, laverton, Ripon, HG4 3sY, UK. Email: [email protected]

Keywords: Bladder ferns, Cystopteris, C. alpina, C. regia, Cyathea incisa, taxonomy, new taxa, Wales, scotland, distribution

aBStRact A taxon in the Cystopteris fragilis (l.) Bernh. complex that occurs in Britain appears up to now to have been overlooked there. It is proposed that this fern, which was recently rediscovered in snowdonia, North Wales, should be referred to a subspecies of C. fragilis, namely subspecies huteri (Hausm. ex Milde) C. Prada & salvo, that occurs in mainland Europe, but had not previously been recognised in the British Isles. The snowdonia fern is distinct in its morphology from other British taxa in the C. fragilis complex and is described here together with its historic records and present distribution, updated from information given in an earlier paper in Watsonia (Tennant, 2010), and its relationship to affiliated taxa is discussed. IntRoDuctIon The Cystopteris fragilis complex is perhaps the most difficult one to resolve of all fern groups. In the British Isles it presently includes the species C. fragilis (l.) Bernh., C. alpina (lam.) Desv., C. diaphana (Bory) Blasdell, and C. dickieana R. sim. It is generally accepted that more than one original diploid parent was probably involved in the ancestry of the present species recognised in the complex. One result of which is the large variation between and within the species, making it especially difficult to recognise the different cytotypes based solely on their gross morphology. Over many decades, a study was made of the species within the complex, and especially their variants, both in the field and in herbarium collections. This led to earlier papers being published on C. alpina and C. dickieana in Britain (Tennant, 1995; 1996; 2010), but two especially distinctive taxa were noted in Britain, which in their morphology did not readily conform to the appearance or descriptions of any of the known presently extant British taxa within the complex. The first of these was found in 2008 in snowdonia, V.C. 49, North Wales, where it was extremely local and scarce. A search in herbarium collections revealed that a morphologically identical taxon had been collected previously there. A paper published in Watsonia (Tennant, 2010) gave most of the records of this fern, which are updated and relisted in this paper, together with its historical references, description, illustrations (Figures 1 & 2), and habitat, and evidence for the presently proposed identity of this snowdonia fern is given. The second distinctive Cystopteris taxon discovered during the course of these studies occurs in northern scotland, and a similar taxon also in North Wales. These ferns appear to be affiliated to certain variants currently included under C. alpina (lam.) Desv., previously as “C. regia” auct., non (l.) Desv. Morphologically these scottish ferns 256 FERN GAZ. 20(6):255-266. 2017 closely match certain Cystopteris forms found by the author recently in alpine regions of mainland Europe in three different countries (Figures 3 & 4). living material and details of these scottish ferns, which were grown from spores, have been donated to the Natural History Museum in london for further study and will be the subject of a later paper.

0 1 2 4 cms

figure 1. Cystopteris fragilis subsp. huteri. Cultivated examples from snowdonia, V.C.49, North Wales illustrating the degree of variation. (Further illustrations were given previously in Tennant (2010)). TENNANT: CYSTOPTERIS FRAGILIS SUBSP. HUTERI 257

figure 2. Cystopteris fragilis subsp. huteri. snowdonia, V.C. 49, North Wales. Cultivated specimen. 258 FERN GAZ. 20(6):255-266. 2017

0 1 2 3 4 cms

figure 3. Cystopteris alpina (lam.) Desv. s. str. From the European Alps, A – D. A. France. B. spain. C. Austria. D. Italy. (British examples from Yorkshire collected by J. Backhouse were illustrated in Tennant (1995)). The Cystopteris variant taxon, which is intermediate between C. alpina and C. fragilis, E – G. E. From the Italian Dolomites. F. French Alps. G. Northern scotland. TENNANT: CYSTOPTERIS FRAGILIS SUBSP. HUTERI 259

figure 4. Cystopteris variant from Northern scotland, V.C. 105, which is intermediate between C. alpina and C. fragilis. 260 FERN GAZ. 20(6):255-266. 2017

matERIalS anD mEthoDS The fieldwork described below on Cystopteris in Britain dates back over many decades through an early interest of the author, particularly in the British records for C. alpina. More recently, following several examinations of herbarium specimens at the Natural History Museum (NHM) london (Bm) and at the Royal Botanic Garden, Edinburgh (E) between 2004 and 2006, details of all relevant specimens were abstracted and compared with recently found examples of Cystopteris. This was followed by further fieldwork in North Wales and in scotland. Permission was obtained from the Countryside Council for Wales to collect a small amount of material on snowdon, V.C. 49, which was undertaken in 2009, following which this snowdonia fern was cultivated in Yorkshire. living examples of the snowdonia Cystopteris were then donated to NHM in london for further studies. This work is being undertaken by Dr F.J. Rumsey at NHM, and so far has included a detailed examination of spores and some plastid DNA studies. Meanwhile, historic publications were examined and information dating back to the 17th century was abstracted, after it was discovered that the snowdonia Cystopteris had first been collected at that time. Field studies on Cystopteris were more recently extended in alpine regions of mainland Europe, namely Norway (Tromsø 2009), France (Vanoise Alps 2010), spain (Central Pyrenees 2012), Italy (Dolomites 2014, 2015), and Austrian Tyrol (2016). Material found there was compared with examples from Britain.

RESultS Description of the Snowdonia Cystopteris This fern was largely described and illustrated in an earlier paper (Tennant, 2010) from samples that had been found in snowdonia in 2008, and cultivated from 2009, although one crucial character was then omitted. During examination of the cultivated material it was noted that the fronds were distinctly glandular, having scattered, capitate glandular hairs on the pinnae, a character unique in British Cystopteris. A further more detailed description is as follows, with illustrations given in Figures 1 and 2. stipe slender, 1.5 – 6 cm. brownish, with scattered pale brown linear scales, frond (lamina) lanceolate, 4 – 10 x 2 – 5 cm, pinnae mainly narrowly ovate, 1.5 – 2.5 x 0.7 – 1.3 cm, with scattered, capitate glandular hairs, which are most visible on the margins, pinnules stalked, the lower oblong-obdeltoid, subtruncate at the apex, often with short, sharply acute points, the upper narrower, linear-obdeltoid, more acute at the apex, the veins mainly terminating in the acute apical points. sori scattered, situated adjacent to the veins, spores echinate, very large, mean c. 50 microns (F.J. Rumsey, pers.comm.). The juvenile sporophytes have well-separated (divaricate), linear-oblong, blunt pinnules, unlike those of typical C. fragilis, but very similar to those of C. alpina. The immature, intermediate fronds usually have either obdeltoid (wedge-shaped) or linear pinnule lobes, again similar to those seen in C. alpina. The mature fronds are more finely dissected, sometimes with pinnae obliquely inclined to the plane of the lamina. The snowdonia ferns, although sharing several characters with C. alpina are readily distinguished from it by the acute points at the apex of the pinnule lobes into which the majority of the veins terminate, and the unique presence of glandular hairs on the pinnae. Plastid DNA examination has also confirmed that the ferns are not C. alpina (F.J. Rumsey, pers. comm.). The relationship of these snowdonia ferns to a described, glandular subspecies of C. fragilis, which occurs in southern, central and western montane regions of mainland Europe is discussed below. TENNANT: CYSTOPTERIS FRAGILIS SUBSP. HUTERI 261 habitat The snowdonia fern is a plant of the alpine zone in Britain, probably not occurring at altitudes below 700m. In the two localities in which it was rediscovered in 2008 it was extremely local and scarce. It occurred on basic rocks in a north-facing vertical crevice and in a recess on the cliff face. No other vascular plants were present, but Cochlearia pyrenaica subsp. alpina (Bab.) Dalby, Minuartia verna (l.) Hiern and Silene acaulis (l.) Jacq. occurred nearby, and typical C. fragilis was not far distant. Records of the Snowdonia Cystopteris specimens in British herbarium collections and references in historical literature showed that the fern that had been rediscovered in snowdonia in 2008 had been collected very much earlier. Identical material was located by the author in 2004-2006 at the Natural History Museum (NHM) in london (Bm) and other relevant Cystopteris specimens were found in the herbarium of the Royal Botanic Garden (RBGE) in Edinburgh (E), and the snowdonia records are given below. This fern was first collected in the late 1680s by Edward lhwyd in snowdonia, and two of lhwyd’s original specimens were recently located by F.J. Rumsey in the sloane herbarium at Bm; these specimens were identical to those found on snowdon by the author in 2008. Further specimens collected by lhwyd, which are probably the same, are present in the herbarium of Oxford University (oXf) (Edgington, 2013). lhwyd’s specimens were also sent to John Ray and details were included in Ray’s Synopsis (Ray, 1724). lhwyd’s record and others from snowdonia were later included by sir J.E. smith (1828; 1834) in his English Flora, but under Cystea regia smith, possibly the first, but illegitimate, binomial name given to this fern, which is discussed later in this paper. The snowdonia Cystopteris would meet the I.U.C.N. Threat Category of ‘critically endangered’, and is especially vulnerable to collecting, therefore precise details of its locations are withheld, but otherwise its presently known records are as follows: snowdonia, sH65, Caernarvonshire, V.C.49 circa 1688, Edward lhwyd (sloane herb., Bm; oXf) snowdon, sH65, V.C.49, 1824, collector unknown, herb.Rev. William Newbould as C. fragilis (Bm) snowdon, 1826, William Wilson (?herb. J.T.I. Boswell-syme), as Cyathea regia (in the C. fragilis folders) (Bm) snowdon, 1847, collector uncertain, herb. F.J. Hanbury, as C. fragilis var. (Bm) Near Glyder Fawr (Cwm Idwal), sH65, V.C.49, 1853, collector’s name illegible, in C. alpina folder (Bm) Cwm Glas, snowdon, 1874, collector uncertain (same sheet gives H. & J. Groves also W.R. linton), as C. fragilis (Bm) snowdon, 1890. J. lloyd Williams, in herb. Arthur Bennett, as C. alpina (later renamed C. fragilis) (Bm) snowdon, 2008, 2009, D.J. Tennant; Cwm Glas area, snowdon, 2008, D.J.T. (herb. DJt; Bm) In several cases not all of the specimens on the sheets for the above records were the same taxon; as few as a single specimen conformed to the snowdonia Cystopteris in question in at least one case. A single specimen on the sheet which represents the 1853 record given above, and those representing the 1853 record are morphologically identical to the others, but require to be re-examined for the presence of glandular hairs. Glandular hairs were present on specimens representing the remaining seven records given above. A specimen marked ‘W.J. Hooker’ in Bm, but undated, was collected at Castell Dinas 262 FERN GAZ. 20(6):255-266. 2017

Bran, Denbighshire, V.C.50, and named Cyathea regia by Hooker, but later renamed as a variety of C. fragilis. This record was listed by Tennant (2010), but is not the snowdonia Cystopteris for which records are given above. This interesting specimen bears resemblance to those found in northern scotland, which were mentioned in the Introduction, and require further investigation. A careful search by the author at Castell Dinas Bran and adjacent rocks failed to locate Hooker’s fern, which is probably extinct there. similarly, a recent but lesser search in Cwm Idwal did not reveal the snowdonia Cystopteris taxon. Following the discovery that the snowdonia taxon was glandular, it was drawn to the attention of the author by another botanist, Brian Burrow, that Flora Iberica (Prada, 1986) describes an infra-specific form of C. fragilis that is distinguished by the presence of glandular hairs, namely subspecies huteri (Hausm. ex Milde) C. Prada & salvo. The description, illustration and spore size given (44-55 (60) microns) for this subspecies matched those of the snowdonia Cystopteris very well. subsp. huteri is a taxon found mainly in montane regions of southern and western mainland Europe, with a general distribution similar to that of C. alpina (lam.) Desv., and has been linked to the latter in scandinavia (F.J. Rumsey, pers. comm.), although the extent of its further geographic limits is not certain. In 2014 and 2015 fieldwork was carried out by the author in the central Italian Dolomites, which is the centre of distribution of subsp. huteri (F.J. Rumsey, pers. comm.). subsp. huteri was found to be frequent there, enabling a comparison to be made with the snowdonia Cystopteris, and material was donated to the NHM for further study by F.J. Rumsey. In the Dolomites C. fragilis subsp. huteri occurred mainly between 1600 and 2400m on dolomitic limestone in open, rocky, alpine woodland, extending to boulders and scree at the base of cliffs well above the tree-line, both habitats with a very rich alpine vascular flora. The other ferns often present were Cystopteris fragilis subsp. fragilis, C. alpina, C. montana (lam.) Desv., Gymnocarpium robertianum (Hoffm.) Newman, Asplenium trichomanes l. subsp. quadrivalens D.E. Meyer emend. lovis, A. trichomanes-ramosum l., A. ruta-muraria l. (including subsp. dolomiticum) and occasionally Woodsia glabella R. Br. ex Richards. Cystopteris fragilis subsp. huteri in the Dolomites was more variable than the snowdonia fern, but close matches to the latter were found there, the majority being intermediate between C. fragilis and C. alpina in general appearance. F.J. Rumsey has agreed that these snowdonia ferns should be referred to C. fragilis subsp. huteri. A very small population of another morphologically distinctive Cystopteris was also found by the author in the Dolomites, very locally at 2300m, and this matched the fern mentioned in the Introduction that occurred in scotland (Figures 3 & 4). The herbarium studies at RBGE (E) showed that this scottish fern had been collected earlier there, and was re-found in Orkney, V.C. 111, in 2010, and later in 2014 by the author in West Ross, V.C. 105, then cultivated from spores. Morphologically similar ferns had also been seen by the author in the French Vanoise and Norwegian Alps. All of these ferns are somewhat intermediate in their morphology between C. fragilis and C. alpina. Those from mainland Europe occur there with C. alpina s. str., although often at lower altitudes, and at least some of these are currently referred to C. alpina. living examples from both recently found localities in scotland that resemble the ferns from mainland Europe have been donated to NHM for biosystematic analyses in order to study their phylogenetic relationships and to establish ploidy levels. TENNANT: CYSTOPTERIS FRAGILIS SUBSP. HUTERI 263

DIScuSSIon Various authors have commented on the difficulties of diagnosing taxa in the Cystopteris fragilis complex by use of morphological characters alone, for example Vida (1974) and Rothfels et al. (2013). Blasdell (1963), however, had successfully developed an evolutionary structure, which in most cases showed a good correlation with recent phylogenetic analyses (Rothfels et al., 2013). The main problems arise because of multiple ancestral origins, the existence of many polyploids, and in Europe, the absence of the ancestral diploid parent. In Europe C. fragilis is tetraploid, hexaploid, or octoploid, but in the British Isles the last is so far unknown. Intermediate ploidy levels have arisen through intraspecific hybridisation, and pentaploid examples are said to be frequent (Vida, 1974). It should therefore be emphasised that the main conclusion in this paper, namely the recognition of C. fragilis subsp. huteri in the British Isles, is very largely based on morphological characters. In this case, however, the morphology of this snowdonia fern is quite distinct from other British variants of C. fragilis; the spores are exceptionally large, and the glandular condition is presently a unique character in European Cystopteris. These characters provide the confidence to refer the snowdonia Cystopteris to this European taxon. As stated above, diploid Cystopteris is not recorded in Europe, but in North America several glandular species of Cystopteris are known and their glandular condition has resulted from the donation of a maternal genome from the diploid plant, C. bulbifera (l.) Bernh. (Rothfels et al., 2003). One glandular North American allohexaploid species, C. laurentiana (Weatherby) Blasdell, which possibly arose as a cross between C. fragilis and C. bulbifera, has been suggested to be synonymous with C. fragilis subsp. huteri (F.J. Rumsey, pers. comm.), but that would imply that C. bulbifera at one time had occurred in Europe, which at present is not proven. The snowdonia fern here referred to C. fragilis subsp. huteri is clearly responsible for all of the previously published erroneous records in Wales for C. alpina s. str. (but see comments above in Results, and below regarding a Welsh fern named by W.J. Hooker). The snowdonia fern does share some characters found in C. alpina, which may have led to the confusion, namely the close similarity of its juvenile sporophytes and the morphology of its immature pinnules (see under Results above and Tennant (2010)). These characters probably arose from an ancestral genome donated from C. alpina, which may therefore have once occurred in Wales. The very large spore size given for C. fragilis subsp. huteri in Flora Iberica was suggested by the authors of this publication to indicate that this taxon is probably octoploid. spores of snowdonia examples were found to be similar in size (F.J. Rumsey, pers. comm.), suggesting that these ferns were likely to be at least hexaploid, if not octoploid, a ploidy level not recorded for C. fragilis in the British Isles. Blasdell (1963) had suggested that spore size in Cystopteris could be used in the determination of the different cytotypes, but Vida (1974) found that, contrary to that opinion, spore size was not always reliable in this respect. R.H. Roberts measured the spores of two different examples of C. alpina for the author. These both had almost identical finely dissected fronds with linear pinnule segments that are associated with classic C. alpina s. str. (Figure 3), but were found to differ widely in spore size (Tennant, 1995), supporting Vida’s findings, which require further investigation using the various cytotypes. Vida (1974) also found that a deliberately induced apogamous diploid of C. fragilis had a much more finely dissected frond than the natural tetraploid plants from which it had been derived, and that can clearly be seen in his Plate 2, fig. 9, which might suggest that degree of frond dissection may not necessarily increase with an increasing 264 FERN GAZ. 20(6):255-266. 2017 ploidy level. Historic Cystopteris nomenclature used by British authors is very relevant to C. fragilis subsp. huteri from snowdonia. This particularly applies to the names ‘incisa’, ‘regia’ and ‘alpina’, and is also relevant to the related taxa from scotland and other taxa from Wales, which are discussed earlier in this paper. smith (1794) in Volume 3, p.163 published the name Cyathea incisa sm., ‘laciniated Cup-fern’ when describing and illustrating a fern collected pre 1790 by E. Forster at a single locality near Walthamstow in south Essex, V.C. 18. smith stated that this fern was not the Polypodium regium of linnaeus and was also different from P. alpinum Jacquin. smith’s description of this fern gives ‘frond bipinnate’, (the pinnule lobes) ‘obtuse with no bristly point’, the last character clearly seeming to distinguish it from the snowdonia subsp. huteri. smith (1828; 1834) later published the name Cystea regia sm., and here he cited lhwyd’s late 17th century snowdon record, two further records from snowdon, namely one by the Rev. Hugh Davies in the early 19th century and one by W. Wilson in 1826, and a further record by Mr. Griffith from Cwm Idwell (Idwal), which all almost certainly referred to the snowdonia subsp. huteri. smith, however, surprisingly also added here the Essex fern (Cyathea incisa and C. regia Forster), thus seeming to suggest that these were synonymous with the snowdonia ferns, even though their descriptions differed. smith also stated that Cystea regia sm. was unquestionably distinct from every other British fern, and from the ‘regia’ of alpine stations (in Europe), presumably meaning the fern now referred to Cystopteris alpina (lam.) Desv., although confusingly he included Aspidium regium sw. (1802) in the synonymy. W.J. Hooker (1842) then grouped together all of smith’s taxa under C. alpina Desv., and added ‘tripinnate’ to smith’s earlier ‘bipinnate’ description of these taxa. Hooker had earlier (before 1842) confirmed the name of one of the specimens from Essex collected by Forster as Cyathea regia (stansfield, 1930), and in the same period had also given the same name to an undated specimen in Bm that had been collected in Denbighshire. V.C. 50. These Essex and Denbighshire records, in the sense of Hooker (1842), therefore represent the first and second British records respectively for C. alpina. J. Backhouse’s record from Upper Teesdale in Yorkshire, V.C. 65 in 1872 (Tennant, 2010) would therefore be the third British record. In spite of smith (1828; 1834) and Hooker (1842) including the snowdonia ferns under the species which is now referred to C. alpina (lam.) Desv., C. fragilis subsp. huteri from snowdonia is shown here to be different, and its first name, Cystea regia sensu sm. (parte) should be retained only in synonymy. The use of the name ‘regia’ for some of these ferns is, however, incorrect on the assumption that the name ‘regia’ was entirely derived from P. regium l. The type sheet of P. regium in lInn contains more than one species and P. regium was therefore a ‘nomen ambiguum’. None of the species on this sheet represent the taxa discussed in this paper, including C. alpina (lam.) Desv.. This has long been known (stansfield, 1929), and much earlier, for example P. regium l. was included by J.T. syme in the synonymy under C. fragilis in the third edition of English Botany, Vol. 12 (1886), which would be correct, although an individual specimen still requires to be selected from the type sheet and formally lectotypified as such. Other authors also considered ‘regia’ to be distinct. Milde made ‘regia’ a variety of C. alpina (Kestner, 1930), and stansfield (1929) proposed the name C. alpina var. obtusa for Forster’s Cystopteris from Essex. After examining the Essex specimen, stansfield pointed out that it was bipinnate, with only shallowly notched pinnule lobes, many of TENNANT: CYSTOPTERIS FRAGILIS SUBSP. HUTERI 265 the veins terminating in an apical point, unlike C. alpina s.str., which is at least tripinnate with virtually all the veins ending in notches. Kestner (1930) had stated that the position of the vein-endings was the only reliable character in the determination of C. alpina, additionally the Cystopteris key given in Murphy and Rumsey (2005) similarly relies on this character for the determination of C. alpina. The first name given to the Essex fern, Cyathea incisa sm. (1794), would be a legitimate name at specific level, and for the Denbighshire Cystopteris as this appears to belong to the same taxon. It is doubtful, however, whether these ferns could be justified as a species distinct from C. alpina, in which case the name would be reduced to synonymy under the latter. The Essex and Denbighshire ferns are both thought to be extinct at their original localities. A similar Welsh taxon from Merionethshire, V.C. 48, has not been collected since 1928, so none of these ferns are presently available for biosystematic studies that might elucidate their correct status. The Cystopteris taxon from scotland discussed briefly above also seems to have affinities with C. alpina. Two examples of this and a morphologically identical fern from Italy are in cultivation by the author, and material is now at NHM for further studies to establish their status. several recently confirmed specimens of C. alpina seen by the author in the Tromsø Museum herbarium had exceptionally broad pinnule lobes. Following the fieldwork by the author, uniformly similar Cystopteris forms were also encountered in alpine regions of France, Italy, and in scotland, some of which are presently determined as C. alpina (lam.) Desv. This might suggest that the concept of C. alpina s.str. as a fern restricted to one with a finely dissected frond and linear pinnule lobes, and with the veins virtually all ending in notches, unlike the ‘regia’ of some authors, may be one that is too narrow. An alternative suggestion may be that these ferns with broader pinnules truly represent a distinct taxon, which is affiliated to C. alpina through the presence of a genome from this species. Hopefully biosystematics will resolve this. Cystopteris fragilis subsp. huteri is presently known in Britain only from two localities in North Wales, where it is very local and scarce. It meets the I.U.C.N. Threat Category of Critically Endangered (CR) and should be considered for inclusion in the British Red Data list, its main vulnerability being one of collecting.

acknoWlEDgEmEntS I would especially like to thank Alison Evans for assistance in the preparation of the manuscript, Clive stace for general advice, Brian Burrow for information on the spanish publication, Clive Jermy for details of previous papers, Eric Meek for organising collection of specimens from one of the scottish sites, Fred Rumsey for information, comments, opinions, and undertaking the preliminary studies at NHM, Arve Elvebakk, Torstein Engelskjøn and Douglas McKean for access to herbarium material, and John Ratcliffe at the Countryside Council for Wales for permission to collect material.

REfEREncES BlAsDEll, R.F. 1963. A monographic study of the fern genus Cystopteris. Mem. Torrey. Bot. Club 21:1-102 EDGINGTON, J. 2013. Who found our ferns? British Pteridological society, special Publication 12. HOOKER, W.J. 1842. The British Flora, Vol.1. longman, london JERMY, A.C. & HARPER, l. 1971. spore morphology of the Cystopteris fragilis 266 FERN GAZ. 20(6):255-266. 2017

complex. British Fern Gazette 10: 211-213. KEsTNER, P. 1930. Cystopteris alpina Desv. British Fern Gazette 6 (2): 33-37. MURPHY, R.J. & RUMsEY, F.J. 2005. Cystopteris diaphana (Bory) Blasdell (Woodsiaceae) – an overlooked native new to the British Isles? Watsonia 25:255-263. PRADA, C. 1986. Cystopteris pp. 115-121 in Flora Iberica, ed. s. Castroviejo. Madrid, C.s.I.C. RAY, J. 1724. synopsis Methodica stirpium Britannicarum, edn. 3 1973 facsimile, Ray society, london. ROTHFEls, C., WINDHAM, M.D., PRYER, K.M. 2013. A plastid phylogeny of the cosmopolitan fern family Cystopteridaceae (Polypodiopsida). systematic Botany 38 (2): 295-306. The American society of Plant Taxonomists. sCHMAKOV, A. 2001. synopsis of the ferns of Russia. Turczaninowia 4: 36-72. sMITH, J.E. 1794 . English Botany, edn. 1, Vol. 3:163. london. sMITH, J.E. 1828. The English Flora, edn. 1, Vol. 4:302. london. sMITH, J.E. 1834. The English Flora, edn. 2, Vol. 4:289. longman, london. sTANsFIElD, F.W. 1929. Cystopteris alpina Desv. British Fern Gazette 6 (1): 15-19. sTANsFIElD, F.W. 1930. Cystopteris alpina Desv. British Fern Gazette 6 (3): 71-72. TENNANT, D.J. 1995. Cystopteris fragilis var. alpina Hook. in Britain. The Naturalist 120: 45-50. TENNANT, D.J. 1996. Cystopteris dickieana R. sim in the central and eastern scottish Highlands. Watsonia 21: 135-139. TENNANT, D.J. 2010. The British records of Cystopteris alpina (lam.) Desv. Watsonia 28: 57-63. VIDA, G. 1974. Genome analysis of the European Cystopteris fragilis complex. 1 Tetraploid taxa. Acta Biologica Academiae scientiarum Hungaricae. Tomus 20 (1-2): 181-192. FERN GAZ. 20(6). 2017 267 268 FERN GAZ. 20(6). 2017

InStRuctIonS foR authoRS papERS should not usually exceed 20 printed pages and are generally expected to be considerably shorter. As well as reports of original research, review articles and short notes, e.g. new records, are encouraged. The senior author should supply an email address to facilitate correspondence. REVIEWS should cover recent developments in any aspect of research on ferns and lycopods. This can include unpublished research by the author, and/or the possible direction of future research. They should be aimed at the general reader who may not be an expert in the chosen topic. Proposals for a review should be discussed first with the review editors. manuScRIptS should be submitted in English (British) in electronic format in 10-point Times New Roman font and single spaced. Electronic versions of text and tables should be compatible with WORD, with figures as TIFF files, and sent as email attachments. All manuscripts will be refereed. thE tItlE should reflect the content of the paper and be in BOlD CAPITAls (11-point) and centrally aligned. Generic and specific names should be in italics and any title containing a generic or specific name should be followed by the family name in brackets e.g. TRICHOMANES SPECIOSUM (hymEnophyllacEaE) In SouthERn SpaIn authoR aBBREVIatIonS should follow Pichi sermolli’s (1996) Authors of scientific names in Pteridophyta, Royal Botanic Gardens, Kew. maIn hEaDIngS: should be in BolD capItalS (10-point) and centrally aligned. SuBSIDIaRy hEaDIngS: should be in bold, the first letter of each word in capitals, the rest in lower case and left-aligned. authoRS’ namES anD full aDDRESSES (including email address): follow the title and are centrally aligned. kEy WoRDS: up to ten. aBStRact: should reflect the content of the paper. fIguRES: there is no distinction between photographs and line drawings in numbering. All should be supplied in mono or colour, TIFF format, 300 dpi and CMYK. (please contact the Production Editor with queries), with a scale bar where appropriate. lettering or numbers (Arabic) should be in the bottom left using uppercase Times Roman. Figure captions should be on a separate sheet. taBlES: can be printed in either portrait or landscape format. Authors should consider this when preparing tables. Authors should ensure that tables fit the printed page size in a legible form. pagESIZE: The Gazette has a useable page size of 123 × 204 mm. This can be used in portrait or landscape mode but any supplied artwork should not be larger than this. mEaSuREmEntS: follow the metric system. chEcklIStS: follow the format of Baksh-Comeau, Fern Gaz. 16(1, 2): 11- 122. REfEREncES: follow the style of a recent issue of The Fern Gazette, e.g.:- HOOKER, W.J. 1864. species Filicum, 5. Dulau & Co., london. MORTON, C.V. 1947. The American species of Hymenophyllum, section Sphaeroconium. Contr. U.s. Natl. Herb. 29(3): 139-201. sTEVENsON, D.W. & lOCONTE, H. 1996. Ordinal and familial relationships of pteridophyte genera. In: CAMUs, J.M., GIBBY, M. & JOHNs, R.J. (Eds) Pteridology in perspective, pp. 435-467. Royal Botanic Gardens, Kew. JouRnal aBBREVIatIonS: should follow Botanico Periodicum Huntianum & supplements. Alterations from the original text at proof stage will be charged for unless they are minor points of detail. A pdf will be provided free to the senior author. FERN GAZ. 20(7): 269-279. 2018 269



Department of Botany, Federal University Lafia, PMB 146, Lafia, Nasarawa State, Nigeria *Correspondence: [email protected], +2348068997606

Key words: Pneumatopteris afra, Nasarawa, Pteridophytes, Taxonomic survey

ABSTRACT The study was carried out to investigate the taxonomic occurrence of Pteridophytes in Lafia and Doma Metropolis of Nasarawa State, Nigeria. A total of 16 locations were sampled in Lafia and Doma respectively. These locations include wetlands, swamps, forest shade and trees. From these locations, ferns were collected, pressed and identified. Their relative abundances in each location were calculated and their ethnobotanical uses by the local dwellers were documented using structured questionnaires. Ferns were observed only in 12 locations in Lafia and nine locations in Doma. Seven species of ferns were identified namely, Pneumatopteris afra, Nephrolepis biserrata, N. undulata, N. cordifolia, Adiantum capillus-veneris, Adiantum sp. and Pityrogramma calomelanos. Pneumatopteris afra was found to be the most widely distributed fern. Of the ferns observed, terrestrial ferns had the highest occurrence (85.7%) in Lafia with only 50% in Doma. Pneumatopteris afra was found to have the highest relative frequency while Nephrolepis biserrata and N. undulata had the lowest relative frequency in Lafia and Doma. All these species have been reported by the local dwellers to have ethnobotanical uses such as in the treatment of ulcer, fever, typhoid, chest pain, stomach pain and diabetes. Also, Pityrogramma calomelanos had the highest percentage awareness by the local dwellers. It is concluded that Lafia is more rich and diverse in fern species than Doma. Locations where ferns were absent are due to the long-term alterations in the microclimatic conditions by various human activities in those places.

INTRODUCTION In most parts of Africa ferns are more diverse in the eastern than in the western mountains (Moran & Smith, 2001). Ecological and physiological factors such as habitat, seasonality, elevation, soils, rainfall, cloud cover and temperature have been identified as the determinants for the diversity, richness and distribution of ferns in most parts of the world (Kornas, 1993). Nigerian’s first biodiversity report stated that ferns and fern allies have been observed to grow in most ecological zones but different habitats (Akinsoji et al., 2016). Although, ferns have wide coverage in Nigeria, due to habitat change by factors such as unplanned urbanization, over-exploitation of minerals and farming, many species have been reduced, endangered or are on the verge of extinction (Dixit, 2000). Pteridophytes are often restricted to specific micro-habitats. However, as a result of serious threats faced by these plants coupled with the fact that they are underutilized 270 FERN GAZ. 20(7): 269-279. 2018 and overlooked in this part of the world because of their relatively small size and crytogamic nature, there is need for adequate survey and documentation of their occurrences, locations and economic values. Therefore, this research aimed to investigate the occurrence, diversity and ethnobotany of ferns in Lafia and Doma metropolis, Nasarawa State, Nigeria.

MATERIALS AND METHODS This study was carried out at Lafia and Doma Local Government areas of Nasarawa State, Nigeria. Habitats assessed for the various diversities of pteridophytes include river banks, streams, forest (under shade), arboreal (top of trees), swamps and some other wet lands. The study was conducted towards the end of rainy season in 2016 and early dry season of 2017. Plants specimens were collected using Random sampling method. Various fern species parts were collected which included , fronds and roots. The samples collected were all recorded in the field. Representative of all collected fern species or specimens, were also photographed. These specimens were later used for identification and to provide permanent records for future use. The geographical coordinates of each sampling location were taken by using a GPS device. Mature fern species were collected in each location and pressed, dried, poisoned then mounted on standard herbarium sheets and classified according to standard procedures. Identifications were made with the aid of taxonomic literature (Oloyede & Odu, 2011). In the field proper care was taken in selecting the plant materials to enable correct identification. Specimens were collected in a good condition, free of insect damage or diseases. Voucher specimens are preserved in the Department of Botany Herbarium, Federal University, Lafia.

Ethnobotanical Uses A total of ninety (90) structured questionnaires were administered to local dwellers of all the locations visited to gather information on the knowledge of the plants by the people, the uses of the plants and the part of the plant that had been used. The relative frequency of occurrence of each fern species was determined to assess the distribution of the species. No of occurences of individual fern species R F = x 100 No of occurences of all ferns Sørensen Similarity Coefficient was calculated to indicate their floristic similarities between the two towns (Lafia and Doma) as follows: 2W Sc = x 100 a + b where Sc is the similarity coefficient, W is the species occurring in both communities under consideration, a is the number of species occurred in Lafia and b is the number of species occurred in Doma. The relative abundance of the ferns at each site was determined according to Bongers et al. (1988) and Kayode (1999): less than five individuals as Rare; 5 to 10 as Occasional; 11 to 30 as Frequent; 31 to 100 as Abundant and over 100 individuals as Very Abundant.

RESULTS Ferns occurrence and distribution in Lafia A total of 16 locations were sampled in Lafia, of which ferns were observed in only 12 AKOMOLAFE & SULAIMON: PTERIDOPHYTES IN NASARAWA STATE 271

Table 1: Sampling Locations In Lafia L.G.A


1 Akurba osanya AKB 8.47104N 8.58747E Present

2 Opposite triple Z farm OTF 8.45695N 8.57489E Present

3 Gandu GAN 8.45116N 8.57304E Absent

4 Agudu AGD 8.51771N 8.52483E Absent

5 Akunzan tabori AKTR 8.4105N 8.56368E Present

6 Beside college of Agric BCA 8.55723N 8.54292E Present

7 Opposite college of Agric OCA 8.51061N 8.52016E Absent

8 Inside college of Agric ICA 8.53292N 8.53659E Present

9 Between lafia and shabu river B/W L$SR 8.58883N 8.5559E Present

10 Between lafia shabu river B/W L$SR2 8.56688N 8.54802E Present

11 Shabu one SHBI 8.57557N 8.55119E Present

12 Shabu two SHB2 8.56688N 8.54802E Present

13 Shabu three SHB3 8.57405N 8.55062 Present

14 Akunzan tabo well AKTW 8.49018N 8.53358E Present

15 FUL Auditorium FUL AUD 8.47066N 8.58688E Present

16 Tudun amba TUA 8.49216N 8.50355E Absent 272 FERN GAZ. 20(7): 269-279. 2018 269-279. 20(7): GAZ. FERN 272 Table 2: The locations occupied by the ferns and the respective abundance status in Lafia S / Species Location / Abundance status N


1 Adiantum sp. X X X X X X X X X X X X X √ A X X

Adiantum 2 X X X X X X X X X X X X X √ A X X capillus-veneris

Pneumatopteris 3 √ VA √ F X X X √ VA X X √ VA √ F X √ A √ A X X X afra

Nephrolepis 4 X X X X X X X X X X X X √ R X X X bisserata

Nephrolepis 5 X X X X X X X X X X X X X X √ F X cordifolia

Nephrolepis 6 X X X X √ F X X √ VA X √ A √ O X √ O X X X undulata

Pityrogramma 7 X X X X √ VA X X X X X X X X √ A X X calomelanos

KEY: √ means present, X means absent A – abundant, VA – very abundant, F – frequent, O – occasional, R – rare AKOMOLAFE & SULAIMON: PTERIDOPHYTES IN NASARAWA STATE 273

Figure 1: Relative frequency of Fern species in Lafia town

Figure 2: Relative frequency of Fern species in Doma town 274 FERN GAZ. 20(7): 269-279. 2018

Table 3: Sampling Locations in Doma LGA


1 Omenza 1 Omz1 8.39514N 8.35005E Present

2 Omenz2 Omz2 8.39017N 8.34801E Present

3 Osota1 OST1 8.39013N 8.3481E Absent

4 Osota 2 OST2 8.39278N 8.35039E Absent

5 GSS Doma GSSD 8.39188N 8.35039E Present

6 Govt. college Doma GCD 8.39024N 8.35762E Absent

7 Arumangye ARG 8.39004N 8.35762E Absent

Federal Science and 8 FSTC 8.39131N 8.34863E Absent Technical College

9 Ogbobula1 OGB1 8.39695N 8.34863E Present

10 Ogbobula2 OGB2 8.39074N 8.34881E Absent

11 Okpuripu OKPU 8.39145N 8.3498E Absent

12 Okussupa OKSP 8.3906N 8.34861E Present

13 Ipugigu IPG 8.39102N 8.3492E Present

GGSSD formal 14 GGDFH 8.4043N 8.36809E Present hostel

15 Campo l CMP1 8.46461n 8.43512E Present

16 Campo 2 CMP2 8.44435N 8.41107E Present AKOMOLAFE & SULAIMON: PTERIDOPHYTES IN NASARAWA STATE 275 X √ A √ CMP2 X √ VA √ CMP1 X √ O GGFH X IPG √ VA √ X √ VA √ OKSP X X OKPU X X OGB2 X √ A √ OGB1 X X FSTC X X ARG Location / Abundance status Location / X X GCD X √ F GSSD X X OST2 X X OST1 X √ A √ OMZ2 X √ VA √ OMZ1 afra The locations occupied by the ferns and the respective abundance status in Doma Species undulata Nephrolepis Pneumatopteris 2 1 N S/ Table 4: Table KEY: √ means KEY: present, X means absent – very abundant, F – frequent, – O abundant, A VA – occasional, R - rare 276 FERN GAZ. 20(7): 269-279. 2018 drying & boiling boiling boiling, MODE OF PREPARATION Drying Drying boiling boiling boiling boiling drying, freshly pounded, boiling boiling Boiling Boiling &air drying air drying Boiling, freshly pounded &drying air drying air drying, freshly pounded, boiling Boiling &air drying Boiling, freshly pounded, air drying leaf PARTS USED PARTS roots & fronds whole plants root Leaflets, fronds root leaflets fronds leaflets whole plants root Whole plants roots whole parts, roots &fronds whole parts leaf whole parts &roots Whole parts &roots leaflets only piles, stomach pain Typhoid, fever, headache, ulcer fever, Typhoid, treatment of chest pain, reducing prolonged labour ETHNOBOTANICAL USES ETHNOBOTANICAL strengthening of bones healing of wounds caused by guinea worm enhancement of male sexual cells healing of stomach pain, fever treating shortage of blood breast cancer diabetes Measles fibroid, shortage of blood, diabetes, abnormal menstruation, stomach pain piles, back pain, waist pain & joint cases sickler cases, high blood pressure, bone fracture, /scorpion bite, skin rashes &diabetes goiter, headache &rheumatism Eyesight issues, bone fracture, goitre, high blood pressure, snake / scorpion bite &diabetes Snake bite, convulsion, high blood pressure, poisoning, goitre, bone fracture, sickle cell anemia Convulsion, diabetes, headache, pile, Gonorrhea, scorpion bite, dysentery 90 40 20 30 30 100 33.3 % AWARENESS % Ethhnobotanical uses of the fern species in Lafia SPECIES Pityrogramma calomelanos Nephrolepis undulata Adiantum capillus-veneris Adiantum sp Nephrolepis cordifolia Nephrolepis biserrata Pneumatopteris afra 1 2 4 5 6 7 3 S/N Table 5: Table AKOMOLAFE & SULAIMON: PTERIDOPHYTES IN NASARAWA STATE 277 MODE OF PREPARATION Boiling Boiling Drying, boiling &freshly pounded Leaf, stem &whole parts Leaf, stem &whole parts Leaf, whole parts PARTS USED PARTS increases male sexual stamina rheumatism, stmach pains, tuberculosis, fever ETHNOBOTANICAL USES ETHNOBOTANICAL Healing of wounds, asthma, pneumonia, cough &ulcer 50 50 % AWARENESS % Ethhnobotanical uses of the fern species in Doma SPECIES Pneumatopteris afra Nephrolepis undulata Nephrolepis 1 2 S/N Table 6: Table 278 FERN GAZ. 20(7): 269-279. 2018 locations (Table 1). Seven species of ferns were identified from the sampled locations in Lafia: Pneumatopteris afra (Christ) Holttum, Nephrolepis undulata (Afzel.) J.Sm., Nephrolepis cordifolia (L.) K.Presl, Nephrolepis biserrata (Sw.) Schott, Adiantum sp, Adiantum capillus-veneris L. and Pityrograma calomelanos (L.) Link (Table 2). Pneumatopteris afra was found to be the most widely distributed fern in Lafia where it was found in seven locations. Of the ferns observed, terrestrial ferns were most frequent with 85.7% occurrence while epiphytes were least frequent at 14.3% . Pneumatopteris afra was found to have the highest relative frequency at 65% while Nephrolepis biserrata and N. undulata had the lowest (5%) (Figure 1). The similarity coefficient between the two towns is 22.2%.

Ferns occurrence and distribution in Doma In Doma, from a total of 16 locations sampled ferns were observed in only nine locations (Table 3). However, in Doma, only two species of ferns were recorded, one terrestrial and the other epiphytic, namely Pneumatopteris afra and Nephrolepis undulata (Table 4). Pneumatopteris afra was found to be the most widespread fern in Doma where it was found in six locations. The relative frequency of Pneumatopteris afra was greater (66.67%) than that of N. undulata (33.34%) (Figure 2).

Ethnobotanical Uses of Ferns in Lafia The results of the survey of ethnobotanical uses and awareness of the ferns in Lafia are presented in Table 5. Every species was discovered to have ethnobotanical uses among the local population. Pneumatopteris afra had 90% percentage awareness and it was used for treating ulcer, fever, typhoid and piles. Pityrogramma calomelanos had the highest percentage awareness (100%) and was used for enhancement of male sexual performance and treating chest pain. The lowest percentage awareness (20%) was recorded for Nephrolepis cordifolia which was used for treating high blood pressure and skin rashes.

Ethnobotanical Uses of Ferns in Doma In Doma, Pneumatopteris afra had 50% percentage awareness and was used for treating rheumatism, stomach pains and tuberculosis (Table 6). Nephrolepis undulata also had 50% percentage awareness and was used for treating wounds, asthma and pneumonia.

DISCUSSSION AND CONCLUSION The occurrence of ferns in the two towns studied had only two species in common. As reported by Jones et al., (2011), some fern species are highly flexible and are found in nearly all habitats and also have various life forms. High species occurrence in areas with suitable environmental conditions showed that such species have adapted to the environment (Richard et al., 2000). Consequently, the relatively high number of ferns species found in Lafia compared with Doma might be the result from more favourable microclimatic conditions than in Doma, where conditions have been affected by human activities such as deforestation and farming. This is supported by Sumesh et al. (2014) who stated that human activities usually increase pressures on plants. The occurrence of some species in Lafia such as Pneumatopteris afra, Nephrolepis undulata and Adiantum capillus-veneris with high relative frequencies suggests that these species will persist in this area if the environmental conditions remain favourable. But species like Adiantum sp., Nephrolepis cordifolia, Nephrolepis biserrata and Pityrogramma calomelanos with low relative frequencies are more likely to become AKOMOLAFE & SULAIMON: PTERIDOPHYTES IN NASARAWA STATE 279 threatened in the future. Also in Doma town, only two ferns were recorded, and Pneumatopteris afra was more frequent than Nephrolepis undulata. The paucity and often total absence of ferns in some locations sampled in Lafia (four sites) and Doma (seven sites) is a clear indication of the accumulated long-term anthropogenic activities in the areas resulting in loss of ferns species at these sites. It is concluded that, in comparison, Lafia has a more rich and diverse ferns flora than Doma, and that despite the presence of relatively few species, all have been used by the local dwellers for various economic purposes. Therefore, conservative measures should be put in place by the Government to avoid future extinction of these ferns.

REFERENCES AKINSOJI, A., AGBOOLA, O.O., ADEONIPEKUN, P.A., OYEBANJI, O.O., ADENIYI, T.A & AJIBODE, M.O. 2016. Distribution of pteridophytes in parts of Lagos and Osun State. Ife J. of Sci. 18(2): 447-453 BONGERS, F., POPMA, J., MEAVE DEL CASTILLO, J. & CARABIAS, J. 1988. Structure and floristic composition of the lowland rainforest of Los Tuxtlos, Mexico. Vegetatio. 74: 55-80. DIXIT, R.D. 2000. Conspectus of pteridophytic diversity in India. Fern J. 17: 77-91. JONES, N.A., ROSS H., LYNAM, T. PEREZ P. & LEITCH, A. 2011. Mental models: an interdisciplinary synthesis of theory and methods. Eco. & Soc. 16(1): 46 KAYODE, J. 1999. Phytosociological investigation of compositae weeds in abandoned farmlands in Ekiti State, Nigeria. Compositae News. 34: 62-68. KORNAS, J. 1993. The significance of historical factors and ecological preferences in the distribution of bibliography of African Pteridophytes. J. of Biogeo. 20: 281-286. MORAN, A.C & SMITH, A.R. 2001. Phytogeographical relationship between Neotropical and African –Madagascar pteridophytes. Brittonia 53: 304-351. OLOYEDE, F.A. & ODU, E.A. 2011. Taxonomic Evaluation of Homosporous Leptosporangiate ferns in Southwestern Nigeria. J. of Curr. Res. 2(11): 009-017 RICHARD, A. F., DEWAR, R. E., SCHWARTZ, M. & RATSIRARSON, J. 2000. Mass change, environmental variability and female fertility in wild Propithecus verreauxi. J. of Human Evol. 39:381-391. SUMESH, N.D., SUBASH, M.D.C. & RAMACHANDRA, T.V. 2014. Conservation of Pteridophytes to maintain vital link between lower and higher group of plants. Energy and Wetlands research group, center for Ecological Sciences, Indian Institute of Science, Bangalore, pp. 560012 280 FERN GAZ. 20(7). 2018


The correct title and citation of this article should be: Gensel, P.G. 2017. Silurian-Devonian origins of ferns and lycophytes - what we know, what we need to find out. Fern Gaz. 20(6): 217-242. FERN GAZ. 20(7): 281-291. 2018 281


Rense Haveman Central Government Estate Agency, Ministry of the Interior and Kingdom Relations, Expert Centre Technique/Nature, P.O. Box 47, 6700 AA Wageningen, the Netherlands. Email: [email protected]

Keywords: Argyll, Melampyro-Holcetea mollis, Dryopteris, Oreopteris, climate, Luzulo luzuloides-Thelypteridetum limbospermae, phytosociology

ABSTRACT On the basis of five phytosociological relevés, the fern-meadow vegetation with Oreopteris limbosperma and Dryopteris cambrensis from the region around Oban, Argyll, West Scotland is described and compared with similar communities in the Netherlands and West Germany. It is concluded that these fern-meadows can be assigned to the Luzulo luzuloides-Thelypteridetum limbospermae, which in Central and continental West Europe is restricted to forest edges. In West Scotland this community is also common in open landscapes, due to the hyperatlantic climate. In comparison with the fern communities in the Netherlands and Germany, the Scottish examples are very rich in fern species, illustrating the optimal development of the community in West Argyll. Possible future development of this community in the Netherlands are discussed in the light of the increase in records of Oreopteris limbosperma and the ecological requirements of Dryopteris cambrensis.

INTRODUCTION For a continental phytosociologist, one of the striking features of the vegetation of the coastal lowlands of West-Argyll is the omnipresence of ferns in open landscapes (Figure 1). Well-known is the invasive character of Pteridium aquilinum (L.) Kuhn in extensively managed grasslands all over the British Isles (Rodwell et al., 1992), but many other fern species are found in the dense tall-forb vegetation on slopes in the hyperatlantic lowland around Oban. After Pteridium, probably Blechnum spicant (L.) Sm. is the most frequent species, but also Dryopteris cambrensis (Fraser-Jenk.) Beitel & W.R.Buck can be found on virtually every slope and this species is much more abundant than Blechnum. Other species which can be found often are, in no particular order, Dryopteris filix-mas (L.) Schott, D. borreri (Newm.) Newm. ex Oberh. & Tavel, D. dilatata (Hoffm.) A.Gray, D.carthusiana (Vill.) H.P.Fuchs, Athyrium filix-femina (L.) Roth, Phegopteris connectilis (Michx.) Watt, and Oreopteris limbosperma Holub. In West-Europe, fern-dominated forest edge communities from lowland, hills and lower mountain areas were recently placed in a newly described alliance, the Holco mollis-Athyrion felicis-feminae (Royer et al.) Haveman & Weeda (class Melampyro- Holcetea mollis Passarge ex Weeda & Haveman; Weeda & Haveman, 2017). The first author who drew the attention to these fern-rich forest edge communities was Wittig (2000), who described Oreopteris-rich forest-edges from the German lower mountain ranges in the Rothhaargebirge, Taunus, and Schwarzwald as Luzulo luzuloides- Thelypteridetum limbospermae Wittig. Apart from Oreopteris limbosperma (syn.: 282 FERN GAZ. 20(7): 281-291. 2018

Thelypteris limbosperma (All.) H.P.Fuchs) also Athyrium filix-femina, Phegopteris connectilis, Blechnum spicant, Gynmocarpium dryopteris (L.) Newman, Dryopteris carthusiana, D. dilatata, and D. filix-mas are fern species mentioned for this vegetation type. This Oreopteris-community is not strictly oreophytic (growing in the mountain belt): Bremer (2016) and Weeda & Haveman (2017) recorded the Luzulo- Thelypteridetum also from the Netherlands, i.e. from the lowlands of Northwest-Europe. Rodwell et al. (1992) described a similar community as Thelyptris limbosperma- Blechnum spicant community (U19) from moist base-poor peaty soils on steep, sheltered banks at low to moderate altitudes through the wetter west and north of Britain. Remarkably enough, other fern species are rare in this community: only Polypodium vulgare L. and Athyrium distentifolium Tausch ex Opiz are mentioned in the table published by these authors. Weeda & Haveman (2017) included this British community in the Luzulo-Thelypteridetum. During a three-weeks summer holiday in July 2017, I was able to study some of the fern-meadow communities in the wider surroundings of Oban, West-Argyll, Scotland. I was especially interested in the communities with the alleged mountain-dwelling Oreopteris limbosperma and Dryopteris cambrensis, because the latter is very rare in the Netherlands. From the species composition, it was immediately clear that the Scottish fern-meadow community bears a very close resemblance to the Luzulo-Thelypteridetum, but also that it had characteristic features of its own. In this paper, I will describe this community on the basis of a few phytosociological relevés. It will be compared with the continental fern communities of the aforementioned Holco-Athyrion and more in particular the Luzulo-Thelypteridetum, in order to gain a better understanding of these communities, both in hyperatlantic Scotland and the subatlantic Netherlands.

Figure 1. Fern-meadow on a lowland hillside in West-Argyll near Loch Fyne (photo: R. Haveman) HAVEMAN: FERN-MEADOW VEGETATION 283

METHODS On July 6th and 7th 2017, I made five phytosociological relevés of fern-rich forest-edges in Ardcastle Forest at the shore of Loch Fyne and the area of Glencruitten House just outside Oban, according to the methods of the French-Swiss school (Mueller-Dombois & Ellenberg, 1974; Westhoff & Van Der Maarel, 1978). In floristically homogeneous parts of the vegetation of 8-20 m2, all species were recorded, and their abundance was estimated using the 9-level extended scale of Braun-Blanquet (Westhoff & Van Der Maarel, 1978) in every single layer (tree, shrub, field, and moss layer). The relevés were digitised and stored in TurboVeg (Hennekens & Schaminée, 2001) in the personal database of the author. After export from TurboVeg, the relevés were imported in JUICE (Tichý 2002) for further analysis. In JUICE, all species were ordered according to their phytosociological position according to Weeda & Haveman (2017). Both a full and a (heavily abbreviated) synoptic table (in frequency percentages) of constant species (frequency > 40%) were exported, and the synoptic table compared with the synoptic tables published by Weeda & Haveman (2017) and Wittig (2000) by arranging them in a longitudinal order (Scotland-the Netherlands-Germany). In the full table, all pteridophytes are included, as well as all species occurring in more than one relevé, or in one relevé with a cover ≥ 5%. In the synoptic tables, only the species with a frequency > 10% are included in at least one column.

RESULTS In the Scottish relevés (Table 1), 11 fern species were recorded in total, and one fern hybrid. The most frequent fern species in the table is Blechnum spicant (in all five relevés), followed by Oreopteris limbosperma, and Dryopteris cambrensis (both in four relevés). In all relevés, Dryopteris species have an abundance prevalence over the other fern species. Other species recorded in at least four out of five relevés are Luzula sylvatica (Huds.) Gaudin, Holcus mollis L., (Hedw.) Schimp., Rhytidiadelphus squarrosus (Hedw.) Warnst. and Polytrichum formosum Hedw.. In all relevés, more than one fern species was found, and relevé 1 in Table 1 is especially rich in ferns: in the vegetation sample of 20 m2 9 pteridophytes were recorded. There are clear differences between the Scottish fern-communities with Oreopteris limbosperma and Dryopteris cambrensis (Table 2, column 1), and the Luzulo- Thelypteridetum in the Netherlands (Table 2, column 2) and Germany (Table 2, column 3). Especially striking is the richness in ferns in the Scottish community: on average, 6.2 fern species are recorded in the Scottish relevés, a number that declines strongly when going to the suboceanic regions in the Netherlands and Germany (viz. 3.9 and 3.4 respectively). Also their absolute number in the Scottish dataset is higher than in the other two (12, vs. 7 and 9 respectively), although the number of Scottish relevés is evidently lower than in the other two groups. Dryopteris cambrensis, D. borreri and Pteridium aquilinum are exclusively or, in case of the latter, mainly recorded in the Scottish relevés, although it cannot be ruled out that Dryopteris affinis Fraser-Jenk., mentioned in one relevé from Germany, is in fact D. borreri. Also Blechnum spicant and Dryopteris filix-mas have a clear optimum in the Scottish relevés, and the same holds true for Holcus mollis, Luzula sylvatica, montanum L., Betula pendula Roth (juveniles), and the mosses Hylocomium splendens and Rhytidiadelphus squarrosus. Typical for the Dutch relevés are Molinia caerulea (L.) Moench and Quercus robur L. (juveniles), whereas the German relevés are differentiated by Luzula luzuloides (Lam.) Dandy & Wilmott. However, the three regional groups of relevés have many taxa in 284 FERN GAZ. 20(7): 281-291. 2018

Table 1. Full table of relevés of fern-rich communities in West-Argyll. Location: G = Glencruitten House, Oban; A = Ardcastle Forest.

Table number 12345 Relevé number (RH 17.) 167 164 165 168 166 Date (July 2017) 76677 Location GAAGG Area (m2) 20 8 10 20 10 Exposition NW NZNW Inclination (degrees) 45 40 45 80 80 Total cover (%) 100 99 95 100 100 Cover herb layer (%) 95 90 90 95 80 Cover moss layer(%) 80 100 10 80 70 Avg. height herb layer (cm) 60 80 60 60 60 Max. height herb layer (cm) 120 120 90 120 80

Pteridophytes Blechnum spicant + 2a +32a Oreopteris limbosperma +32b 2a . Dryopteris cambrensis 2a 33. 2b Pteridium aquilinum 2b 2b 2a .. Dryopteris filix-mas + 2a 2b .. Dryopteris dilatata 3+. . 2a Dryopteris borreri +. . 2a . Athyrium filix-femina 2b ...2a Dryopteris carthusiana ...34 Phegopteris connectilis 2a .... Polypodium vulgare .+... Dryopteris x deweveri ....2b Melampyro-Holcetea Holcus mollis ++2a +1 Luzula sylvatica 2b + 2b 4+ Oxalis acetosella +. . . + Epilobietea Epilobium montanum +. ++1 Digitalis purpurea 1. 1. + Scrubs and juvenile trees Rubus subgen. Rubus 2a +. . 2b Betula pendula .+.++ Rubus spectabilis 2a .... Mosses Hylocomium splendens 432a 2b 2a Rhytidiadelphus squarrosus 2b 1 2m 4 2b Polytrichum formosum 2m 3 2a .1 Lophocolea heterophylla 2m 1. . . Sphagnum capillifolium .3.2a . Peltigera didactyla ...+1 Lophocolea bidentata 2m .... Dicranum scoparium 2m .... pyriformis ..2a .. Kindbergia praelonga ....2b Other species Deschampsia cespitosa +. . +. Ranunculus repens +. . . + Potentilla erecta .++.. Agrostis capillaris .1.+. HAVEMAN: FERN-MEADOW VEGETATION 285

Table 2. Synoptic table of Oreopteris limbosperma communities in Scotland (column 1, source: Table 1), the Netherlands (column 2, source: Weeda & Haveman 2017), and Germany (column 3, source: Wittig 2000).

Column 123 Country Sc NL Ge Number of relevés 5 11 21

Pteridophytes Dryopteris cambrensis 80 .. Dryopteris borreri 40 .. Polypodium vulgare 20 .. Dryopteris x deweveri 20 .. Pteridium aquilinum 60 9. Blechnum spicant 100 64 24 Oreopteris limbosperma 80 100 100 Dryopteris dilatata 60 55 57 Athyrium filix-femina 40 64 76 Dryopteris carthusiana 40 64 14 Phegopteris connectilis 20 36 33 Dryopteris filix-mas 60 . 14 Gymnocarpium dryopteris ..14 Dryopteris affinis ..5

Hylocomium splendens 100 .. Pseudoscleropodium purum 20 55 . Potentilla erecta 40 45 . Holcus mollis 100 27 43 Atrichum undulatum 20 45 10 Oxalis acetosella 40 9 43 Digitalis purpurea 60 9 43 Rhytidiadelphus squarrosus 100 18 14 Polytrichum formosum 80 64 43 Rubus species 60 73 52 Agrostis capillaris 40 45 67 Juncus effusus 20 45 33 Luzula sylvatica 100 . 52 Epilobium montanum 80 . 24 Betula pendula 60 .5 Rubus idaeus 20 . 71 Molinia caerulea . 73 . Quercus robur . 55 . Deschampsia flexuosa . 27 51 Dicranella heteromalla . 45 5 Galium saxatile . 18 48 epiphylla . 55 14 Luzula luzuloides ..81 286 FERN GAZ. 20(7): 281-291. 2018 common, like Dryopteris dilatata, Athyrium filix-femina, Phegopteris connectilis, Atrichum undulatum (Hedw.) P.Beauv., Polytrichum formosum, Rubus L. subgen. Rubus, Agrostis capillaris L., and Juncus effusus L. Several other species are common to two regions, whereas they are lacking from the third. Potentilla erecta (L.) Raeusch. is an example, which was found in both the Scottish and the Dutch communities, and not in the German.

DISCUSSION Phytosociological Assignment The here described Scottish fern-meadows are at least for the greater part to be assigned to the Luzulo luzuloides-Thelypteridetum limbospermae, on the basis of the presence of the characteristic species Oreopteris limbosperma as well as the constant species, with many pteridophytes and other species which are found in both the Scottish relevés as well as in the German and/or the Dutch plots. The last relevé, without Oreopteris, probably represents a fragmentary example of this association. As was shown though, the Scottish fern-meadows have their own characteristics, with several species not found in the suboceanic forms of the association. Especially typical is Dryopteris cambrensis. According to Fraser-Jenkins (2007), this species has its distribution centre in oceanic Europe. In the British Isles it is common in Scotland, contrary to its name less so in Wales, and rather rare elsewhere (Page 1997; Trewren 2014). Considering this, the Scottish fern-meadows could probably be described as a separate subassociation (Luzulo luzuloides-Thelypteridetum dryopteridetosum cambrensis prov.) but, at this stage, a formal description of such a subassociation is rejected, since the available dataset is too small for a proper assessment.

Figure 2. Abundant fern growth in a forest edge, Oban, Argyll. Apart from Pteridium aquilinum, the vegetation is formed mainly by Dryopteris species and Oreopteris limbosperma (photo: R. Haveman) HAVEMAN: FERN-MEADOW VEGETATION 287

Ecology In the German lower mountain belt, the Luzulo-Thelypteridetum is almost strictly confined to forest edges (Wittig, 2000), and the same applies to the situation in the Netherlands (Weeda & Haveman, 2017). In Scotland, the community has a much broader ecological amplitude. Although it is frequently found in forest edges (relevés 3-5 in Table 1, Figure 2), it also develops frequently at forest clearings on suitable soils (relevés 1 and 2 in Table 1, Figure 3). Typical species of woodland clearings (grouped under the heading of Epilobietea and Scrubs and juvenile trees in Table 1) are not confined to the latter environment: they are equally present in the relevés from forest edges, and also in the relevés from Germany (Table 2 column 3, less so in the Dutch relevés). Unfortunately, no relevés were made in the stands on the third environment where the community is found, viz. abandoned and extensively managed grasslands (Figure 1). However, several short checks of the stands in this situation made clear that the species composition of the vegetation here is very similar to the community which develops in forest edges or at forest clearings. The abundant occurrence of the combination of Oreopteris limbosperma, Blechnum spicant, Athyrium filix-femina, and several Dryopteris species outside the buffered forest micro-climate indicate the high air-humidity and annual rainfall in this part of the UK, where water availability is never a problem (see also Knaben 1950, who drew the same conclusion after study of the fern-communities of West-Norway). According to the climate maps of the Met Office (www.metoffice.gov.uk), the region directly around Oban has an average annual rainfall of 1,700-2,200 mm, which is 2.5 to 3 times the amount in the Netherlands. The German lower mountains receive more rain than the Netherlands, but not as much as the Oban region: the higher regions of the Rothaargebirge e.g., have an average annual rainfall of 1,000 mm (www.wetter.de). Where the Luzulo-

Figure 3. A species rich fern community in a forest clearing in Glencruitten House forest, Oban, Argyll (photo R. Haveman) 288 FERN GAZ. 20(7): 281-291. 2018

Thelypteridetum grows in zonation with Pteridium-dominated communities, the first is confined to the steep banks, or the shelter of woodlands (Figure 4). Probably this is caused by difference in water demand, which is higher in Oreopteris than in Pteridium (Weeda & Haveman, 2017). In this respect, a striking description of the edaphic demands of Oreopteris limbosperma is given by Page (1997): "... niches where trickling acidic peaty water provides the most constant surface run-off or subterranean seepage, creating cool and constantly moist but well-aerated edaphic conditions ..." In West-Argyll, the Luzulo-Thelypteridetum can form complexes with different types of grasslands. On the least fertile situations, the grasslands can be assigned to the class Nardetea Rivas Goday et Borja Carbonell. On soils with a somewhat better availability of nutrients, the main grassland type that is found in complex with the fern-meadow community is the Lolio-Cynosuretum Tüxen (alliance Cynosurion cristati Tüxen, class Molinio-Arrhenatheretea Tüxen). Usually, both Nardion and Cynosurion grasslands are grazed by cattle or sheep. Where the grassland is mown, especially at the bottom of valleys, the grassland vegetation resembles the Crepido-Juncetum acutiflori Oberdorfer, a grassland community characteristic for peaty soils with laterally water movement and seepage. It is remarkable that this last grassland type is also mentioned by Bremer (2016) when he discusses the landscape in which the Luzulo-Thelypteridetum is found. In The vegetation of Scotland, the classic book edited by Burnett (1964), Oreopteris limbosperma is mentioned only in the context of sub-alpine scrubs and fern-meadows (p. 162-163, and p. 516). Only one remark in the chapter on the history and regional patterns in Scottish vegetation (p. 574) associates the species with burning on north- exposed slopes in the Northern Highlands. The occurrence of fern-dominated forest edges

Figure 4. Abandoned hillside meadow with fern encroachment. Large areas are covered by Pteridium aquilinum (in the background and in the lower right corner). Steep banks are invaded by Oreopteris limbosperma and Dryopteris species (lower left corner, and the linear structure in the centre of the photo). Oban, Argyll (photo: R. Haveman) HAVEMAN: FERN-MEADOW VEGETATION 289 and fern-meadows in the lowlands (other than those dominated by Pteridium aquilinum) like those in the studied part of Argyll, are completely neglected. The same holds true for the description of the fern-dominated communities in Rodwell's British Plant Communities (Rodwell et al., 1992). The already mentioned Thelypteris limbosperma- Blechnum spicant community (U19), which is placed in the Dryoptero-Calamagrostidion Nordh. alliance (class Betulo-Adenostyletea Br.-Bl.) by the authors with some reserve, is also considered to have its main distribution at higher altitudes, although it was recorded as low as 245 m above sea level. According to Page (1997), Oreopteris limbosperma might reach its highest abundance in western Scotland anywhere on a world scale, and here it can be found nearly from sea-level to about 880 m.

The Dutch Oreopteris Community In Comparison Bremer (2016) and Weeda & Haveman (2017) characterised the Luzulo luzuloides- Thelypteridetum limbospermae in the Netherlands as an association in development (an association in statu nascendi; Westhoff 1990). The current distribution of Oreopteris limbosperma in the Netherlands is the result of a rather recent increase (Figure 5). In the period before 1950, the species was only found in 45 standard grid cells (5x5 km). This increased in a short time to 71 in 1955, possibly as a result of a better recognition of the species or intensified surveys. Until 2000, the number of grid cells in which the species was found increased gradually to 103, in order to increase exponentially in the following years, to 176 in 2015 (verspreidingsatlas.nl). This expansion of Oreopteris can be attributed to the development of the landscape in the 20th Century. Most of the recent population growth of Oreopteris in the Netherlands is observed in forest plantations (mostly of Scots pine, Pinus sylvestris) in former heathland areas from the late 19th and early 20th Century (Bremer, 2016; Weeda & Haveman, 2017). Apparently, site conditions

Figure 5. Cumulative numbers of grid cells (5x5 km) with Oreopteris limbosperma in the Netherlands between 1940 and 2015. (Data source: verspreidingsatlas.nl) 290 FERN GAZ. 20(7): 281-291. 2018 are developing into a favourable state for the establishment of Oreopteris in these landscapes after about 100 years, especially in ditch sides in these plantations. In most sites where the species occurs it is only present in very small populations, sometimes only consisting of one single plant. This is in sharp contrast with the situation in West Scotland, where the species usually forms dense stands, and where a mature fern community is developed. The difference between the mature Scottish fern-meadows and the juvenile Dutch Oreopteris community is expressed in particular in the average number of fern species: 6.2 in the Scottish relevés, and only 3.9 in the Dutch ones. Remarkably, in the only relevé that was published by Bremer (2016) which can compete with the Scottish one in this respect (relevé 2003.20 in the table published by Bremer), Dryopteris borreri was recorded, the only observation of a species of the Dryopteris affinis complex in the association in the Netherlands. Considering all available data, including vegetation structure, fern species numbers, as well as the ominipresence of the described fern-meadow vegetation, the Luzulo- Thelypteridetum may be optimally developed in the lowlands and low mountain areas of West Scotland. In the Central European lower mountain regions, O. limbosperma can form dense stands locally, but the number of fern species drops notably in comparison with the fern species numbers in the Scottish fern-meadows. In the Netherlands, the distribution area of Oreopteris is still developing, and it is only rarely found in a dense fern vegetation rich in pteridophytes. The major absent in the Dutch relevés published by Bremer (2016) and Weeda & Haveman (2017) is Dryopteris cambrensis, which is so prominently present in the Scottish relevés. This species was discovered only recently in the Netherlands, viz. in 2010 by Koos Ballintijn in the Purmerbos just north of Amsterdam. The species is recorded in eight grid cells now, although their identity is not yet verified in all cases (oral comm. Sipke Gongrijp). Whether the species is really as rare as the online map (verspreidingsatlas.nl) suggests is not clear. The apomicts in the Dryopteris affinis aggregate are difficult to separate, although it is possible after some experience. Considering the distribution pattern in the UK, where the species seems to be much more restricted in the east than in the west, D. cambrensis may be and remain a rare species in the Netherlands. It remains an open question whether this species will be able to establish in the Luzulo-Thelypteridetum. Its dispersion might be adequate to reach the sites eventually, but whether the conditions for the establishment of the species are appropriate is not certain. The current occurrence of the species in dense (young) forest plantations suggest that D. cambrensis is confined to rather high levels of air humidity. In this respect, the environmental conditions in which the Luzulo-Thelypteridetum develops might be favourable also for the growth of D. cambrensis too. The future will show whether this and other fern species can become established in this association in the Netherlands. I expect however, that the Luzulo-Thelypteridetum never will become as species rich and luxuriant as in the coastal areas of Argyll.

REFERENCES BREMER, P. 2016. Een nieuwe plantengemeenschap voor Nederland: het Luzulo luzuloides-Thelypteridetum limbospermae Wittig 2000. Stratiotes 49: 11-22. BURNETT, J.H. (ed.) 1964. The vegetation of Scotland. Oliver & Boyd, Edinburgh & London. FRASER-JENKINS, C. 2007. The species and subspecies in the Dryopteris affinis group. HAVEMAN: FERN-MEADOW VEGETATION 291

Fern Gazette 18: 1-26. HENNEKENS, S.M. & SCHAMINÉE, J.H.J. 2001. TURBOVEG, a comprehensive data base management system for vegetation data. J.Veg.Sci. 12: 589-591. KNABEN, G. 1950. Botanical investigations in the middle districts of western Norway. Årbok for Universitetsmuseet 8: 1-177. MUELLER-DOMBOIS, D. & ELLENBERG, H. 1974. Aims and methods of vegetation ecology. Wiley, London. PAGE, C.N. 1997. The ferns of Britain and Ireland. Cambridge University Press Cambridge. RODWELL, J.S., PIGOTT, C.D., RATCLIFFE, D.A., MALLOCH, A.J.C., BIRKS, H.J.B., PROCTOR, M.C.F., SHIMWELL, D.W., HUNTLEY, J.P., RADFORD, E., WIGGINTON, M.J. & WILKINS, P. 1992. British Plant Communities. Volume 3. Grasslands and montane communities. Cambridge University Press, Cambridge. TICHÝ, L. 2002. JUICE, software for vegetation classification. J.Veg.Sci. 13: 451-453. TREWREN, K. 2014. Some taxa within the Dryopteris affinis complex. A field guide. British Pteridological Society, London. WEEDA, E.J. & HAVEMAN, R. 2017. Melampyro pratensis-Holcetea mollis. Stratiotes 50/51: 46-71. WESTHOFF, V. 1990. Neuentwicklung von Vegetationstypen (Assoziationstypen in statu nascendi) an naturnahen neuen Standorten, erläutert am Beispiel der Westfriesischen Inseln. Ber. Reinhold-Tüxen-Ges. 2: 11-23. WESTHOFF, V. & VAN DER MAAREL, E. 1978. The Braun-Blanquet approach. In: WHITTAKER, R.H. (red.), Ordination and Classification of Communities, Springer, The Hague: 287-373. WITTIG, R. 2000. Das Luzulo luzuloidis-Thelypteridetum limbospermae, eine azidokline Saumgesellschaft der hoheren Mittelgebirge. Tuexenia 20: 131-142. 292 FERN GAZ. 20(7). 2018 FERN GAZ. 20(7): 293-300. 2018 293


Armin Jagel1 & Marcus Lubienski2

1 Danziger Str. 2, 44789 Bochum, Germany; [email protected] 2 Am Quambusch 25, 58135 Hagen, Germany; [email protected]

Key Words: Marsilea aegyptiaca, Elafonisos, Greece, European species

ABSTRACT The water-clover species Marsilea aegyptiaca was first detected on the Mediterranean island of Elafonisos (Peloponnese, Greece) nearly 25 years ago. This was the first record for the species as part of the European flora. Recent work has shown that M. aegyptiaca still occurs at the site, and data are presented concerning its identification, habitat and distribution. Morphological characters of all known European species within the genus are compared.

INTRODUCTION Water-clovers (Marsilea L.) are heterosporous ferns and the most species rich group within the Marsileaceae. The family additionally comprises the pillworts (Pilularia L.) and the monotypic genus Regnellidium Lindm. All three genera are aquatic to semi- aquatic rhizomatous plants with roots and leaves born at nodes and sori arranged in sporocarps (Kramer, 1990; Nagalingum et al., 2006). Recent phylogenetic studies have revealed that the Clover ferns (Marsileaceae) together with the Floating ferns (Salviniaceae; Salvinia Seg. and Azolla Lam.) represent a monophyletic group of heterosporous ferns within the fern clade, which evolved in the Mesozoic (Pryer, 1999; Smith et al., 2006; Nagalingum et al., 2006). Twentieth century treatments of the genus mainly focussing on morphology have been published for Africa (Launert, 1968, 1970, 1971, 1984), Australia (Jones, 1998), India (Gupta, 1962), and the Americas (Johnson, 1986). Recent molecular studies have thrown some light on the phylogenetic relationships within Marsilea (Nagalingum et al., 2007, 2008; Whitten et al., 2012). During extensive field work on the island of Elafonisos in 1991 (Laconia, Peloponnese, Greece; see Jagel, 1992) the semi-aquatic Water-clover species Marsilea aegyptiaca Willd. was found growing abundantly in a seasonal pool. This was not only the first record for Greece but also the first true European occurrence of the species known at that time; its only known station at the time being from the Lower Volga in Russia (Jalas & Suominen, 1972; Tutin et al., 1993; Tzvelev, 2012) and an erroneous record for Spain (Alcober et al., 1980). The site was reinvestigated in 2016 and M. aegyptiaca confirmed to be still thriving at its obviously undisturbed habitat. In regard to the biogeographical importance of the record, more detailed and comprehensive data are given below.

BIOLOGY AND IDENTIFICATION Water-clover species (Marsilea L.) are in general a variable and frequently confusing group of plants and depending on the life phase, are nearly impossible to separate from each other. Most members of the genus inhabit seasonally dry areas inundated only 294 FERN GAZ. 20(7): 293-300. 2018 during the rainy season and like most aquatic plants are characterized by a phenotypic plasticity, which renders identification on a species level quite difficult. This is emphazised when plants are in their sterile, vegetative growth stage with submerged rhizomes and floating leaves on the water surface. Sporocarps are formed only in the terrestrial life stage, but are essential for species determination. Sterile, aquatic plants lacking sporocarps are nearly impossible to identify, because the different species in that life stage are morphologically very similar. Marsilea aegyptiaca (Figure 1) belongs to a group of xerophytic members within the genus and is adapted to extreme dry conditions. Crouch et al. (2011) report that the species in Southern Africa inhabits arid semideserts and is obviously able to exist for several years without being submerged. Consequently M. aegyptiaca is quite small in its overall appearance (stipes in the dry land form max. 5 cm long with pinnae max. 1 cm long) and shows hairy leaves (Figure 2 a-d), a feature often displayed by xerophytic Marsilea species. Nevertheless this is a very unstable and variable character, because hairs on the leaves and petioles readily occur in hydrophytic species too, when being in their terrestrial life stage. The pinnae of the land form are narrowly cuneate and pilose with the distal margin being slightly truncate and shallowly bilobed. The of M. aegyptiaca is quite distinct (Figure 3): usually appearing in clusters of two or more on short, free pedicels (2-8 mm), almost squarish in lateral view, with a distinct lateral furrow, densely pilose at first, becoming subglabrous later, lacking an inferior but mostly having a prominent superior tooth. In its aquatic life stage M. aegyptiaca is less distinct (see above). The floating leaves have stipes up to 30 cm long, broadly obovate, glabrous and larger pinnae (max. 2.5 cm) with rounded and shallowly sinuate distal margins (Figure 4 a, b).

Figure 1. Marsilea aegyptiaca at its growing site on the island of Elafonisos. JAGEL & LUBIENSKI: MARSILEA AEGYPTIACA 295

Table 1 shows the morphological characters of all known European species within the genus Marsilea. The former listed Azorean endemic species M. azorica Launert & Paiva has recently been shown to be a misidentification and synonymous with the invasive M. hirsuta R. Br. from Australia and Asia (Schaefer et al., 2011) and therefore is excluded here.

GEOGRAPHY, HABITAT, AND ECOLOGY Elafonisos is located in the region of Laconia, an administration unit of the Peloponnese. The island lies just off the Southwest coast of Malea Peninsula and is separated from it by a 600 m wide strait. The history of Elafonisos as an island is short, being part of the peninsula in former times. Therefore, it is not surprising that the largest part of the flora is typical for the South Peloponnese. However, some species show relations to the floristic region of the South Aegean (Jagel, 1992). In the Northwest part of Elafonisos at Leptos Kavos a seasonally moist habitat of about 1000 m² exists (Figure 5 a, b), at a distance of not more than 100 m from the Mediterranean sea. From late autumn until very early spring the shallow depression is

Figure 2 a, b, c, d. Marsilea aegyptiaca, fertile dry land plant in terrestrial life stage with hairy leaves. 296 FERN GAZ. 20(7): 293-300. 2018

Table 1: Macromorphological characters of the European species of Marsilea based on Gupta (1962), Launert (1968, 1983, 1984), Castroviejo et al. (1986), Johnson (1986), Prelli (2001), Tutin et al. (1993), Delgado Vázquez & Plaza Arregui (2010), Crouch et al. (2011), Schaefer et al. (2011). M. quadrifolia M. strigosa M. batardae M. aegyptiaca clustered, 2-3, mostly solitary solitary or in clustered, usually rarely solitary crowded in two loose groups of 2- 2, rarely solitary regular 3, not crowded Arrangement imbricating rows along the rhizome along the rhizome stalked sessile stalked stalked oblong-ellipsoid, oblique-elliptic, irregularly squarish beanshaped obliquely squarish to squarish or subcircular rectangular, obovoid Shape longer than high longer than high as long as high as long as high not laterally laterally usually laterally usually laterally flattened flattened flattened flattened Size 3-6 mm 3-5 mm 3-4.5 mm 2-3 mm superior tooth 0-1 superior tooth 1 superior tooth 1 superior tooth 1

Sporocarp (short to (short to (prominent) (prominent, blunt indistinguishable) indistinguishable) to acute) Teeth inferior tooth 0 inferior tooth 0-1 inferior tooth 0-1 inferior tooth 0

glabrous (hairy strigose or silky strigose or silky densely pilose only when pubescent, later pubescent, later later subglabrous young) subglabrous subglabrous Surface without a furrow without a furrow without a furrow with a distinct on the lateral on the lateral on the lateral curved vertical sides sides sides furrow on the lateral sides 2-20 mm 1-2 mm 2-8 mm 2-8 mm Pedicel (stalk of sporocarp) 2- to 4-branched, unbranched unbranched unbranched rarely unbranched long, creeping, short, caespitose, long, creeping, long, creeping to profusely sometimes profusely subcaespitose branched branched branched internodes more nodes crowded internodes 5-20 internodes 5-10 Rhizome than 10 mm mm mm (sometimes longer) obovate-cuneate obdeltate-cuneate obdeltate-cuneate narrowly cuneate to obdeltate Shape apex entire to apex entire to apex entire to apex deeply undulate, rounded crenulate, crenate crenate to sinuate

Pinnae rounded or entire, rounded glabrous glabrous to glabrescent hairy or glabrous Pubescence sparsely hairy JAGEL & LUBIENSKI: MARSILEA AEGYPTIACA 297 filled with fresh water not influenced by salt water. Marsilea aegyptiaca is very frequent in this small area, with hundreds or thousands of fronds being found. A smaller population exists more or less 50 m east of the pond at the border of a scrub. The sub-surface of the site is formed by limestone rocks (Symeonidis, 1969), covered by a shallow layer of sandy fine soil. After the beginning of the rainy season in autumn e.g. when Narcissus serotinus L. is flowering and when the shallow depression is filled up with rainwater, Ranunculus peltatus Schrank subsp. fucoides (Freyn) Munoz Garmendia (= Ranunculus saniculifolius Viv.) can be found as a water plant in the pond. At this time of year the Marsilea grows in its aquatic life stage (Figure 4). The pond is framed by tufts of Juncus heldreichianus Parl. and Schoenus nigricans L. At the end of winter or in early spring, usually at the end of March, the pond rapidly dries out within a few weeks. Species found growing together with M. aegyptiaca are Anagallis foemina Miller, Briza minor L., Isoetes phrygia (Boiss.) Hausskn., Isolepis cernua (Vahl) Roem. & Schult., Juncus capitatus Weigel, Juncus hybridus Brot., borysthenicum (Schrank) Litv., Lythrum hyssopifolia L., Mentha pulegium L., Plantago weldenii Rchb., Polypogon maritimus Willd., Ranunculus muricatus L., Romulea ramiflora Ten. Sagina maritima G.Don, Solenopsis laurentia (L.) C.Presl, Trifolium lappaceum L. Trifolium resupinatum L., and Triglochin barrelieri Loisel. Most of these are highly specialized and characteristic for such sites in the Peloponnese.

DISTRIBUTION Marsilea aegyptiaca is mainly an African species, recorded for Ethiopia, Botswana,

Figure 3 a, b, c, d. Marsilea aegyptiaca showing characteristic clustered, squarish, pilose sporocarps with lateral furrow and blunt superior tooth on short pedicels. 298 FERN GAZ. 20(7): 293-300. 2018

Namibia, , Sudan, Egypt, Tunisia, Algeria, Libya, and Madagascar (Launert, 1984; Crouch et al., 2011). The species also extends its range to the east with occurrences on the Indian subcontinent (Gupta, 1962; Sharma, 2005). Until the 1990s M. aegyptiaca was known from Europe only from the river Volga and its delta near the Caspian Sea in Russia (Jalas & Suominen, 1972; Tutin et al., 1993; Tzvelev, 2012). A record for Spain (Alcober et al., 1980) turned out to be a misidentification and the plants are now known to belong to a different species, Marsilea batardae Launert, occurring in Southwest Spain and Southern Portugal (Launert, 1983; Castroviejo et al., 1986; Rosselló-Graell et al., 2000; Prelli, 2001; Delgado Vázquez & Plaza Arregui, 2010). The discovery of M. aegyptiaca on the Peloponnesian island of Elafonisos in 1991 therefore was a remarkable addition to the knowledge concerning the range of the species now extending into the Eastern Mediterranean (Jagel, 1992) and representing the first record for Greece and the European Union. Later a second occurrence for the region has been reported from Cyprus (Hand, 2011), validating the species as an element of the European mediterranean flora.

HABITAT AND THREATS In former times sites of M. aegyptiaca on Elafonisos were used for agricultural purposes

Figure 4 a, b. Marsilea aegyptiaca, sterile plant with floating leaves in aquatic life stage.

Figure 5 a, b. Habitat of Marsilea aegyptiaca on Elafonisos, dried up in spring (a) and flooded in winter (b). JAGEL & LUBIENSKI: MARSILEA AEGYPTIACA 299 perhaps because of the good water supply, with evidence of several low walls of collected field stones. Nowadays there is no noticeable human influence, neither huts, houses nor stables are in the proximity so no current threat for the fern is perceived. The fact that the species has been known there for nearly 25 years now with no obvious change to the site supports this assessment. A large area of similar seasonally wet ponds is situated nearby on the mainland in Viglafia north of the beach of Punta. The area seems to be comparable with the pond of Elafonisos but is quite overgrazed and in parts more brackish. Although it has been investigated intensively over several years no plants of the Water-clover could be found although most accompanying plants of the Elafonisos site were present. However the site is nevertheless remarkable for hosting some very rare plant species which could not be found on Elafonisos: Callitriche brutia Petagna, (L.) Del., Crassula vaillantii (Willd.) Roth, Isoetes gymnocarpa (Gennari) A.Braun, Bory, and Myosurus heldreichii Heldr. ex H.Lév. Additionally a third Quillwort species, Isoetes phrygia (Boiss.) Hausskn., is present here (as it is at the Marsilea site on Elafonisos), together with a hybrid probably involving this taxon.

ACKNOWLEDGEMENTS The authors would like to thank Dr. H. W. Bennert (Ennepetal [formerly of Ruhr- University of Bochum], Germany) for the initial confirmation of Marsilea aegyptiaca in 1992. He also contributed with literature and helpful discussions. Dr. V. M. Dörken (University of Konstanz, Germany) also supplied us with literature. Dr. A. Troia (University of Palermo, Italy) is gratefully acknowledged for his help with the determination of the different Isoetes species at the sites.

REFERENCES ALCOBER, J.A., IBARS, A., MANSANET, J., MATEU, I. & PUCHE, M.F. 1980. Marsilea aegyptiaca Willd. nueva cita para España. Anales Jard. Bot. Madrid 36: 55- 59. CASTROVIEJO, S., LAíNZ, M., LóPEZ GONZALES, C., MONTSERRAT, P., MUñOZ GARMENDIA, F., PAIVA, J. & VILLAR L. 1986. Flora Iberica: Plantas vasculares de la Peninsula Iberica e Islas Baleares. Vol. 1. Lycopodiaceae- Papaveraceae. Real Jardín Botánico, C.S.I.C., Madrid. CROUCH, N.R., KLOPPER, R.R., BURROWS, J.E. & BURROWS, S.M. 2011. Ferns of Southern Africa. A comprehensive guide. Struik Nature, Cape Town. DELGADO VáZQUEZ, A.J. & PLAZA ARREGUI, L. 2010. Helechos Amenazados de Andalucía: Avances en Conservación. Consejería de Medio Ambiente. Junta de Andalucía, Sevilla. GUPTA, K.M. 1962. Botanical Monograph No. 2. Marsilea. Council of Scientific & Industrial Research, New Delhi. HAND, R. 2011. Supplementary notes to the flora of Cyprus VII. Willdenowia 41: 341- 355. JAGEL, A. 1992. Zur Flora und Vegetation der Insel Elafonisos (Lakonien, Griechenland). Diploma thesis. Spezielle Botanik, Ruhr-University, Bochum. JALAS, J & SUOMINEN, J. (eds.) 1972. Atlas Florae Europaeae. 1. Pteridophyta (Psilotaceae to Azollaceae). The Committee for Mapping the Flora of Europe and Societas Biological Fennica Vanamo, Helsinki. JOHNSON, D.M. 1986. Systematics of the New World Species of Marsilea (Marsileaceae). Syst. Bot. Monographs 11. Ann Arbor. JONES, D.L. 1998. Marsileaceae. In: Orchard, A. E. & McCarthy, P. M. (eds.) Flora of 300 FERN GAZ. 20(7): 293-300. 2018

Australia, Vol. 48, Ferns, gymnosperms and allied groups, pp. 166-173, ABRS/CSIRO Australia, Melbourne. KRAMER, K.U. 1990. Marsileaceae. In: KUBITZKI, K. (Ed) The Families and Genera of Vascular Plants. Volume I. Pteridophytes and Gymnosperms, pp. 180-183, Springer, Berlin/Heidelberg/New York/London/Paris/Tokyo/Hong Kong/Barcelona. LAUNERT, E. 1968. A monographic survey of the genus Marsilea Linnaeus. Senckenbergiana biol. 49: 273-315. LAUNERT, E. 1970. Marsilea Linnaeus: Variation on a theme. Senckenbergiana biol. 51: 433-435. LAUNERT, E. 1971. A remarkable new African species of Marsilea Linnaeus. Senckenbergiana biol. 52: 449-452. LAUNERT, E. 1983. A new species of Marsilea from Portugal. Bol. Soc. Brot., Sér. 2, 56: 99-104. LAUNERT, E. 1984. A revised key to and new records of African species of the genus Marsilea. Garcia de Orta, Sér. Bot., Lisboa 6: 119-140. NAGALINGUM, N.S., NOWAK, M.D. & PRYER, K.M. 2008. Assessing phylogenetic relationships in extant heterosporous ferns (), with a focus on Pilularia and Salvinia. Bot. J. Linn. Soc. 157: 673-685. NAGALINGUM, N.S., SCHNEIDER, H. & PRYER, K.M. 2006. Comparative morphology of reproductive structures in heterosporous water ferns and a re- evaluation of the sporocarp. Int. J. Plant Sci. 167: 805-815. NAGALINGUM, N.S. SCHNEIDER, H. & PRYER, K.M. 2007. Molecular phylogenetic relationships and morphological evolution in the heterosporous fern genus Marsilea. Syst. Bot. 32: 16-25. PRELLI, R. 2001. Les fougères et plantes alliées de France et d’Europe occidentale. Belin, Paris. PRYER, K.M. 1999. Phylogeny of marsileaceous ferns and relationships of the fossil Hydropteris pinnata reconsidered. Int. J. Plant Sci. 160: 931-954. ROSSELLO-GRAELL, A., DRAPER, D., TAULEIGNE GOMES, C. & CORREIA, A.I.D. 2000. Distribuição de Marsilea batardae Launert em Portugal e determinação do seu estatuto de ameaça. Portugaliae Acta Biol. 19: 219-224. SCHAEFER, H., CARINE, M.A. & RUMSEY, F.J. 2011. From European priority species to invasive weed: Marsilea azorica (Marsileaceae) is a misidentified alien. Syst. Bot. 36: 845-853. SHARMA, O.P. 2005. Pteridophytic flora of Bundi district, Southeastern Rajasthan. Zoos’ Print Journal 20: 1836-1837. SMITH, A.R., PRYER, K.M., SCHUETTPELZ, E., KORALL, P., SCHNEIDER, H. & WOLF, P.G. 2006. A classification for extant ferns. Taxon 55: 705-731. SYMEONIDIS, N. 1969: Stratigraphisch-Paläontologische Untersuchungen über die Neogenablagerungen von Lakonien, insbesondere der Gebiete von Neapolis (Eparchia Epidaurus-Limira) und gegenüber liegenden Insel Elaphonisos. Ann. Geol. Pays Helleniques 21: 531-553. TUTIN, T.G., BURGES, N.A., CHATER, A.O., EDMONDSON, J.R., HEYWOOD, V.H., MOORE, D.M. VALENTINE, D.H., WALTERS, S.M. & WEBB, D.A. (eds.) 1993. Flora Europaea. Volume 1. Psilotaceae to Platanaceae. 2nd ed. Cambridge University Press, Cambridge. TZVELEV, N.N. (ed.) 2012. Conspectus Florae Europae Orientalis. Tomus 1., Consociatio editionum scientificarum KMK, Petropoli/Mosqua. WHITTEN, W.M., JACONO, C.C. & NAGALINGUM, N.S. 2012. An expanded plastid phylogeny of Marsilea with emphasis on North American species. Amer. Fern J. 102: 114-135. FERN GAZ. 20(7): 301-303. 2018 301



The state of Gujarat is divided into five zones viz., Kutch, Saurastra, North, Central and South Gujarat. These zones are based on climate and geographic location. The district Junagadh is located on the Kathiawar peninsula in western Gujarat (Saurastra zone). It contains the Gir National Park and Girnar wildlife sanctuary, which is the only home for Asiatic lions. Girnar hills are also declared as a forest reserve for bamboos (Nakar & Jadeja, 2015), and ‘Girnari Giddh’, the long-billed vulture, is found only in the Girnar hills. The Girnar region alone accounts for about 25 per cent of the species in Gujarat state (Patel et al., 1961). However, lycophyte and fern diversity of the whole of the Gujarat state including Saurashtra regions (particularly Junagadh) is less understood, although recently there have been a few reports on the pteridophyte diversity of Central, North and South Gujarat (Kachhiyapatel et al., 2015, 2016; Rajput et al., 2016). The present investigation was carried out to study pteridophytes from Girnar hills. During this survey an interesting pteridophyte was collected which, after critical observation of morphological characters, was identified as Hypodematium crenatum (Forssk.) Kuhn. subsp. crenatum and it is reported here as new distributional record for Gujarat state. The identification was confirmed by comparing generated nucleotide sequences with the GenBank database, and the sequence data were submitted to BOLD. The genus Hypodematium in the family Hypodematiaceae is represented by 20 species in the world (PPG-I, 2016). A sole species of Hypodematium (divided into three subspecies viz., H. crenatum subsp. crenatum, H. crenatum subsp. loyalii Fraser-Jenk. & Khullar and H. crenatum subsp. mehrae Fraser-Jenk.) has been reported from India (Fraser-Jenkins, 2008). We have found a single subspecies, H. crenatum subsp. crenatum, for Gujarat state. Its description, range of distribution, ecology, conservation status and specimen examined is given.

Systematic Treatment Hypodematium crenatum (Forssk.) Kuhn, subsp. crenatum, Fraser-Jenkins, Taxon. Revis. Indian Sub-continental Pteridophytes., 204 - 206, 612. 2008; Patil, Systematic Studies on Pteridophytes of Satara District, 111-112. 2014. Hypodematium crenatum (Forssk) Kuhn. Manickam and Irudayaraj, Pterid. FI. W. Hypodematium hirsutum (Don) Ching. Indian Fern J. 1(1-2): 49.1985. Plant lithophytic, perennial herb; rhizome long creeping, densely red-brown scaly, giving woolly look to rhizome; scales 1-2 cm × 1-3 mm, red-brown, oblong lanceolate- lanceolate, glossy, apex long acuminate, margin entire; frond 20-30 cm × 15-20 cm, green-dark green, bipinnatified-tripinnatifid; stipe 10-18 cm long, brown, densely scaly at base, grooved, glabrous above; lamina 5-12×15-20 cm, bi-pinnatifid-tri-pinnatifid, ovate, apex acute, base cuneate, basal half tri-pinnatifid, distal half base bi-pinnatifid, distal most part simply pinnatifid; pinnae 6-10 pairs, subopposite, lanceolate, apex acute, base decurrent, pale brown, stiff, hairs sparsely-densely distributed; texture herbaceous; vein forked 3-4 times, indistinct above and below, free, reaching up to margin; sori median on the vein lets in two rows; indusia reniform, entire, densely covered with short stiff hair; spores 45-55 µm in dia., reniform to ellipsoid, exine with prominent 302 FERN GAZ. 20(7): 301-303. 2018 protuberances (and see Figure 1). Distribution: World: Africa, China, India, Japan, Malaysia, Myanmar, Philippines and Thailand India: Himachal Pradesh, North East India, Punjab, Gujarat, Maharashtra, Kerala. Gujarat: Junagadh

Figure 1. A: Habit of Hypodematium crenatum subsp. crenatum. B: Frond C: Rhizome D: Stipe base showing dense scales E: Stipe base scale F: Pinna G: Pinnules showing arrangement of sori H: Enlarged view showing hairs on costules I: Sorus J: Indusium K: Sporangium L: Spore. Scale bar: A-3 cm, B-3 cm, C- 1cm, D-0.5 cm, E-2 mm, F- 2 cm, G-5 mm, H-1mm, I-0.5 mm, J-0.5 mm, K-100 µM, L-25 µM. KACHHIYAPATEL ET AL: HYPODEMATIUM CRENATUM 303

BOLD ID: MIPDG013-15 Phenology: Vegetative phase: June-September; Reproductive phase: October- December Ecology: Lithophytic, collected from rock crevices at Girnar hill slopes (on the way to Sitavan), between 650-700 m. Specimens Examined: INDIA-Maharashtra: Kolhapur Dt., Pankala, alt 1000 m., 26/10/1962, Bhandare, BLAT; 02/01/1952, V. D. Vartak (BLAT205), BLAT; Satara Dt., Wai Tehasil; Panchagani, alt. 1100 m., Oct. 1918, Blatter (BLAT1211), BLAT; Satara Dt., Panchgani, alt. 1200 m., Sept. 2014, (SMP10071) SUK; Gujarat: Junagadh dt, Girnar hills, on the way to Sitavan, 680 m, (21°32’03.50” N, 70°31’28.10” E, at 668 m), R.N. Kachhiyapatel & K.S. Rajput 129, 26/12/2014, BARO.

ACKNOWLEDGMENTS The authors are grateful to the Gujarat Biodiversity Board (GBB) and Forest Department, Government of Gujarat for providing necessary permission to visit the area and permit collection of the specimens, and to an anonymous reviewer and Dr Mary Gibby for valuable suggestions on the previous version of the manuscript.

REFERENCES FRASER-JENKINS, C.R. 2008. Taxonomic Revision of Three Hundred Indian Subcontinental Pteridophytes with a Revised Census-List. Bishen Sing Mahendra Pal Sing Dehara Dun. KACHHIYAPATEL, R.N., SINGH, A.P., RAOLE, V.M. & RAJPUT, K.S. 2015. Distribution and occurrence of some pteridophytes in Gujarat: a new record for the state. J. of Indian Bot. Soc. 94 (3&4): 236-244. KACHHIYAPATEL, R.N., PATEL, S.K. & RAJPUT, K.S. 2016. Reporting Adiantum capillus-veneris L. and Thelypteris prolifera (Retz.) C.f. Reed as new record for Gujarat state together with their molecular identity. Indian Fern J. 33: 69-80. NAKAR, R.N. & JADEJA, B.A. 2015. Flowering and fruiting phenology of some herbs, shrubs and under-shrubs from Girnar Reserve Forest, Gujarat, India. Curr. Sci. 108(1):111- 118. PATEL, G.D., RAJYAGOR, S.B., TRIPATHY, S. & CHOKSHI, U.M. 1961. Gujarat state gazetteers. Directorate of Govt. Print., Stationery and Publications. PATIL, S. 2014. Systematic studies on pteridophytes of Satara districts (Maharashtra). A Ph. D. Thesis submitted to Shivaji University, Kolhapur. RAJPUT, K.S., KACHHIYAPATEL, K.N., PATEL, S.K. & RAOLE, V.M. 2016. Assessment of Pteridophyte Diversity and their status in Gujarat state, Western India. Plant Sci. Today’s. 3(4):337-348. THE PTERIDOPHYTE PHYLOGENY GROUP, 2016. A community-derived classification for extant lycophytes and ferns. J. Syst. Evol. 54(6):563-603.

R. N. KACHHIYAPATEL, S. M. PATIL & K. S. RAJPUT Laboratory of Plant Anatomy & Pteridology, Department of Botany, The M. S. University of Baroda, Vadodara – 390 002. *Address for correspondence: Email: ks.rajput[email protected] 304 FERN GAZ. 20(7). 2018 FERN GAZ. 20(7). 2018 305



The preparation of a checklist of pteridophytes from Gunung Mulu National Park, Sarawak, necessitates eleven new combinations to bring the generic treatment into line with PPG 1 [Pteridophyte Phylogeny Group] (2016).


DAVALLIACEAE 1. Davallia grammatosora (Copel.) Parris comb. nov., based on Davallodes grammatosora Copel., Philipp. J. Sci. Bot. 3: 34, t. 6 (1908), syn. Davallodes hirsuta (C.Presl) Copel. non Davallia hirsuta Sw. (1801). Type: Philippines, Mindanao, Copeland 1724 (BM, P).

2. Davallia pusilloides (Copel.) Parris comb. nov., based on Humata pusilloides Copel., Sarawak Mus. J. 2: 338 (1917). Type: Philippines, Mindanao, Zamboanga, San Ramon, May 1910, E B Copeland 153 (BM, K, MU, P, PNH, SING, UC).

3. Davallia subvestita (C.Chr.) Parris comb. nov., based on Humata kinabaluensis var. subvestita C.Chr., Gardens’ Bulletin Straits Settlement 7: 232 (1934). Type: Borneo, Sabah, Mt Kinabalu, near Lobang, 18 Nov. 1931, R E Holttum SFN 25549 (BM, BO, K, SING).

HYMENOPHYLLACEAE 1. Abrodictyum saxatile (T.Moore) Parris comb. nov., based on Trichomanes saxatile T.Moore, Gard. Chron. 1862: 45 (1862). Type: Borneo, collector not specified (K?).

2. Abrodictyum setigerum (Backh. ex T.Moore) Parris comb. nov., based on Trichomanes setigerum Backh. ex T.Moore, Gard. Chron. 1862: 45 (1862). Type, Borneo, collector not specified (K?).

3. Crepidomanes novoguineense (Blume) Parris comb. nov., based on Trichomanes novoguineense Brause, Bot. Jahrb. Syst. 49: 7 (1912). Type: Papua New Guinea, Lager “Hochmoos” 65 km südwärts der Tami-Mündung, 1600 m, Juli 1910, L Schulze 33/35 (B).

4. Crepidomanes teysmannii (Bosch) Parris comb. nov., based on Trichomanes teysmannii Bosch, Ned. Kruid. Arch. 5: 142 (1861). Type: Sumatra, Teysmann s. n. (L).

5. Hymenophyllum album (Blume) Parris comb. nov., based on Trichomanes album Blume, Enum. Pl. Javae: 226 (1828). Type: Java, Blume s. n. (L).

POLYPODIACEAE 1. Leptochilus bolsteri (Copel.) Parris comb. nov., based on Polypodium bolsteri Copel., Philipp. J. Sci. 1, Suppl. 4: 257, pl. 4a (1906). Type: Philippines, Mindanao, Surigao, April 1906, Bolster s. n. (MICH). 306 FERN GAZ. 20(7). 2018

THELYPTERIDACEAE 1. Amblovenatum immersum (Blume) Parris comb. nov., based on Aspidium immersum Blume, Enum. Pl. Javae: 156 (1828). Type: Java, Blume s. n. (L).

2. Amblovenatum paraphysophorum (Alderw.) Parris comb. nov., based on Dryopteris paraphysophora Alderw., Bull. Bot. Buitenzorg, ser. 3, 2: 143 (1920). Type: Sumatra, Lebong Tandai, C J Brooks 250S (BM, BO).

BARBARA S. PARRIS Fern Research Foundation, 21 James Kemp Place, Kerikeri, Bay of Islands, New Zealand 0230 Email: barbara2parr[email protected] FERN GAZ. 20(7). 2018 307 308 FERN GAZ. 20(7). 2018

INSTRUCTIONS FOR AUTHORS PAPERS should not usually exceed 20 printed pages and are generally expected to be considerably shorter. As well as reports of original research, review articles and short notes, e.g. new records, are encouraged. The senior author should supply an email address to facilitate correspondence. REVIEWS should cover recent developments in any aspect of research on ferns and lycopods. This can include unpublished research by the author, and/or the possible direction of future research. They should be aimed at the general reader who may not be an expert in the chosen topic. Proposals for a review should be discussed first with the review editors. MANUSCRIPTS should be submitted in English (British) in electronic format in 10-point Times New Roman font and single spaced. Electronic versions of text and tables should be compatible with WORD, with figures as TIFF files, and sent as email attachments. All manuscripts will be refereed. THE TITLE should reflect the content of the paper and be in BOLD CAPITALS (11-point) and centrally aligned. Generic and specific names should be in italics and any title containing a generic or specific name should be followed by the family name in brackets e.g. TRICHOMANES SPECIOSUM (HYMENOPHYLLACEAE) IN SOUTHERN SPAIN AUTHOR ABBREVIATIONS should follow Pichi Sermolli’s (1996) Authors of scientific names in Pteridophyta, Royal Botanic Gardens, Kew. MAIN HEADINGS: should be in BOLD CAPITALS (10-point) and centrally aligned. SUBSIDIARY HEADINGS: should be in bold, the first letter of each word in capitals, the rest in lower case and left-aligned. AUTHORS’ NAMES AND FULL ADDRESSES (including email address): follow the title and are centrally aligned. KEY WORDS: up to ten. ABSTRACT: should reflect the content of the paper. FIGURES: there is no distinction between photographs and line drawings in numbering. All should be supplied in mono or colour, TIFF format, 300 dpi and CMYK. (please contact the Production Editor with queries), with a scale bar where appropriate. Lettering or numbers (Arabic) should be in the bottom left using uppercase Times Roman. Figure captions should be on a separate sheet. TABLES: can be printed in either portrait or landscape format. Authors should consider this when preparing tables. Authors should ensure that tables fit the printed page size in a legible form. PAGESIZE: The Gazette has a useable page size of 123 × 204 mm. This can be used in portrait or landscape mode but any supplied artwork should not be larger than this. MEASUREMENTS: follow the metric system. CHECKLISTS: follow the format of Baksh-Comeau, Fern Gaz. 16(1, 2): 11- 122. REFERENCES: follow the style of a recent issue of The Fern Gazette, e.g.:- HOOKER, W.J. 1864. Species Filicum, 5. Dulau & Co., London. MORTON, C.V. 1947. The American species of Hymenophyllum, section Sphaeroconium. Contr. U.S. Natl. Herb. 29(3): 139-201. STEVENSON, D.W. & LOCONTE, H. 1996. Ordinal and familial relationships of pteridophyte genera. In: CAMUS, J.M., GIBBY, M. & JOHNS, R.J. (Eds) Pteridology in perspective, pp. 435-467. Royal Botanic Gardens, Kew. JOURNAL ABBREVIATIONS: should follow Botanico Periodicum Huntianum & Supplements. Alterations from the original text at proof stage will be charged for unless they are minor points of detail. A pdf will be provided free to the senior author. FERN GAZ. 20(8): 309-333. 2018 309



*216 Lincoln Heights Road, Ottawa, Ontario Canada K2B A8A, [email protected]

**Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche, Sezione di Botanica ed Ecologia Vegetale, Università degli Studi di Palermo, Via Archirafi 38, I-90123, Palermo Italy, [email protected]

Key Words: Isoetes, lycophytes, taxonomy, global research, biogeography, conservation

ABSTRACT With the advent of cytological analysis, Scanning Electron Microscopy, the recognition of sterile hybrids, improved access to wild populations and increasingly comprehensive molecular investigations, there has been a revolution in Isoetes taxonomy in recent times. The last 40 years have seen an almost 100% increase in the diversity documented during the previous two centuries. Four geographic areas account for over 90% of this diversity: northern-central South America, eastern North America, western Eurasia (including the northern Mediterranean) and South to Tropical Africa. There is a significant global conservation concern for Isoetes, with many taxa being rare (known from one to a handful of populations) and some known only from herbarium specimens. The taxa of the extraordinary rock-outcrop pools and the ephemeral wetlands found in disjunct areas around the world appear to be particularly vulnerable to habitat destruction. We expect 100 or more new taxa to be described in coming years. Such taxonomic productivity will require more systematic cytological and molecular investigations to be undertaken. It also requires the assembly of a larger global inventory of high-quality voucher specimens to provide the material for such investigations.

INTRODUCTION Speaking of quillworts (Isoetes, Isoetaceae), the late geneticist and pteridologist, Donald Britton, said only half-jokingly “…. this is a crazy genus! Just add water and stir” (D. M. Britton in lit, 1989). In the following, we undertake a review of the dramatic changes that have occurred in our global understanding of the taxonomy of this ‘crazy genus’ in recent decades as a result of the work of Britton and many others. Isoetes (Isoetaceae) is an ancient heterosporous lycophyte dating back to the Period (Pigg, 2001). With few exceptions, species exhibit a similar simple form and structure (Figure 1). A spirally-arranged, typically small (3 - 30 cm tall), set of

1 The authors dedicate this paper to the memory of Donald M. Britton (1923-2012) and Enrico Bellini (1940-2002) who in addition to being fine gentlemen and scholars, inspired and developed our interest in Isoetes. 310 FERN GAZ. 20(8): 309-333. 2018 unbranched quill-like leaves with usually partially velum-covered sporangia embedded into their basal abaxial faces, is developed from a shallowly buried two or three-lobed . At a gross scale there is not much to separate one taxon from another. Just the same, a high degree of taxonomic diversity as well as a high degree of (Jermy, 1990) exists with approximately 200 species, subspecies and varieties currently recognized (Troia et al., 2016). Spore morphology is the primary character employed for distinguishing individual Isoetes taxa. The demonstrate a remarkable diversity of perispore (surface) ornamentation expressions (Figure 2), ranging from laevigate (virtually featureless) to tuberculate (covered in broad, low, symmetrical projections) to echinate (spiny) or cristate-reticulate (intricate patterns of muri (walls), tubercles and/ or spines) (Hickey, 1986b; Punt et al., 2007). These ornamentation patterns are typically expressed consistently throughout the range of each taxon. The notable exception to consistent morphological representation is with sterile hybrids. The megaspores of such taxa have been the source of much confusion for over a century (Engelmann, 1882; Motelay, 1892; Eaton, 1900), exhibiting a range of sizes and morphological features intermediate between their putative parents. Deformed (often flattened) individual megaspores with congested ornamentation patterns likened to brain coral and/ or with irregular, even ‘dumb-bell’ shapes (Figure 3) are frequent and are strongly indicative of sterile hybrid status. are typically less boldly ornamented, varying from plain to tuberculate to echinate (Figure 4). While consistent in size in relation to the cytology of the particular taxon (Kott & Britton, 1983; Taylor et al., 1993; Musselman, 2002; Macluf et al., 2006), they often vary widely in appearance, even within individual populations. Microspores of hybrids are variable in size. Typically they are amphibious and misshaped and, as with megaspores, exhibit ornamentation patterns intermediate between those of the putative parents. All Isoetes occupy sites which are at least seasonally wet and grow in fresh (rarely brackish) water, typically being amphibious or purely aquatic, most growing in sterile or at least nutrient poor, typically acidic to circumneutral substrates. These habitats vary widely from deep-water oligotrophic lakes (growing as deeply as 4-5 metres), permanently flowing rivers and high elevation bog pools, to open woodland seepages, shallow ephemeral swales and briefly inundated pools in open bedrock outcrops.

Figure 1: A typical Isoetes plant: A) in situ, and B) excavated, also showing corm and roots (Isoetes minima A.A.Eaton, Salmo, British Columbia, Canada, 11 June 2017 and 28 June 2008, respectively) (Photos: D. F. Brunton). BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 311

Through all of this, be they like the more than half metre long, flexulose aquatic species Isoetes japonica A.Braun of eastern Asia, the more typically less than 30 cm long tall, reflected, emergent I. drummondii A.Braun of southern Australia, or the stiffly-erect, 5-15 cm tall rock pool ephemeral I. piedmontana (N.Pfeiffer) C.Reed of the southeastern United States (USA), virtually all Isoetes retain this simple gross-scale

Figure 2: Variations in Isoetes ornamentation: A) diploid I. nuttallii A.Braun, B) diploid I. cubana Engelm., C) decaploid I. lacustris L.(s. str.). (SEM images A & C - P. Sokoloff; B - D. M. Britton).

Figure 3: Aborted, sterile Isoetes hybrid megaspores: A) triploid I. engelmannii A.Braun x hyemalis D.F.Brunton, b) heptaploid I. ×harveyi A.A.Eaton, pro sp. (I. echinospora ssp. muricata (Durieu) Löve & Löve x lacustris) (SEM image A - P. Sokoloff; B - D. M. Britton).

Figure 4: Variation in Isoetes ornamentation: A) tetraploid I. septentrionalis D.F.Brunton., B) I. longissima Bory (s. str.) (SEM image A - P. Sokoloff; B - Carmela Di Liberto, University of Palermo, Italy (with Angelo Troia). (Scale bar = 10 µm) 312 FERN GAZ. 20(8): 309-333. 2018 appearance. There has been a substantial change in our global understanding of the taxonomic diversity of Isoetes, however, since it was last enumerated almost 100 years ago (Pfeiffer, 1922). Change has been particularly dramatic in the latter half of that period (Troia et al., 2016), which is the focus of this review. For our purpose, ‘recent’ commences ca. 1980 when Scanning Electron Microscope (SEM) and cytology were first applied in comprehensive reviews of Isoetes in North America (Boom, 1979; Kott, 1980; Kott & Britton, 1983), Europe (Berthet & Lecoq, 1977) and Australia (Marsden, 1979).

METHODS The following assessments are based upon a comprehensive reading of the Isoetes literature as well as the authors’ experience from investigations of the genus since the 1980s. This includes on-site ecological and population investigations in southern Europe (A.T.) and across North America (D.F.B.). In concert with various research associates and supplemented by extensive investigations of herbarium material, SEM imagery and the application of cytological data, these studies have clarified or described a substantial number of taxonomic issues (see References, below). We pay special attention to the literature concerning systematics, phylogeny, karyology and biogeography. We also considered the literature in other areas of investigation such as ecology, vegetation science, physiology and conservation biology. For the nomenclatural purposes of the present study we follow Troia et al. (2016).

DISCUSSION Historical Context Pfeiffer (1922) conducted the most comprehensive global review of the genus, producing a mostly herbarium-based enumeration of the traditional 19th Century - early 20th Century understanding of Isoetes diversity. Her study advanced the more modest reviews of Baker (1880) and Motelay (1883) and relied especially upon the work of Braun (1846), Durieu (1864), Engelmann (1867; 1888) and Eaton (1900). Working independently, she described 77 species and varieties globally. The publication of her remarkably insightful review coincided with an independent description of 15 new South American species (Weber, 1922), resulting in a total of 92 taxa being accepted at a species, subspecies or varietal level by the end of that year. That fewer than 25 species were described worldwide in the subsequent 50 years indicates how the technology and raw data available to researchers in that earlier era had largely been exploited by Pfeiffer’s time. After a long period of relative nomenclatural inactivity following Pfeiffer (1922), Isoetes has experienced a surge in taxonomic innovations. In effect, there has been a late 20th Century - early 21st Century Isoetes taxonomic ‘renaissance’. This results in part from geographically expanded investigations of previously poorly explored regions and habitats. More substantially, however, it reflects the application of new investigative tools for the reconsideration of both long-standing interpretations and new data.

Cytology and SEM Following the pioneering work of Manton (1950) on various pteridophyte genera, the connection between chromosome number and Isoetes spore size was investigated in detail in northern North America (Kott, 1980; Taylor et al., 1985; Taylor & Hickey, 1992). These break-through investigations provided confident determination of both the consistency and taxonomic significance of physical distinctions that had previously been BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 313 thought to represent only insignificant morphological variations within broadly circumscribed species. Cytological data quickly became a major line of evidence in subsequent global taxonomic investigations. Cytological investigations revealed that genetically distinct polyploid taxa are common in Isoetes, derived from basic diploid species (2x=2n=22) through hybridization and subsequent chromosome doubling (Taylor et al., 1985; Taylor & Hickey, 1992; Taylor et al., 1993). Significant progress was made in documenting Isoetes cytology in this period, with chromosome counts for over 80 taxa reported in Takamiya (1999) alone. The chromosome numbers for over 46% of all named taxa are presently known (Troia et al., 2016). The production of detailed, clear illustrations of seemingly subtle variations of various Isoetes morphological features through the systematic application of SEM became common in the 1980s and 1990s (Kott & Britton, 1983; Prada, 1983; Ferrarini et al., 1986; Schelpe & Anthony, 1986; Srivastava et al., 1993; Musselman & Knepper, 1994). For the first time this permitted commonly occurring but minute features to be confidently and reliably related to larger-scale characteristics such as velum size, sporangium shape and colouring, sporangium-topping ligule characteristics and site ecology. In the late 1980s the application of cytological data and SEM imagery in concert with comprehensive reviews of preserved herbarium material and significantly expanded field investigations, lead to the detection and description of sterile hybrids in North America. The existence of natural Isoetes hybrids had been identified in Canada 50 years earlier (Jeffrey, 1937) in a publication that was overlooked by researchers for some decades thereafter (Britton & Brunton, 1992). Independently, Matthews and Murdy (1969) reported probable naturally occurring hybrids in the southeastern USA. Other possible hybrids were subsequently reported in the eastern USA (Boom, 1979, Taylor et al., 1985). The first sterile hybrid Isoetes to be formally named was I. ×hickeyi W.C.Taylor & N.Luebke (Taylor & Luebke, 1988). The certain identification of sterile hybrids is taxonomically important. Their existence confirms that genetic barriers are in place between previously described and morphologically similar species. They also indicate the existence of previously unsuspected species. Although research has not been undertaken evenly throughout the globe, over a dozen hybrids have now been formally described in North America (Taylor et al., 1985; Taylor & Luebke, 1988; Britton & Brunton, 1989; Musselman & Knepper, 1994; Brunton, 2015), with others named in Japan (Takamiya et al., 1997), Russia (Mochalova et al., 2015) and India (Singh et al., 2018). Undescribed sterile hybrids have been detected in western Europe (A. C. Jermy, pers. comm., Prada & Rolleri, 2003; D.F.B., pers. obs.) and northern South America (J. R. Hickey, pers. comm.) and are suspected in South Africa (Musselman & Roux, 2002), Australia (Tasmania) (pers. obs.) and New Zealand (Hofstra & de Winton, 2016).

A Case Study of Recent Diversity The combination of cytological data, SEM imagery and hybrid detection has confirmed the existence of distinct taxa previously thought only to represent clusters of populations demonstrating insignificant morphological variation within broadly circumscribed species. The widespread North American Isoetes engelmannii s.l provides an excellent example of how dramatic (and complex) this recent clarification has been. The specific distinction of diploid I. engelmannii s.str. (Figure 5A and C) and I. valida (Engelm.) 314 FERN GAZ. 20(8): 309-333. 2018

W.Clute (Luebke, 1992, Brunton & Britton, 1996a) was confirmed by the recognition of their sterile diploid hybrid I. ×haltonharvillii Musselman & R.Bray (Musselman et al., 1995). A widespread tetraploid (4x=2n=44) derivative, I. appalachiana D.F.Brunton & D.M.Britton (Brunton & Britton, 1999) was subsequently described (Figure 5B and D), apparently the fertile product of doubled I. ×haltonharvillii (Brunton & Britton, 1997). The publication of Isoetes appalachiana was followed in turn by the determination of hexaploid (6x=2n=66) I. microvela D.F.Brunton, suspected to be the fertile doubling of triploid (3x=2n=33) I. appalachiana x engelmannii (Brunton & Britton, 1998). Tetraploids I. hyemalis, I. riparia Engelm. s.str. and I. septentrionalis (Brunton et al., 1994, Caplen & Werth, 2002; Brunton & McNeill, 2015), as well as hexaploids I. boomii N.Luebke and I. georgiana N.Luebke (Luebke, 1992; Brunton & Britton, 1996b), all represent recently described or clarified taxa within or connected to I. engelmannii s.l. Three additional sterile hybrids were identified in this period from within the Isoetes engelmannii s.l. : tetraploids I. ×brittonii D.F.Brunton & W.C.Taylor (I. appalachiana x septentrionalis) (Brunton & Taylor, 1990; Brunton, 2015), I. ×bruntonii D.Knepper & Musselman (I. appalachiana x hyemalis) (Musselman et al., 1996; Brunton, 2015) and hexaploid I. ×fairbrothersii J.D.Montgomery & W.C.Taylor (I. engelmannii x lacustris) (Montgomery & Taylor, 1994). In addition, 19th Century taxa I. ×foveolata A.A.Eaton, pro. sp. (I. engelmanii Dur. x tuckermanii A.Braun) and I. × eatonii R.Dodge, pro. sp. (I. echinospora x engelmannii and its synonym I. ×gravesii A.A.Eaton, pro sp.) were recognized to represent sterile I. engelmannii hybrids (Taylor et al., 1985; Jermy, 1990). Thus since the mid 1980s, over a dozen taxa in eastern North America were recognized to have been derived in whole or in part from I. engelmannii s.l.!

Figure 5: Megaspore (A) and microspore (C) of diploid Isoetes engelmannii (St Louis, Missouri, USA, G. Engelmann s. n., 1842 (MO 100901 - Syntype) compared with megaspore (B) and microspore (D) of tetraploid I. appalachiana (Huntington County, Pennsylvania, D. F. Brunton & K. L. McIntosh 11,171, 6 July 1992) (SEM images - D. M. Britton). BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 315

Recent Diversity Trends Approximately 80 new taxa have been described in the recent era (Troia et al., 2016). The early to mid 1980s was a particularly productive time with over 20 species or subspecies being described globally, followed by eight new taxa in 1986 and four in each of 1991 and 1994 (Figure 6). A second even larger surge in taxonomic activity occurred early in the 21st Century (almost 25 taxa), with five new taxa recognised in each of 2005 and 2010. Figure 7 illustrates our contemporary understanding of Isoetes diversity, with four global ‘hot spots’ centred on 1. northern-central South America (64 taxa), 2. eastern North America (45 taxa), 3. western Eurasia (including the northern Mediterranean) (39 taxa), and 4. South to Tropical Africa (35 taxa) (adapted from Troia et al., 2016). Together these account for over 180 taxa, or more than 90% of global diversity. That is a radically different picture from the traditional global understanding of Isoetes diversity. Based on

Figure 6: taxonomic activity (number of taxa described) since 1960s (adapted from Troia et al. 2016); particularly active periods shaded.

21-26 taxa 16-20 taxa 11-15 taxa 6-10 taxa 1-5 taxa 0 taxa

Figure 7: Global Isoetes distribution centres of diversity (Troia et al., 2016). 316 FERN GAZ. 20(8): 309-333. 2018

Pfeiffer (1922) and Weber (1922), the traditional five primary centres of diversity would have been identified as western Eurasia (including the northern Mediterranean) (19 taxa - 21% of global diversity), western North America (16 taxa - 17% of global diversity), eastern North America (12 taxa - 13% of global diversity), northern South America (12 taxa - 13% of global diversity), and Australia-New Zealand (9 taxa - 10% of global diversity). While the prominence of these contemporary ‘hot spots’ is undoubtedly biased towards regions where ‘Isoetologists’ have lived and/ or could most easily access, we believe the overall pattern at least broadly represents global reality. Recent developments within the major Isoetes diversity areas identified in Figure 7 are summarized below.

Northern-central South America (31% of global diversity) The expansion of known documented Isoetes diversity in northern South America in recent years is remarkable, with more than a doubling of the number of taxa that were known to exist prior to 1930. Most of these are from Brazil, with a particular focus in the Atlantic Rainforest region where almost 40% of pteridophytes are considered endemic (Prado, Sylvestre & Labiak et al., 2015; Prado, Sylvestre & Pereira, 2015). This total includes at least seven Isoetes taxa (Pereira, Mittelbach & Labiak, 2015). A number of new species represent clarifications of taxa first described (though frequently not legitimately so) in the works of Fuchs-Ekert (1982; 1992). Most new taxa are rare, with several being known from only a single (or handful of) site(s) (Pereira et al., 2016). At least one is known only from 19th Century herbarium specimens (Pereira & Stutzel et al., 2017). This recent neotropical work was substantially built upon the foundation of taxonomic research established by James Hickey and his associates. Their work focused particularly on species diversity in the northwest of the continent (Hickey, 1984; 1985; 1986a; 1989). The taxonomy of Isoetes in the northwestern portion of the continent remains substantially unresolved (Pinto-Zarata et al., 2017), being hampered by the limited availability of herbarium material and a confused nomenclatural history. A number of new species have been described in this region (e.g. five of nine species reported from Peru (Hickey, 1994) have been described since 1979 as well as others in the high elevation I. karstenii A.Braun complex (Small & Hickey, 2001)). Undescribed species and hybrids remain to be discriminated and documented, however (R. J. Hickey, pers. comm.).

Eastern North America (22% of global diversity) The recent expansion of our knowledge of Isoetes diversity on the eastern side of the North American continent commencing with the investigation of species diversity in the southeastern USA by Boom (1979; 1982) has been simply explosive. The systematic review of herbarium collections in the 1980s and 1990s and subsequent field investigations supported by cytological analysis of morphologically, ecologically and/ or distributionally distinctive elements detected within long-established species, has led to the recognition of numerous new taxa. While 32 taxa (24 species or subspecies and eight named hybrids) were enumerated in the definitive Flora of North America (Taylor et al., 1993), the total now stands at 51 taxa (35 species and subspecies and 16 described hybrids). A number of additional candidate species, subspecies and hybrids are under active investigation. This growth represents an almost 60% increase in total North American Isoetes diversity over the last 25 years. BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 317

Somewhat akin to the geographic concentration witnessed in the Atlantic Coastal Rainforest region of South America (see Northern-central South America, above), the Atlantic Coastal Plain of the southeastern USA is particularly species rich and supports a number of rare and endemic taxa (Reed, 1965; Musselman et al., 2001; Brunton & Britton, 2006; Brunton, 2016). Considerable attention has been focussed in recent years on species of shallow, ephemeral pools occurring on open, acidic, granitic or sandstone bedrock outcrops (flat- rocks) in the southeastern USA (Matthews & Murdy, 1969; Heafner & Bray, 2005; Brunton, 2016). Comparable species have been described from central and southern Africa (Hall, 1971; Schlepe & Anthony, 1986; Roux et al., 2009), the Mediterranean region (Troia & Raimondo, 2009; Troia & Greuter, 2015b), southern and central Australia (Johnson, 1984; Chinnock, 1993; 1998) and Mexico (Mora-Oivo, 2013) from similar outcrop pools and ephemeral swales. Such pools are otherwise referred to as erosion pits, !gua, gnammas and vleis. These outcrop taxa include the only known distichous-leaved Isoetes, the diminutive and remarkably similar Rury of Georgia, USA (Rury, 1985; Allison, 1993) and I. australis Williams of western Australia (Williams, 1943). Outcrop taxa are frequently rare and are confined to widely disjunct sites. Many species, such as the aforementioned Isoetes tegetiformans and I. australis or I. melanospora Engelm. of the southeastern USA and I. pusilla C.R.Marsden & R.J.Chinnock of eastern Australia (Entwhisle, 1994), bear remarkable ecological and morphological similarities (Figure 8) despite being hugely separated geographically.

Western Eurasia (including northern Mediterranean) (19% of global diversity) Pfeiffer (1922) initially enumerated 13 taxa in this region. Although Berthet & Lecocq (1977) made an important contribution to European Isoetes research with their SEM- based review of French taxa, taxonomic reconsiderations in the period began with the work of Carmen Prada (Prada, 1979; 1980; 1983) focussed on the Iberian peninsula. Beyond that, only occasional taxonomic investigations (including karyology) were undertaken (Schneller, 1982; Britton & Brunton, 1996). With the start of the new Millennium, however, dedicated Isoetes research was initiated in Italy (Troia & Bellini, 2001; Cesca & Peruzzi, 2001; Peruzzi et al., 2003), including the review and reconsideration of known diversity and the description of new species (Troia & Raimondo, 2009; Troia & Azzella, 2013). In this period Prada and associates expanded their studies beyond Spain (Prada & Rolleri, 2005; Rolleri & Prada, 2007). Romero and associates focused on critical Spanish taxa (Romero et al., 2004; Romero & Real, 2005) while Musselman and associates (Musselman, 2002; Bolin et al., 2008; 2011) studied Isoetes in the eastern part of the Mediterranean basin. Troia and associates concentrated their studies on expanding and clarifying previously documented Isoetes taxonomy in the central and eastern Mediterranean region. This included national syntheses for Italy (Troia & Greuter, 2014, 2015a) and Greece (Troia & Greuter, 2015b). In addition, a nomenclatural clarification of the taxonomically important but perplexing I. longissima-velata complex of the Mediterranean has been prepared (Troia & Rouhan, 2018). The treatment of Isoetes in Flora Europaea (Jermy & Akeroyd, 1993) lists 14 taxa. Jermy also studied aquatic taxa in northwestern Europe, providing the first confirmation of a European sterile hybrid (I. echinospora s.str. x lacustris - A. C. Jermy, pers. comm.). Documentation was also provided at that time for triploid plants of I. echinospora (s. 318 FERN GAZ. 20(8): 309-333. 2018

Figure 8: Morphologically similar bedrock outcrop pool species Isoetes pusilla of eastern Australia, LEFT (top to bottom): full plant; megaspore and microspore from this specimen (SEM image P. Sokoloff); habitat at Ingram’s Rock, Victoria (Photo: N. Walsh), and I melanospora of the southeastern USA, RIGHT (top to bottom): full plant; megaspore from this specimen (SEM image D. M. Britton); habitat at Mount Arabia, DeKalb County, Georgia (Photo: D. F. Brunton). Scale bar = 100 µm. BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 319 str.) in England that were morphologically indistinguishable from the otherwise diploid plants of this species but showed no evidence of hybridization (Rumsey et al., 1993); this unique circumstance for at least a Isoetes species remains unresolved (F.J Rumsey, pers. comm.). Vegetation inventories of subalpine Isoetes communities in the Pyrenees Mountains of France, Spain and Andorra have led to the unexpected discovery and description of I. creussensis J.J.Lazare & S.Riba (Lazare & Riba, 2010; Aymerich & Saez, 2013), probably the duodecaploid (12x=2n=132) detected earlier in this region by Taylor & Hickey (1992). A hexaploid (6x=2n=66) or heptaploid (7x=2n=77) I. ×hickeyi-like sterile hybrid has also has been reported (Taylor & Hickey, 1992) here. A reconsideration of the rare and ambiguous (possibly hybrid) taxon I. brochonii M.L.Motelay, and its relationship to other Pyrenean taxa, is on-going (D.F.B., pers. obs.).

South to Tropical Africa (17% of global diversity) Pfeiffer (1922) enumerated only four taxa in this region, probably because she was limited by the availability of voucher material and the restricted nature of botanical explorations in the region up to that time. That number of documented taxa has increased steadily over the last century, with at least 10 taxa now known. The increase to date is entirely from southern Africa (Wanntorp, 1970; Schelpe & Anthony, 1980; Musselman & Roux, 2002). Most new taxa are found in briefly-flooded ephemeral ponds (vleis) or bedrock outcrop solution pits (Roux et al., 2009). Tropical African species remain less well known, presumably due to continuing limited field exploration opportunities and relatively limited herbarium collections. A variety of amphibious species in ephemeral grassy wetlands and perhaps also in flat rock-like habitats over sandstone bedrock, are known here (Halls, 1971).

Areas of Lesser Diversity A number of areas of lesser diversity have also received particular attention in recent years. These are described below in descending order of the documented diversity that is apparent. Australia - New Zealand (16 - 23 species; 8 to 11% of global diversity): Isoetes of that region, along with those in a major area of eastern Asia, were enumerated in a comprehensive investigation by Marsden (1979). In that foundational study a number of new taxa from Australia were described, several of which are confined to ephemeral open bedrock pools (Chinnock, 1993). These share remarkably close morphological and ecological affinities with flat rock species in the southeastern USA (see Eastern North America, above). Approximately double the traditional number of Australian Isoetes known to Pfeiffer (1922) are now recognized (Chinnock, 1998) - a total of 16 species, with at least one Tasmanian species remaining undescribed (Garrett, 1996). Much remains to be resolved here, however, with possible hybrids and cryptic species suggested by abnormal morphological characteristics in several species (referred to by Marsden (1979) as Type I, Type II and Type III megaspores) and multiple ploidy levels reported from other taxa such as apparently apomictic I. muelleri A.Braun s.l. (Chinnock, 1998). The taxonomy of New Zealand taxa was unresolved in Marsden’s (1979) study and remains so today. There appear to be up to five aquatic taxa represented here, probably including hybrids (Hofstra & de Winton, 2016). Eastern Asia (Japan/ China/ Korea) (12 taxa; 6% of global diversity): Research over the last three decades has doubled the known diversity of Isoetes taxa in this region. 320 FERN GAZ. 20(8): 309-333. 2018

All the newly described taxa are aquatics and most are very rare (Liu et al., 2005), being known from one to a handful of sites. Two (including I. ×michinokuana M.Takamiya, M.Watanabe & K.Ono, the first Asian hybrid to be described) result from the especially comprehensive morphological and cytological investigations of Japanese Isoetes by M. Takamiya and associates (Takamiya et al., 1997; Takamiya, 2001). Another two new species are found on a single island off the coast of Korea (Choi et al., 2008). Central America - Caribbean (10 taxa; 5% of global diversity): Known diversity in this floristically transitional area between North and South America is moderate in extent but demonstrates wide ecological variation and significant endemism (e.g. four species confined exclusively to Mexico). Known diversity has increased in recent years with the description of three new endemic species, including I. jamaicensis J.R.Hickey from its namesake island (Hickey, 1981), the lowland I. pallida J.R.Hickey of the South American I. triangula Weber s.l. complex found in southern Mexico (Hickey, 1988), and a bedrock outcrop pool taxon I. tamaulipana A.Mora-Olivio, A.Menoza-Ruiz & J.G.Martinez-Ávalos, known from a single site in north-eastern Mexico (Mora-Olivio et al., 2013). India (6-23 taxa; 3 to 11% of global diversity): Ironically, while the endemic Isoetes coromandalina L.f. s.str. was one of the first Isoetes to be described (Linnaeus 1781), it was the only known member of the genus from India known to Pfeiffer (1922). The 18 species now reported from the subcontinent (Shukla et al., 2002, Pantil & Rajput, 2017) were almost all described after 1960 (Troia et al., 2016). The disposition of a number of recently described taxa and the various subspecies and varieties within several species complexes (Pant & Shrivastava, 1962; Gena & Bhardwaja, 1984; Shrivastava et al., 1993; Srivastava, 1998; Shukla et al., 2002) is controversial and remains unresolved. This is due to uncertainties from the reports of multiple and irregular chromosome counts and possibly apomictic reproduction amongst widely scattered, rarely occurring taxa with distinctions dependent on variable morphological characteristics. At least six of the 23 described taxa (Pantil & Rajput, 2017), however, seem solidly established at a species level (Fraser-Jenkins et al., 2016; Troia et al., 2016). The first Indian sterile hybrid, I. xgopalkrishnae S.K.Singh, P.K.Shukla & N.K.Dubey, has also recently been described (Singh et al. 2018). North Pacific Beringia (ca. 7 taxa; 3% of global diversity) - The Isoetes taxa of the area between Far East Asia (Siberia, Russia) and northwest North America (Alaska, USA and Yukon, Canada) have received attention in recent years due to their location within the phytogeographically significant Beringian region (Brunton et al., 2015), and one sterile hybrid, I. ×gopalkrishnae S.K.Singh, P.K.Shukla & N.K.Dubey (Singh et al., 2018) has recently been described. Britton and Brunton (1999), Mochalova (2006) and Mochalova et al. (2015) document at least seven taxa (three hybrids) intermixing across this North Pacific transition zone. One of these, Isoetes ×paratunica D.F.Brunton, O.Mochalova & A.A.Bobrov (I. asiatica (Makino) Makino x I. maritima Underw.) (Mochalova et al., 2015), represents the first hybrid reported between putative parents, that occur predominantly on separate continents (Asia and North America, respectively). Research continues on the intercontinental relationships of these taxa and their phytogeographic implications (A. Bobrov, pers. comm.). Kim et al. (2009) and Kim and Choi (2016) have conducted DNA analyses that further refine the origins and taxonomic distinctions of this Beringian complex. BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 321

Molecular Investigations Molecular investigations of Isoetes commenced with the Hoot & Taylor (2001) assessment of the value of these data for species determination and the clarification of Isoetes phylogeny. Although employed in support of some morphologically and cytologically determined specific taxonomic decisions (e.g. Bolin et al., 2011) and to examine the genetic diversity of particular taxa (Chen et al., 2005), molecular research to date has largely been applied at a higher level. Molecular data have also been employed in many areas of the world over the last 15 years to investigate genetic diversity within and between populations of rare and endangered Isoetes. This research has particular importance for the development of conservation management strategies aimed at establishing sustainable populations with such species as I. hypsophila H.vonHandel-Mazzetti (Chen et al., 2005; Li et al., 2012), I. sinensis T.C.Parker (Kang et al., 2005; Li et al., 2012) and I. yunguiensis Q.F.Wang & W.C.Taylor (Chen et al., 2007; Dong et al., 2018) in China, I. coreana Y.H.Chung & H.K.Choi (Kim et al., 2008) in Korea and I. malinverniana V.deCesati & G.DeNotaris (Gentili et al., 2010; Abeli et al., 2017) in Italy. It has been similarly applied for clarifying diversity in undetermined rare taxa in New Zealand (Hofstra & de Winton, 2016) and with both I. butleri G.Engelmann in the United States (Vander Stelt et al., 2017) and I. durieui J.Bory in Italy (Troia, 2003). Carrying on from fossil and morphological interpretation-based studies such as Retallack (1997) and Taylor and Hickey (1992), molecular research has focussed on broader questions of phylogeny and the evidence for extinct lineages (Rydin & Wikstrom, 2002; Hoot et al., 2004; Taylor et al., 2004; Larsen & Rydin, 2016; Pereira et al., 2017). This permitted Hoot et al. (2006), for example, to identify six major well- supported Isoetes clades globally. In recent years Xiang Lui and associates have applied a significant focus of Isoetes molecular research in China on regional origins and prehistoric dispersal patterns (X. Liu, pers. comm.). Bolin at al. (2018) employed DNA flow cytometry to consider the utility of C Values for the identification of particular species and Freund et al. (2018) combined molecular phylogeny data with morphological documentation to clarify the evolution of particular character states (e.g. the number of corm lobes). Molecular investigations, both at a high level and in regards to particular taxa represent a new and powerful tool that will be fundamental to future taxonomic studies of Isoetes, as they now are to virtually all groups of vascular plants.

Other Areas of Investigation While the limited scope of our review directs focus onto recent taxonomic accomplishments, it is important to note that significant strides in our understanding of other aspects of Isoetes were made during this period. The following briefly notes some of the major achievements of other areas of biological investigation. Vegetation classification involving Isoetes communities was conducted extensively in Eurasia, with considerable work being undertaken recently in the Mediterranean region. A preliminary synthesis of the Isoetes - related vegetation classification in this region was made by Quézel (1998). Studies on the ecology and vegetation science of Isoetes communities are now available for most (though not all) species around the Mediterranean: northwest Africa (Boutin et al., 1982), Azores (Pietsch, 1994), Spain (Romero & Amigo, 1995; Molina, 2005; Molina et al., 2011), France (Médail, 2003; M. Rhazi et al., 2004; L. Rhazi et al., 2009), Italy (Abeli et al., 2014), Sardinia (Bagella & Caria, 2013) and Turkey (Kürschner & Parolly, 1999). These studies are clearly 322 FERN GAZ. 20(8): 309-333. 2018 conservation-oriented in some cases (e.g. Médail, 2003; M. Rhazi et al., 2004; L. Rhazi et al., 2009; Gentili et al., 2010). Troia (2016) investigated the dispersal and possible migration routes of some of the current Mediterranean taxa, especially those with African affinities and particularly considering faunal and climatic vectors. Amongst climatic issues, severe wind storms were speculated to be a distributional vehicle for bedrock outcrop Isoetes taxa in the southern USA (Brunton, 2001) as they are for other types of terrestrial and wetland vascular plants (Matthews et al., 1991; Brunton et al., 2018). Presumably discouraged by long-standing taxonomic uncertainty in regards to many species, other contemporary investigations of Isoetes biogeography have been relatively few. Studies in East Asia employing geological, cytological and molecular data, however, have examined the origins and original dispersal patterns of the genus in that region (Liu et al., 2004; Taylor et al., 2004). Other investigations on a more local scale considered possible recent Trans-Pacific dispersal between North America and Asia (Brunton et al., 2015). Ecological and physiological investigations of Isoetes have been relatively few, despite being initiated early in the recent era in the pioneering studies of Matthews & Murdy (1969) regarding rock outcrop communities in the southern USA. Indeed, the benefit of considering ecological characteristics as an aid to taxonomic clarification was demonstrated earlier with recognition of the importance of substrate chemistry for distinguishing the morphologically similar ephemerals I. melanopoda Gay & Durieu s.str. and I. butleri Engelm. in the southern USA (Taylor et al., 1975). Substrate considerations have subsequently been applied to investigations of many aquatic and amphibious species in North America and elsewhere. Karrfalt and Hunter (1980) examined how the uniquely mucilage-covered sporophylls of South American Isoetes andicola (Amstutz) L.D.Gomez (Stylites andicola Amstutz) were literally squeezed out of the alpine vegetation in which they developed by the expansion of new plant growth, thus aiding the dispersal of sporangia. Other physiological and morphometric investigations in this period relate to leaf structure (ligule) functions (Kristen et al., 1982; Sharma & Singh, 1984; Singh, 1984), the early development of sporophytes (Hilger et al., 2002) leaf (intrastelar canal) anatomy (Romeo et al., 2000), and the early development of sporophytes (Hilger et al., 2002). The only extensive in situ ecological investigations of purely aquatic Isoetes to date were undertaken in central and northern Europe. Margrit Voge conducted underwater studies of numerous deep-water populations of I. lacustris at this time (Voge, 1997; 2003). She focussed especially on the importance of water quality parameters for controlling the health and size of particular populations. The importance of water quality to population sustainability was also investigated in China by Wen et al. (2003). More recently, Chappuis et al. (2015) examined the relative importance of light and organic elements in the stability (or instability) of southern European I. lacustris populations. Sharma (1998) and Sudova et al. (2011) examined the apparently symbiotic role of mycorrhizal fungi connected with the roots of Isoetes taxa in India and central Europe, respectively. The most dramatic physiological Isoetes investigations in recent decades resulted from the remarkable work of Jon Keeley and associates who determined that the CAM photosynthetic process typically employed by desert plants was also widely employed by Isoetes (Keeley, 2014). Their field and laboratory studies determined that CO2 uptake of Isoetes plants was at least substantially through their roots, enabling these plants to BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 323 prosper in sterile, otherwise prohibitively challenging habitats (Keeley & Busch, 1984; Keeley & Sandquist, 1991). Those investigations focussed mostly on ephemeral pool taxa in semi-arid habitats in southern California, USA but also included subalpine American taxa and South American tropical alpine species (Keeley et al., 1994).

Conservation Biology and Management Conservation concerns are increasingly significant motivators of Isoetes investigations, with many global conservation initiatives focussing on rare species of precarious status that are in danger of extinction. Taxa known from only a single population are known around the world, including I. pallida in southern Mexico (Hickey, 1988), I. viridimontana M.A.Rosenthal & W.C.Taylor in the northeastern USA (Rosenthal et al., 2014), I. sabatina A.Troia & M.M.Azzella (Troia & Azzella, 2013) in central Italy, I. hallasanensis H-K.Choi, Ch.Kim & J.Jung in Korea (Choi et al., 2008), I. libanotica Musselman, J.F.Bolin & R.D.Bray in Lebanon (Bolin et al., 2011) and I. junciformis D.F.Brunton & D.M.Britton in the southeastern USA (Brunton & Britton, 1999). Liu et al. (2005) note that all Isoetes taxa in China are in danger of extinction and several eastern Mediterranean taxa are similarly at risk (Bolin et al., 2011; Troia & Greuter, 2015a). Amphibious bedrock outcrop and ephemeral pool taxa may be the most threatened class of Isoetes globally, these being considered to be of even greater conservation value by sharing habitat with other specialized (often endemic) flora and fauna (Allison, 1993; Bauder & McMillian, 1998; Bagella & Caria, 2013; Ernandes & Marchiori, 2013; Bagella et al., 2016; Brunton, 2016).

FUTURE POSSIBILITIES Based on the rate of nomenclatural innovation in recent years (Figure 6) and the incentive provided by new research techniques supplementing traditional databases, we speculate that 100 or more additional Isoetes taxa will be identified in future years. The richest, perhaps globally most important areas for investigation would appear to be the bedrock outcrop and ephemeral pool habitats of the USA, Australia and South Africa and the less well investigated areas of Tropical Africa and interior South America. In addition to potentially providing taxonomic insights, the remarkably strong ecological and morphological similarities of geographically widely dispersed bedrock outcrop taxa (Figure 8) suggest an exceptional opportunity for the study of . Despite no Isoetes being known from the area, a strong case can be made for including the Caucasus region of central Asia amongst those having a high potential for future taxonomic contributions. The extraordinarily rich biodiversity of this region is known to support the highest level of Temperate Zone endemism in the Northern Hemisphere (Batsatsachvii et al., 2013). Molecular clarification of the subtle and even cryptic relationships within Isoetes species complexes could have major conservation management implications in many parts of the world. Again, this is especially true in regards to bedrock outcrop and ephemeral wetland taxa. Particular regions such as India, central and northern South America, South to Tropical Africa, central Asia and the eastern Mediterranean appear to be particularly in need of the initiation or continuation of systematic cytological and molecular taxonomic investigations and clarification. That applies as well to a number of unusually challenging species complexes including Isoetes muelleri s.l. in Australia, I. coromandelina s.l. in India, I. longissima s.l. in the Mediterranean region of Europe, I. karstenii s.l. in South 324 FERN GAZ. 20(8): 309-333. 2018

America, and both I. riparia s.l. and I. melanopoda s.l. in North America. Although considerable improvements have been seen in recent decades in the growth of Isoetes herbarium collections globally, more systematic gathering of high quality material is needed to facilitate future physiological and molecular investigations, particularly with morphologically challenging species groups. This is especially important in geographic regions (identified above) which traditionally have been deficient in preserved plant material. Similarly, cytological determinations remain outstanding for over half of the described taxa (Troia et al., 2017). These determinations are fundamental to understanding polyploid taxa and interspecfic hybrid relationships. And of course, the application of rapidly evolving and increasingly sophisticated molecular investigative tools is required in a systematic basis, not just in regards to individual taxa or species complexes. Despite the laudable achievements of pioneering 19th and 20th Century investigators and the remarkable advances of recent decades, the potential for the identification of new taxa remains high. Indeed, one could accurately state that such potential exits because of those pioneering efforts. Regardless, it is clear that previously undetected Isoetes taxa can be expected just about anywhere in the world where these curious - and ‘crazy’ - plants are to be found.

ACKNOWLEDGMENTS Our thanks to W. Carl Taylor, Botany, Smithsonian Institution, Washington, DC, USA for providing background information and encouragement for this project. We appreciate permission for use of the SEM images produced by Paul Sokoloff, Botany, Canadian Museum of Nature, Ottawa, Ontario, Canada (with Daniel Brunton), the late Donald M. Britton, University of Guelph, Guelph, Ontario, Canada and Carmela Di Liberto, University of Palermo, Italy (with Angelo Troia). We are grateful as well to Alexander Bobrov, I. D. Papanin Institute for Biology of Inland Waters, Borok, Russia, R. James Hickey, University of Miami, Ohio, USA and Xiang Lui, Laboratory of Plant Systematics and Evolutionary Biology, Wuhan University, Wuhan, China for their contributions of regional information. Neville Walsh, Royal Botanic Gardens, Melbourne, Australia also provided regional information and photographic material and offered helpful comments on the treatment of Australian taxa. We are particularly appreciative of the invaluable observations that James R. Hickey, Miami University, Oxford Ohio USA. Jim possibly has the longest continuous record of and perspective on Isoetes research in the world. And finally our sincere thanks to Editor Bridget Laue, without whose patience and persistence this paper would never have seen the light of day. REFERENCES ABELI, T., ORSENIGO, S., ARDENGHI, N.M.G., LUCASSEN, E.C.H.E.T. & SMOLDERS, A.J.P. 2014. Hydrochemical characterization of a stand of the threatened endemic . Amer. Fern J. 103: 241-244. ALLISON, J.R. 1993. Recovery plan for three granite outcrop plant species: black- spored quillwort (), mat-forming quillwort (Isoetes tegetiformans), and little amphianthus (Amphianthus pusillus). United States Fish and Wildlife Service, Jackson, Mississippi. AYMERICH, P. & SAEZ, L. 2013. Noves dades pteridològiques dels Pirineus catalans. Orsis 27: 175-194. BAGELLA, S. & CARIA, M.C. 2013. Sensitivity of ephemeral wetland swards with Isoetes histrix Bory to environmental variables: implications for the conservation of BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 325

Mediterranean temporary ponds. Aquatic Conservation 23: 277-290. BAGELLA, S., GASCON, S., FILIGHEDDU, R., COGONI, A. & BOIX, D. 2016. Mediterranean temporary pools: new challenges from a neglected habitat. Hydrobiologia 782: 1-10. BAKER, J.G. 1880. A Synopsis of the species of Isoetes. J. Bot. Br. Foreign 9 65-70, 105-110. BATSATSACHVII, K., SCHATZ, G.E. & SCHULKINA, T. (Eds) 2013. Caucasus Plant Initiative: A Regional Plant Conservation Strategy. https://www.bgci.org/files/Worldwide/News/2013/caucasus_strategy.pdf BAUDER, E.T. & MCMILLAN, S. 1998. Current distribution and historical extent of vernal pools in southern California and northern Baja California, Mexico. In: WITHAM, C.W., BAUDER, E.T., BELK, D., FERREN, W.R. & ORNOFF, R. (Eds) Ecology, Conservation and Management of Vernal Pools, pp. 56-70. Proceedings from a 1996 Conference. California Native Plant Society, Sacramento. BERTHET, P. & LECOCQ, M. 1977. Morphologie sporale des espèces françaises du genre Isoetes L. Pollen & Spores 19: 329-359. BOLIN, J.F., BRAY, R.D., KESKIN, K. & MUSSELMAN, L.J. 2008. The genus Isoetes L. (Isoetaceae, Lycophyta) in South-Western Asia. Turk. J. Bot. 32: 447-457. BOLIN, J.F., BRAY, R.D. & MUSSELMAN, L.J. 2011 A new species of diploid quillwort (Isoetes, Isoetaceae, Lycophyta) from Lebanon. Novon 21: 295-298. BOLIN, J.F., HARTWIG, C.L., SCHAFRAN, P. & KOMARNYTSKY, S. 2018. Application of DNA flow cytometry to aid species delimitation in Isoetes. Castanea 83: 38- 47. BOOM, B.M. 1979. Systematic Studies of the Genus Isoetes in the Southeastern United States. Master’s Thesis, University of Tennessee, Knoxville. BOOM, B.M. 1982. Synopsis of Isoetes in the southeastern United States. Castanea 47: 38-59. BOUTIN, C., LESNE, L. & THIÉRY, A. 1982. Ecologie et typologie de quelques mares temporaires à isoètes d’une région aride du Maroc occidental. Ecol. Medit. 8: 31– 56. BRAUN, A. 1846. Uber die nordamericanischen Isoetes Arten. Flora oder Bot. Zeit. (Regensburg) 12: 177-180. BRITTON, D.M. & BRUNTON, D.F. 1989. A new Isoetes hybrid ( x riparia) for Canada. Canad. J. Bot. 67: 2995-3002. BRITTON, D.M. & BRUNTON, D.F. 1992. Isoetes ×jeffreyi, hyb. nov.: a new Isoetes (I. macrospora x riparia) from Quebec, Canada. Canad. J. Bot. 70: 447-452. BRITTON, D.M. & BRUNTON, D.F. 1996. Isoetes ×pseudotruncata, a new triploid hybrid from western Canada and Alaska. Canad. J. Bot. 74: 51-59. BRITTON, D.M. & BRUNTON, D.F. 1999. Isoetes in Alaska and the Aleutians. Amer. Fern J. 79: 133-141. BRUNTON, D.F. 2001. Quillwort dispersal: which way is the wind blowing? Chinquapin 9: 20; 24-26. BRUNTON, D.F. 2015. Key to the quillworts (Isoetes: Isoetaceae) of the southeastern United States. Amer. Fern J. 105: 86-100. BRUNTON, D.F. 2016. Flat Rock Quillwort, Isoetes graniticola sp. nov. (Isoetaceae), a new lycophyte from the southeastern United States. Rhodora 118: 261-275. BRUNTON, D.F. & BRITTON, D.M. 1996a. The status, distribution and identification of Georgia Quillwort (Isoetes georgiana; Isoetaceae). Amer. Fern J. 86: 105-113. 326 FERN GAZ. 20(8): 309-333. 2018

BRUNTON, D.F. & BRITTON, D.M. 1996b. Taxonomy and distribution of (Isoetaceae). Amer. Fern J. 86: 16-25. BRUNTON, D.F. & BRITTON, D.M. 1997. Appalachian Quillwort, (Isoetes appalachiana, sp. nov.; Isoetaceae), a new pteridophyte from the eastern United States. Rhodora 99: 118-133. BRUNTON, D.F. & BRITTON, D.M. 1998. Isoetes microvela (Isoetaceae), a new quillwort from the southeastern coastal plain of the United States. Rhodora 100: 261- 275. BRUNTON, D.F. & BRITTON, D.M. 1999. Rush Quillwort (Isoetes junciformis, sp. nov.), a new pteridophyte from southern Georgia. Amer. Fern J. 79: 187-197. BRUNTON, D.F. & BRITTON, D.M. 2006. spp. silvatica (subsp. nov.), a new quillwort (Isoetaceae) from eastern North America. Castanea 71: 15- 30. BRUNTON, D.F., BRITTON, D.M. & TAYLOR, W.C. 1994. Isoetes hyemalis, sp. nov. (Isoetaceae); a new quillwort from the southeastern United States. Castanea 59 (1): 12-21. BRUNTON, D.F. & MCNEILL, J. 2015. Status, distribution and nomenclature of Northern Quillwort, Isoetes septentrionalis (Isoetaceae), in Canada. Canad. Field- Nat. 129: 174-180. BRUNTON, D.F., MOCHALOVA, O.A. & BOBROV, A.A. 2015. Isoëtes (Isoëtaceae) in the northern Pacific: diversity, taxonomy and distribution. In: Problems of taxonomy and geography of aquatic plants, pp. 90-91. Proceedings of the International Conference (Borok, Russia, 21-24 October, 2015),Yaroslavl, Filigran. BRUNTON, D.F. & TAYLOR, W.C. 1990. Isoëtes ×brittonii hyb. nov. (Isoëtaceae): A naturally occurring hybrid (I. engelmannii ´ I. riparia) in the eastern United States. Amer. Fern J. 80: 82-89. CAPLEN, C.A. & WERTH, C.R. 2000. Isozymes of the complex, II. Ancestry and relationships of polyploids. Syst. Bot. 25: 260-280. CESCA, G. & PERUZZI, L. 2001. Isoetes (Lycophytina, Isoetaceae) with terrestrial habitat in Calabria (Italy). New karyological and taxonomical data. Flora Medit. 11: 303-309 CHAPPUIS, E., LUMBRERAS, L., BALLESTEROS, E. & GACIA, E. 2015. Deleterious interaction of light impairment and organic matter enrichment on (Lycopodiophyta, Isoetales). Hydrobiologia 760: 145-158. CHEN, J.-M., LUI, X., WANG, J.-Y., GITURU, W.R. & WANG, Q.F. 2005. Genetic variation within the endangered quillwort Isoetes hypsophila (Isoetacae) in China as evidenced by ISSR analysis. Aquatic Bot. 82: 89-98. CHINNOCK, R.J. 1993. Notes on Isoetes and Tmesipteris in Victoria. Muelleria 8: 57- 60. CHINNOCK, R.J. 1998. Isoetaceae. In: , Volume 4. Australian Biological Resources Study, Department of the Environment and Energy, Canberra. CHOI, H, JUNG, J. & KIM, C. 2008. Two new species of Isoetes (Isoetaceae) from Jeju Island, South Korea. J. Pl. Biol. 51: 354-358. DURIEU DE MAISONNEUVE. 1864. Notes sur quelques espèces nouvelles d’Isoetes de l’Amérique du Nord et de l’Australie. Bull. Soc. Bot. France 11(4): 100-105. EATON, A.A. 1900. The genus Isoetes in New England, In: Fernwort Papers, pp. 1-16. W. N. Clute & Co., Binghamton. ENGELMANN, G. 1867. Isoetaceae. In: GRAY, A. Manual of the Botany of the BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 327

Northern United States, 5th Edition, pp. 698-700. Iverson, Blakeman & Company, New York. ENGELMANN, G. 1882. The Genus Isoetes in North America. Trans. Acad. Sci. St. Louis 4: 358-390. ENTWISLE, T.J. 1994. Ferns and allied plants (Psilophyta, Lycopodiophyta, Polypodiophyta). In: WALSH, N.G. & ENTWISLE, T.J. (Eds), Flora of Victoria Vol. 2, Ferns and Allied Plants, Conifers and Monocotyledons. Inkata Press, Melbourne. ERNANDES, P. & MARCHIORI, S. 2013. Mediterranean temporary ponds in Puglia (South Italy): a “joyau floristique” to protect. Acta Bot. Gallica 160: 53-64. FERRARINI, E., CIAMPOLINI, F., PICHI SERMOLLI, R.E.G. & MARCHETTI, D. 1986. Iconographia Palynologica Pteridophytorum Italiae. Webbia 40: 1-202. FRASER-JENKINS, C.R., GANDHI, K.N., KHOLIA, B.S. & BENNIAMIN, A. 2016. Isoeteaceae, In: An annotated checklist of Indian pteridophytes Part I (Lycopodiaceae to Thelypteridaceae), pp. 54–60. Bishen Singh Mahendra Pal Singh, Dehradun, India. FREUND, F.D., FREYMAN, W. & ROTHFELS, C. 2018. Inferring the evolutionary reduction of corm lobation in Isoetes using Bayesian model-averaged ancestral state reconstruction. Amer. J. Bot. 105: 275-286. FUCHS-ECKERT, H.P. 1982. Zur heutigen Kenntnis von Vorkommen und Verbreitung der sudamerianischen Isoetes-Arten. Proceedings of the Koninklijke Nederlandse Akademie va Weetenschappen, Series C 85: 2015-260. FUCHS-ECKERT, H.P. 1992. Supplementum ad indicem Isoetalium. Bionature 12: 99- 159. GARRETT, M. 1996. The Ferns of Tasmania, their ecology and distribution. Tasmanian Forest Research Council, Hobart. GENA, C.B. & BHARDWAJA, T.N. 1984. Three new species of genus Isoëtes from Rajasthan. J. Bombay Nat. Hist. Soc. 81: 165-168. GENTILI, R., ABELI, T., ROSSI, G., LI, M., VAROTTO, C. & SGORBATI, S. 2010. Population structure and genetic diversity of the threatened quillwort Isoetes malinverniana and implication for conservation. Aquatic Bot. 93: 147-152. HALLS, J.B. 1971. Observations on Isoetes in Ghana. Bot. J. Linn. Soc. 64: 117-139. HEAFNER, K.D. & BRAY, R.D. 2005. Taxonomic reassessment of North American granite outcrop Isoetes species with emphasis on vegetative morphology and I. piedmontana (Pfeiffer) Reed sensu lato. Castanea 70: 204-221. HICKEY, R.J. 1981. A new Isoetes from Jamaica. Amer. Fern J. 71: 69 - 74. HICKEY, R.J. 1984. Chromosome numbers of Neotropical Isoetes. Amer. Fern J. 74: 9- 13. HICKEY, R.J. 1985. Revisionary Studies of Neotropical Isoetes. University of Connecticut, Storrs University of Adelaide [Phd Thesis]. HICKEY, R.J. 1986a. The early evolutionary and morphological diversity of Isoetes, with descriptions of two new Neotropical species. Syst. Bot. 11: 304-321. HICKEY, R.J. 1986b. Isoetes megaspore surface morphology: nomenclature, variation and systematic importance. Amer. Fern J. 76: 1-16. HICKEY, R.J. 1988. Isoetes pallida, a new species from Mexico. Amer. Fern J. 78: 35- 36. HICKEY, R.J. 1989. A new species of Isoetes from Territorio Federal Amazonas, Venezuela. Ann. Missouri Bot. Gard. 76: 1160-1162. HICKEY, R.J. 1994. Pteridophyta of Peru, Part VI - Family 28. Isoetaceae. Fieldiana 34: 88-97. 328 FERN GAZ. 20(8): 309-333. 2018

HILGER, H.H., WEIGEND, W. & FREY, W. 2002. The gametophyte-sporophyte junction in Isoetes boliviensis Weber (Isoetaceae, Lycopodiophyta). Phyton 42: 149- 157. HOFSTRA, D. & DE WINTON, M. 2016. Geographic scales of genetic variation amongst Isoëtes in New Zealand. Aquatic Bot. 131: 28-37. HOOT, S.B., NAPIER, N.S. & TAYLOR, W.C. 2004. Revealing unknown or extinct lineages with Isoetes (Isoetaceae) using DNA sequences from hybrids. Amer. J. Bot. 91: 899-904. HOOT, S.B. & TAYLOR, W.C. 2001. The Utility of nuclear ITS, a LEAFY homolog intron, and chloroplast atpB-rncL spacer region data in phylogenetic analyses and species delimitation in Isoetes. Amer. Fern J. 91: 166-177. HOOT, S.B., TAYLOR, W.C. & NAPIER, N.S. 2006. Phylogeny and biogeography of Isoetes (Isoetaceae) based on nuclear and chloroplast DNA sequence data. Syst. Bot. 31: 449-460. JEFFREY, E.C. 1937. The cytology of a heterosporous Isoetes. Bot. Mag. (Tokyo) 521: 203-209. JERMY, A.C. 1990. Isoetaceae, In: KRAMER, K.U. & GREEN, P.S. (Eds) The Families and Genera of Vascular Plants Volume 1: Pteridophyes and Gymnosperms, pp. 24- 31. Springer-Verlag, Berlin. JERMY, A. C. & AKEROYD, J. R. 1993. Isoetes L. In: TUTIN, T.G., BURGES, N.A., CHATER, O., EDMONDSON, J.R., HEYWOOD, V. MOOR, D., VALENTINE, D.H., WALTERS, S.M. & WEBB, D.A. Flora Europaea, Volume 1 Psilotaceae to Platanaceae, 2nd ed., pp. 6-7. Cambridge University Press, Cambridge. JOHNSON, E.R.L. 1984. Taxonomic revision of Isoetes L. in Western Australia. J. Roy. Soc. Western Australia 66, Part 4: 28-43. KARRFALT, E.E. & HUNTER, D.M. 1980. Notes on the natural history of Stylites gemmifera. Amer. Fern J. 70: 69-72. KEELEY, J.E. 2014. Aquatic CAM Photosynthesis: a brief history of its discovery. Aquatic Bot. 118: 38-44. KEELEY, J.E. & BUSCH, G. 1984. Carbon assimilation characteristics of the aquatic CAM plant . Pl. Physiol. 76: 525-530. KEELEY, J.E. & SANDQUIST, D.R. 1991. Diurnal photosynthesis Cycle in CAM and non-CAM seasonal-pool aquatic macrophytes. Ecology 72: 716-727. KEELEY, J.E., DEMASON, D.A., GONZALEZ, R. & MARKHAM, K.R. 1994. Sediment-based nutrition in tropical alpine Isoetes. In: RUNDEL, P.W., SMITH, A.P. & MIENZER, F.C. (Eds) Tropical Alpine Environments, pp.167-194. Cambridge University Press, Cambridge. KIM, C. & CHOI, H.-K. 2016. Biogeography of North Pacific Isoetes (Isoetaceae) inferred from nuclear and chloroplast DNA sequence data. J. Pl. Biol. 59: 386-396. KIM, C., NA, N.R., SHIN, H. & CHOI, H.-K. 2009. Systematic evaluation of Isoetes asiatica Makino (Isoetaceae) based on FLAP, nrITS and chloroplast DNA sequences. J. Pl. Biol. 52: 501-510. KOTT, L.1980. Chromosome numbers for Isoetes in northeastern North America. Canad. J. Bot. 58: 980-984. KOTT, L. & BRITTON, D.M. 1983. Spore morphology and taxonomy of Isoetes in northeastern North America. Canad. J. Bot. 61: 3140-3164. KRISTEN, U., LIEBEZEIT, G. & BIEDERMANN, M. 1982. The ligule of Isoetes lacustris: ultrastructure, mucilage composition. and a possible pathway of secretion. BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 329

Ann. Bot. 49: 569-584. KÜRSCHNER, H. & PAROLLY, G. 1999. On the occurrence of Isoëtes histrix in the Menderes Massif of western Turkey - a synecological study and the first record of an Isoëtion community for Turkey. Bot. Jahrb. Syst. 121: 423-451. LARSÉN, E. & C. RYDIN. 2016. Disentangling the phylogeny of Isoetes (Isoetales), using Nuclear and Plastid Data. Int. J. Pl. Sci. 177: 157–174. LAZARE, J.J. & RIBA, S. 2010. Les Isoètes de l’Estany de Creussans (Andorre). J. Bot. Soc. France 30: 19-26. LINNAEUS, C. 1781. Supplementum Plantarum Systematis Vegetabilium Editionis Decimae Tertiae, Generum Plantarum Editiones Sextae, et Specierum Plantarum Editionis Secundae. Brunsvigne. LIU, X., GITURU, W.R. & WANG, Q.-F. 2004. Distribution of basic diploid and polyploid species of Isoetes in East Asia. J. Biogeogr. 31: 1239-1250. LIU, X., WANG, J.-Y. & WANG, Q.-F. 2005. Current status and conservation strategies for Isoetes in China: a case study for the conservation of threatened aquatic plants. Oryx 39: 335-338. LUEBKE, N.T. 1992. Three new species of Isöetes for the southeastern United States. Amer. Fern J. 82: 23-26. MACLUF, C.C., MORBILLI, M.A. & GIUDICE, G.E. 2006. Microspore morphology of Isoetes Species (Lycophyta) from southern South America. Bot. Review 72: 121- 124. MANTON, I. 1950. Problems of cytology and evolution in the Pteridophyta. Cambridge University Press, New York. MARSDEN, C.R. 1979. Morphology and Taxonomy of Isoetes in Australia, India, North- East and South-East Asia, China and Japan. Department of Botany, University of Adelaide [Phd Thesis]. MATTHEWS, J.F., FAIRCLOTH, W.R. & ALLISON, J.R. 1991. Portulaca biloba Urban (Portulacaeae), a species new to the United States. Syst. Bot. 130: 53-61. MATTHEWS, J.F. & MURDY, W.M. 1969. A study of Isoetes common to the granite outcrops of the southeastern Piedmont, United States. Bot. Gaz. 130: 53-61. MÉDAIL, F. 2003. La colonisation des mares temporaires méditerranéennes par les ligneux menace la survie à court terme d’une ptéridophyte très rare et menacée, l’isoète sétacé (Isoetes setacea Lam.). Ecol. Medit. 29: 251-252. MOCHALOVA, O.A. 2006. The genus Isoetes (Isoetaceae) in the northern Far East of Asia. Bot. Zhurn. (Moscow & Leningrad) 91: 94-98 [in Russian]. MOCHALOVA, O.A., BOBROV, A.A. & BRUNTON, D.F. 2015. Isoetes in Kamchatka (northern ) with description of a new hybrid I. ×paratunica (I. asiatica x I. maritima). Amer. Fern J. 105: 101-112. MOLINA, J.A. 2005. The vegetation of temporary ponds with Isoetes in the Iberian Peninsula. Phytocoenologia 35: 219-230. MOLINA, J.A., LUMBRERAS, A., GALLARDO, T., AGOSTINELLI, E., CASERMEIRO, M.A. & PRADA, C. 2011. Isoetes distribution pattern in a Mediterranean vernal pool system. Acta Bot. Gallica 58: 27-36. MONTGOMERY, J.D. & TAYLOR, W.C. 1994. Confirmation of a hybrid Isoetes from New Jersey. Amer. Fern J. 84: 115-120. MORA-OLIVO, A., MENDOZA-RUIZ, A. & MARTÍNEZ-ÁVALOS, J.-G. 2013. Isoetes tamaulipana (Isoetaceae), a new species from Mexico. Phytotaxa 267: 113- 120. 330 FERN GAZ. 20(8): 309-333. 2018

MOTELAY, M.L. 1883. Monographie des Isoëteae. Actes Soc. Linn. Bordeaux 36: 309‐404. MOTELAY, M.L. 1892. Isoetes brochoni Motelay, spec. nov. Actes Soc. Linn. Bordeaux 45: 45-48 + Plate II. MUSSELMAN, L.J. 2002. Ornamentation of Isoetes (Isoetaceae, Lycophyta) microspores. Bot. Review 68: 474-487. MUSSELMAN, L.J., BRAY, R.D. & KNEPPER, D.A. 1996. Isoetes ×bruntonii (Isoetes engelmannii ´ I. hyemalis), A new hybrid quillwort from Virginia. Amer. Fern J. 86: 8-15. MUSSELMAN, L.J. & KNEPPER, D.A. 1994. Quillworts of Virginia. Amer. Fern J. 84: 48-68. MUSSELMAN, L.J., KNEPPER, D.A., BRAY, R.D., CAPLEN, C.A. & BALLOU, C. 1995. A new Isoetes hybrid from Virginia. Castanea 60: 245-254. MUSSELMAN, L.J. & ROUX, J.P. 2002. Isoetes toximontana (Isoetaceae), a new quillwort with green megaspores from Northern Cape of South Africa. Novon 12: 504-507. MUSSELMAN, L.J., TAYLOR, W.C. & BRAY, R.D. 2001. Isoetes mattaponica (Isoetaceae), a new diploid quillwort from freshwater tidal marshes of Virginia. Novon 11: 200-2004. PANT, D.D. & SRIVASTAVA, G.K. 1962. The genus Isoetes in India. Proc. Natl. Inst. Sci. India 28 (Part B): 242-280. PATIL, S.M. & RAJPUT, K.S. 2017. The genus Isoetes from India: An Overview. Plant Science Today 4: 213-226. PEREIRA, J.B.S., LABIAK, P.H., STUTZEL, T. & SHULTZ, C. 2017. Origin and biogeography of the ancient genus Isoetes with focus on the neotropics. Bot. J. Linn. Soc. 185: 253-271. PEREIRA, J.B.S., MITTELBACH, M. & LABIAK, P. 2015. Studies on chromosome numbers and spore size in Brazilian Isoetes. Amer. Fern J. 105: 226-237. PEREIRA, J.B.S., SALINO, A., ARRUDA, A. & STUTZEL, T. 2016. Two new species of Isoetes (Isoetaceae) from northern Brazil. Phytotaxa 272: 141-148. PEREIRA, J.B.S., STUTZEL, T. & SCHULZ, C. 2017. Isoetes nana, a new species from the coastal mountains of southeastern Brazil. PhytoKeys 89: 91-107. PERUZZI, L., CESCA, G. & PUNTILLO, D. 2003. Isoetes (Isoetaceae), Ophioglossum and Botrychium (Ophioglossaceae) in Calabria (S. Italy): more karyological and taxonomical data. Caryologia 56: 359-363. PFEIFFER, N.E. 1922. Monograph on the Isoetaceae. Trans. Acad. Sci. St. Louis 9: 79- 233. PIETSCH, W. 1994. Isöetes azorica Durieu ex Milde - ein Endemit der Azoren - Vegetations-und Standortsverhältnisse, Gefährdung und Schutzmaßnahmen. Phytocoenologia 24: 649-665. PIGG, K.B. 2001. Isoetalean lycopsid evolution: from the Devonian to the present. Amer. Fern J. 91: 99-114. PINTO-ZÁRATE, J.H., OROZCO-RINCÓN, E. & MARQUÍNEZ-CASAS, X. 2017. Panorama de la investigación sobre los Isoëtes de Colombia (Isoëtaceae, Lycopodiopsida). In: MORENO-GAONA, D.A. (Ed) Memorias IX Congreso Colombiano de Botánica, 30 de julio al 3 agosto de 2017, Tunja-Boyacá. Ciencia en Desarrollo 8 (1, Supl. Esp.): 304-305, Asociación Colombiana de Botánica y Universidad Pedagógica y Tecnológica de Colombia. BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 331

PRADA, C. 1979. Estudio de la anatomia foliar de las eespecies Espanolas del genero Isoetes L. Lagascalia 9: 107-113. PRADA, C. 1980. Estudios palinologicos y cromosomicos en las especies Espanolas del genero Isoetes (Isoetaceae). Palinologia 1: 211-225. PRADA, C. 1983. El genero Isoetes L. en la Peninsula Iberica. Acta Bot. Malac. 8: 73- 100. PRADA, C. & ROLLERI, C.H. 2003. Caracteres diagnosticos foliares en taxones Ibericos de Isoetes L. (Isoetaceae, Pteridophyta). Anales Jard. Bot. Madrid 60: 371- 386. PRADA, C. & ROLLERI, C.H. 2005. A new species of Isoetes (Isoetaceae) from Turkey, with a study of microphyll intercellular pectic protuberances and their potential taxonomic value. Bot. J. Linn. Soc. 147: 213-228. PRADO, J., SYLVESTRE, L., LABIAK, S. H., WINDISCH, P.G., SALINO, A., BARROS, I.C.L., HIRAI, R.Y., ALMERIA, T.E., SANTIAGO, A.C.P., KLIELING- RUBIO, M.A., N. PEREIRA, A., OLLGAARD, B., RAMOS, C.G.V., MICKEL, J.T., DIETRICH, V.A.O., MYNSSEN, C.M., SCHWARTSBURD, P., CONDACK, J.P.S., PEREIRA, J.B.S. & MATOS, F.B. 2015. Diversity of ferns and lycophytes in Brazil. Rodriguesia 66: 1073-1083. PRADO, J., SYLVESTRE, L. & PEREIRA, J.B.S. 2015. Isoetaceae, In: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro.

ZIMMER, E. 2014. Isoetes viridimontana: a previously unrecognized quillwort from Vermont, USA. Amer. Fern J. 104: 7-15. ROUX, J.P., HOPPER, S.D. & SMITH, R.J. 2009. Isoetes eludens (Isoetaceae), a new endemic species from Kamiesburg, Northern Cape, South Africa. Kew Bull. 64: 123- 126. RUMSEY, F.J., THOMPSON, P. & SHEFFIELD, E. 1993. Triploid Isoetes echinospora (Isoetaceae: Pteridophyta) in northern England. Fern Gaz. (UK) 14: 215-221. RURY, P.M. 1985. A new and unique, mat-forming Merlin’s-grass (Isoetes) from Georgia. Amer. Fern J. 68: 102-104. RYDIN, C. & WIKSTROM, N. 2002. Phylogeny of Isoëtes (Lycopsida): resolving basal relationships using rbcL sequences. Taxon 51: 83-89. SCHELPE, E.A.C.L.E. & ANTHONY, N.C. 1986. Pteridophyta. In: LEISTER, O.A. (Ed) Flora of Southern Africa. Botanical Research Institute, Pretoria. SCHNELLER, J.J. 1982. Cytological investigations on Isoetes malinverniana. Webbia 35: 307-309. SHARMA, B.D. 1998. Fungal associations in the roots of three species of Isoetes L. Aquatic Bot. 61: 33-37. SHARMA, B.D. & SINGH, R. 1984. The ligule In Isoetes. Amer. Fern J. 74: 22-28. SHRIVASTVA, G.K. 1998. Isoetaceae in India: morphology and taxonomy. Indian Fern J. 15: 165-177. SHRIVASTVA, G.K., PANT, D.D. & SHUKLA, P.D. 1993. The genus Isoetes L. in India. Amer. Fern J. 83: 105-119. SHUKLA, P.K, SHRIVASTAVA, G.K. & SHUKLA, S.K. 2002. The quillworts (Isoetes) of India: distribution, endemism and species radiation. Biodivers. & Conservation 11: 959-973. SINGH, S.K., SHUKLA, S.K., DUBY, N.K. & SHUKLA, P.K. 2018. Isoetes ×gopalkrishnae, a new interspecific hybrid from central India. Phytotaxa 362: 68- 76. SMALL, R.L. & HICKEY, R.J. 2001. Systematics of the North Andean Isoetes karstenii Complex. Amer. Fern J. 91: 41-69. SUDOVA, R., RYDLOVA, J., CTVRTILKOVA, M., HAVRANEK, P. & ADAMEC, L. 2011. The incidence of arbuscular mycorrhiza in two submerged Isoetes species. Aquatic Bot. 94: 183-187. TAKAMIYA, M. 1999. Natural history and taxonomy of Isoetes (Isoetaceae). Acta Phytotax. Geobot. 50: 101-138. TAKAMIYA, M. 2001. Isoetes sinensis var. sinensis in Korea (Isoetes: Pteridophyta). Fern. Gaz. (UK) 16: 169-176. TAKAMIYA, M., WATANABE, M. & ONO, K. 1997. Biosystematic studies on the genus Isoetes in Japan IV. Morphology and anatomy of sporophytes, phytogeography and taxonomy. Acta Phytotax. Geobot. 48: 89-122. TAYLOR, W.C. & HICKEY, R.J. 1992. Habitat, evolution and speciation in Isoetes. Ann. Missouri Bot. Gard. 79: 613-622. TAYLOR, W.C., LEKSCHAS A.R., WANG, Q.-F., LIU, X., NAPIER, N.S. & HOOT, S.R. 2004. Phylogenetic relationships of Isoetes (Isoetaceae) in China as revealed by nucleotide sequences of nuclear ribosomal ITS region and second intron of a LEAFY homolog. Amer. Fern J. 94: 196-205. TAYLOR, W.C. & LUEBKE, N.T. 1988. Isoetes ×hickeyi: A naturally occurring hybrid between I. echinospora and I. macrospora. Amer. Fern J. 78: 6-13. BRUNTON & TROIA: TAXONOMIC RESEARCH INTO ISOETES 333

TAYLOR, W.C., LUEBKE, N.T., BRITTON, D.M., HICKEY, R.J. & BRUNTON, D.F. 1993 Isoetaceae - Quillwort family. In: FLORA OF NORTH AMERICA EDITORIAL COMMITTEE (Eds) Flora of North America, volume 2: Pteridophytes and Gymnosperms, pp. 64-75. Oxford University Press, New York. TAYLOR, W.C., LUEBKE, N.T. & SMITH, M.B. 1985. Speciation and hybridization in North American Quillworts. Proc. Royal Soc. Edinburgh 86B: 259-263. TAYLOR, W.C., MOHLENBROCK, R.H. & MURPHY, J.A. 1975. The spores and taxonomy of and I. melanopoda. Amer. Fern J. 65: 33-38. TROIA, A. 2016. Dispersal and colonization in heterosporous lycophytes: palynological and biogeographical notes on the genus Isoetes in the Mediterranean region. Webbia 71: 277-281. TROIA, A. & AZZELLA, M.M. 2013. Isoetes sabatina (Isoetaceae, Lycopodiophyta), a new aquatic species from central Italy. Pl. Biosyst. 147: 1052-1058. TROIA, A. & BELLINI, E. 2001. Karyological observations on Isoetes durieui Bory (Lycophyta, Isoetaceae) in Sicily. Bocconea 13: 397-400. TROIA, A. & GREUTER, W. 2014. A critical conspectus of Italian Isoetes (Isoetaceae). Pl. Biosyst. 148: 13-20. TROIA, A. & GREUTER, W. 2015a. A conspectus of and key to Greek Isoetes (Isoetaceae) based on a reassessment of Haussknecht’s gatherings of 1885. Willdenowia 45: 391-403. TROIA, A. & GREUTER, W. 2015b. Flora Critica d’’Italia: Isoetaceae. Fondazione per la Flora Italiana, Firenze. http://www.floraditalia.it/pdf/Isoetaceae.pdf [accessed 29 April 2018]. TROIA, A., PEREIRA, J.B.S., KIM, C. & TAYLOR, W.C. 2016. The genus Isoetes (Isoetaceae): a provisional checklist of the accepted and unresolved taxa. Phytotaxa 277: 101-145. TROIA, A. & RAIMONDO, F.M. 2009. [published 2010]. Isoetes todaroana (Isoetes, Isoetaceae), a new species from Sicily (Italy). Amer. Fern J. 99: 238-243. TROIA, A. & ROUHAN, G. 2018. Clarifying the nomenclature of some Euro- Mediterranean quillworts (Isoetes, Isoetaceae): Indicator species and species of conservation concern. Taxon, https://doi.org/10.12705/675.10. VOGE, M. 1997. Plant size and fertility of Isoetes lacustris L. in 20 lakes of Scandinavia. Arch. Hydrobiologia 139: 171-185. VOGE, M. 2003. Environmentally related demography: field studies on Isoetes lacustris L. (Lycophyta, Isoetaceae) in Europe. In: CHANDRA, S. & SHRIVASTAVA, M. (Eds) Pteridology in the New Millennium, pp. 233-260, Kluwer Academic Publishers, Dordrecht. WAGNER, W.H., WAGNER, F.S. & TAYLOR, W.C. 1986. Detecting abortive spores in herbarium specimens of sterile hybrids. Amer. Fern J. 76: 129-140. WANNTORP, H-E. 1970. The genus Isoetes in South West Africa. Svenska Botanisk Tidskrift 64: 141-157. WEBER, U. 1922. Zur anatomie und systematik der gattung Isoetes L. Hedwigia 63: 219-262. WEN, M.C., PANG, X.-A., WANG, Q.-F. & TAYLOR, W.C. 2003. Relationship between water chemistry and the distribution of the endangered aquatic quillwort Isoetes sinensis Palmer in China. J. Freshw. Ecol. 18: 361-367. WILLIAMS, S. 1943. On Isoetes australis S. Williams, a new species from Western Australia. Proc. Roy. Soc. Edinburgh, Sec. B, 62: 1-11. 334 FERN GAZ. 20(8). 2018


Daniel Brunton was born in Ottawa, Canada on 17 May 1948. He grew up in that area and graduated with a Geography degree from Carleton University, Ottawa, in 1973. A keen naturalist from an early age, he translated that interest into summer employment and then permanent work in provincial parks in Ontario and Alberta (1967-1979), involved in the areas of environmental interpretation, ecological inventories and systems planning. The lessons learned in parks prepared him for a career as an independent ecological consultant from which he retired (mostly) in 2017. Encouragement from federal government scientists in Ottawa and insights gained from expert associates in those field endeavors encouraged his interest in natural history generally and botany in particular.

Isoetologists (left to right) Carl Taylor, Angelo Troia and Daniel Brunton visiting bedrock outcrop Isoetes sites (I. piedmontana is abundant in the flooded erosion pits visible behind Taylor) at Clover, South Carolina, USA, 2 April 2016 (Photo: Gerry Taylor). FERN GAZ. 20(8). 2018 335

An extensive series of field based studies across Canada resulted in numerous publications on rare flora and fauna, especially pteridophytes, from the 1970s through the 1990s and the development of a very large (20,000 specimen) private herbarium. Association with the late Donald M. Britton, foremost Canadian pteridologist, drew Brunton into the challenging - perplexing! - world of ‘Isoetology’ in the late 1980s. Beginning with their discovery of an undescribed hybrid, the Britton and Brunton team explored Isoetes throughout North American and beyond. They generated numerous papers between 1989 and 2006, describing over a dozen new species, hybrids and subspecies. To date, Brunton has produced 49 publications dealing with the taxonomy, diversity, distribution, ecology and conservation of Isoetes in North America, Asia and Europe.

Angelo Troia was born in Palermo, Italy on 3 September 1967 and grew up in Sicily. After obtaining a degree in Natural Sciences at the University of Palermo in 1991 and doing his (obligatory) service in the Italian Navy, he undertook PhD studies in Plant Biosystematics and Ecology between Florence and Bologna (Italy). His doctoral thesis was on the systematics and biogeography of the rare insular endemic Cytisus aeolicus Guss. (Fabaceae). Returning to Sicily in 1997, he was introduced to the fantastic world of Mediterranean quillworts by the late Prof. Enrico Bellini, his studies focussing on caryological, taxonomic and ecological aspects of various species. At the IX OPTIMA Meeting in Paris, in May 1998, Troia and Bellini presented a poster about the (previously unknown) chromosome number of the Mediterranean Isoetes durieui. After a period as Director of the Trapani Saltworks Nature Reserve in western Sicily, he returned to academic research in 2007, working as a botanical researcher at the University of Palermo. There he prepared the treatments for the three lycophyte families for the project “Flora Critica d’Italia”. Troia’s ongoing research is focused on the diversity, ecology and biogeography of plants, with a particular focus on the genus Isoetes (and other lycophytes and ferns), freshwater plants and their habitats, the role of islands in the ecology of the Mediterranean, and a wide variety of conservation issues. In the course of his studies, he has produced 27 publications involving the taxonomy, biogeography, cytology, ecology and conservation of Isoetes. His most recent area of research is the study of Characeae (green algae). 336 FERN GAZ. 20(8). 2018



Two new combinations in New Guinea Grammitid ferns (Polypodiaceae) are proposed: Prosaptia ledermannii (Brause) Parris and Radiogrammitis habbemensis (Copel.) Parris. Herbarium abbreviations follow Thiers (2018).

Prosaptia ledermannii (Brause) Parris, comb. nov., based on Davallia ledermannii Brause, Bot. Jahrb. Syst. 56: 123 (1920). Type: Nordöstl. Neu-Guinea, Kaiserin-Augusta- Fluss-(Sepik-)Gebiet, Schraderberg, 2070 m alt., 31 May 1913, Ledermann 11842 (BM! image). Syn. Polypodium subhamatopilosum Alderw., Bull. Jard. Bot. Buitenzorg, sér. 3, 5: 220 (1922), nomen novum pro Davallia ledermannii Brause, non Polypodium ledermannii Brause (1920); Prosaptia subhamatopilosa (Alderw.) Ching, Bull. Fan Mem. Inst. Biol. 10: 242 (1941); Ctenopteris ledermannii (Brause) Copel., Univ. Calif. Publ. Bot. 18: 225 (1942); Grammitis subhamatopilosa (Alderw.) Christenh., Global Flora GLOVAP Nomenclature Part 1, Vol. 4: 55 (2018).

Radiogrammitis habbemensis (Copel.) Parris, comb. nov., based on Grammitis habbemensis Copel., Univ. Calif. Publ. Bot. 18: 223 (1942). Type: Netherlands New Guinea, 9 km east of Lake Habbema, 2860 m, Oct. 1938, Brass 10520 (holotype MICH!; isotypes A!, BO!, BRI, K! L! Syn. Oreogrammitis habbemensis (Copel.) Parris, Gard. Bull. Singapore 58(2): 260 (2007). A detailed examination of the second collection of this species (Kessler 14049 & Abrahamczyk, Indonesia, Papua Barat, road from Wamena to Lake Habbema, 6 km before summit, 4°08’S 138°47’E, 3000 m, 20 March 2011, UC) shows that the rhizome is radial with stipes arranged in whorls, characteristic of Radiogrammitis Parris rather than dorsiventral, with stipes in two rows, characteristic of Oreogrammitis Copel., so the species needs to be transferred to the former from the latter.

ACKNOWLEDGEMENTS I wish to thank the Keepers of A, K, L, MICH and UC for loan of material, the Keeper of BO for permission to examine Grammitid holdings in 2013 and Ewen Cameron (AK) for expediting the loans.

REFERENCES ALDERWERELT VAN ROSENBURGH, C. R. W. K. 1922. New or interesting Malayan ferns 12. Bull. Jard. Bot. Buitenzorg, sér. 3, 5: 179-240. BRAUSE, G. 1920. Bearbeitung der von C, Ledermann von der Sepik-(Kaiserin-Augusta-) Fluss-Expedition 1912-bis 1913 und von anderen Sammlern aus dem Papuagebiete früher mitgebrachten Pteridophyten, nebst Übersicht über alle bis jetzt aus dem Papuagebiet bekannt gewordenen Arten derselben. Bot. Jahrb. Syst. 56: 31-250. CHING, R. C. 1941. New family and combinations of Ferns. Bull. Fan. Mem. Inst. Biol. 10: 235-256. Continued on page 347 FERN GAZ. 20(8): 337-346. 2018 337


V. C. Pence Center for Conservation and Research of Endangered Wildlife (CREW) Cincinnati Zoo & Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220, USA email: [email protected]

Key words: cryopreservation, ex situ conservation, gametophyte; in vitro; long-term storage

ABSTRACT In vitro grown gametophytes of six species of ferns, which had been cryopreserved using the encapsulation dehydration procedure, were evaluated for survival after 20 yrs of storage in liquid nitrogen. Tissues were rewarmed and transferred to a recovery medium with the same methods originally used to test pre-storage viability. All six species resumed growth. Post-storage viability was not consistently higher or lower than pre-storage viability of LN exposed tissues, likely reflecting the small sample sizes. However, these results demonstrate that long-term storage in liquid nitrogen is a viable option for preserving gametophytes of at least some fern species and could be utilized as an additional tool for preserving valuable gametophyte collections and for the ex situ conservation of fern biodiversity.

INTRODUCTION For many species of ferns, gametophyte tissues have proven to be highly adaptable to growth in vitro (Table 1). Most of these have been initiated through the aseptic germination of spores, although the aseptic germination of gemmae has also been demonstrated (Raine & Sheffield, 1997). As in vitro cultures, gametophytes can provide tissues for research and for propagation, both for ornamental ferns as well as for ferns of conservation concern. The ex situ conservation of ferns has traditionally relied on living collections and spore banks (Ballesteros, 2011). However, there are situations where it may be desirable to conserve other types of fern tissues, particularly if spores are few or are difficult to access. If only a few spores are available, they can be germinated in vitro and multiplied to provide a theoretically unlimited supply of gametophytic tissue, which can be used to generate sporophytes. Tissues of such in vitro-grown gametophytes have also been shown to be adaptable to storage using cryopreservation, or storage in liquid nitrogen (LN) (Barnicoat et al., 2011; Makowski et al., 2016, 2015, Mikula et al., 2010; Mikuła, Jata & Rybczyski, 2009; Pence, 2015; 2000; Wilkinson, 2002). This can serve as an alternative to maintaining active cultures, which requires a constant input of labor and resources. Gametophytes appear to be particularly adaptable to cryostorage, largely due to their simplicity of form and high regenerability (Maeda & Ito, 1981). However, results thus far have demonstrated the ability of gametophytic tissues to survive only short-term exposure to LN, generally 1 hr-3 days (Mikula et al., 2010; Pence, 2000). In this lab in the 1990s, tissues from in vitro cultures of gametophytes of several fern species were shown to survive LN exposure (Pence, 2000). These common species were chosen because they provided readily accessible material for experimentation, but three 338 FERN GAZ. 20(8): 337-346. 2018 Reference 2011 Pence, 2000 Somer et al. , 2009 Somer et al. , 2009 Pence, 2000 Kaźmierczak, 2003 Somer et al. , 2009 Pence, 2015 Kamachi & Noguchi, 2012 Makowski et al. , 2015 Mikula et al. , 2010; Pence, 2000 Mikula et al. , 2010 Barnicoat et al., 2009 Mikula et al. , 2010; Mikuła, Jata & Rybczyski, Mikula et al. , 2010 Mikula et al. , 2010 Mikula et al. , 2010 Somer et al. , 2009 Mikula et al. , 2010 Pence, 2000 if a Method LN Exposed ED ED ED V ED, EV, V ED, EV, V ED, EV, ED, EV, V ED, EV, V ED, EV, V ED, EV, ED ED V ED, EV, ED TABLE 1. TABLE Family Parkeriaceae Cyatheaceae Adiantaceae Adiantaceae Adiantaceae Schizaeaceae Aspleniaceae Parkeriacae Dicksoniaceae Dicksoniaceae Dryopteridaceae Cyatheaceae Cyatheaceae Cyatheaceae Davalliaceae Davalliaceae Dicksoniaceae Aspleniaceae Aspleniaceae L. Species (Kaulf.) Holttum Sw. Adiantum capillus-veneris Anemia phillitidis (L.) Sw. Asplenium adiantum-nigrum L. Brongn. Ceratopteris richardii (L.) Brongn. Ceratopteris thalictroides Ceterach officinarum DC. Cibotium schiedei Schltdl. & Cham. Cyathea australis Domin. Cyathea smithii Hook.f. Davallia canariensis (L.) Sm. Davallia fejeensis Hook. Colenso Dicksonia fibrosa Adiantum tenerum Arn. Cibotium glaucum (Sm.) Hook. & Cyathea delgadii Sternb. Adiantum trapeziforme L. Asplenium scolopendrium L. Ctenitis pauciflora Cyathea dealbata (G.Forst.) Sw. , some of which have also survived exposure to LN. Species of ferns that have been grown as gametophytes in vitro PENCE: FERN GAMETOPHYTES AFTER 20 YEARS IN LIQUID NITROGEN 339 2009) 2011 2011 1999; Somer et al., Marimuthu & Manickam, 2011 Pence, 2000 Somer et al. , 2009 2002 Wilkinson, Barnicoat et al., Barnicoat et al. , 2011 1992 Rogers & Banister, Makowski et al. , 2016 Mikula 2010 Hollingsworth et al. , 2012 Pence, 2000 Mikula et al. , 2010 Bertrand et al., Whittier & Steeves, 1960 Barnicoat et al., Sara & Manickam, 2007 Marimuthu & Manickam, 2011 DeSoto et al. , 2008 Fiori, Santos & Randi, 2009 Somer et al. , 2009 Ashmore et al. , 2011 1997 Raine & Sheffield, ED ED V ED, EV, ED V ED, EV, ED ED ED Dryopteridaceae Osmundaceae Dicksoniaceae Dryopteridaceae Marattiaceae Polypodiaceae Polypodiaceae Pteridaceae Dryopteridaceae Thelypteridaceae Thelypteridaceae Hymenophyllaceae Blechnaceae Polypodiaceae Hymenophyllaceae Polypodiaceae Adiantaceae Osmundaceae Aspleniaceae Dennstaedtiaceae Thelypteridaceae (Rac.) Campbell (Blume) Holttum Sw. Sun Tuff’ ‘ Sun Methods: ED = encapsulation dehydration; EV = encapsulation vitrification; V = vitrification V = encapsulation vitrification; Methods: ED = encapsulation dehydration; EV Osmundastrum cinnamomeum (L.) C.Presl devexa (Kunze) Copel. Tectaria speciosum Willd. Trichomanes (Pr.) Hook. Dicksonia sellowiana (Pr.) (L.) J.Sm. Drynaria quercifolia A.Gray Dryopteris dilatata (Hoffm.) Dryopteris filix-mas (L.) Schott Lepisorus longifolius smithii Macroglossum Notholaena R.Br. L. Osmunda regalis (L.) J.Sm. Phlebodium aureum Phyllitis (Asplenium) scolopendrium (L.) Newman Pteridium aquilinum (L.) Kuhn Pteris adscensionis unitus (L.) Holttum Sphaerostephanos Thelypteris confluens (Thunb.) C.V.Morton radicans (L.) Sm. Woodwardia Hymenophyllum tunbrigense (L.) Sm. Polypodium cambricum L. Holttum triphyllum (Sw.) Pronephrium a 340 FERN GAZ. 20(8): 337-346. 2018 of the species are also congeneric with rare species (Kang et al., 2008; Kishida, 2015; Yu et al., 2017). In those studies, in addition to testing the initial survival of the gametophytes (Pence, 2000), samples were banked in the Frozen Garden™ of CREW’s CryoBioBank™ for long-term storage. In a recent study to evaluate the viability of numerous samples from the Frozen Garden after years of storage, several samples of gametophytes of these six species were removed and evaluated for their viability after 20 yrs in LN. The results of that study are presented here.

METHODS In vitro gametophyte cultures of Davallia fejeensis Hook., Drynaria quercifolia (L.) J. Sm., Cibotium glaucum (Sm.) Hook & Arn., Adiantum trapeziforme L., Adiantum tenerum Sw., and Phlebodium aureum L. were originally initiated by the aseptic germination of spores in vitro, as previously described (Pence, 2000), and maintained on a medium of half-strength Murashige and Skoog salts and minimal organics (Linsmaier and Skoog, 1965) with 1.5% sucrose and 0.22% Phytagel (gellan gum, Sigma Chemical Co., St. Louis, MO) (= ½ MS medium), in 60 x 15 mm Petri plates (Corning™ Falcon™, Durham, NC), approximately 15 ml of medium/plate. Cultures were maintained at 26oC under cool white fluorescent lights at 10 – 20 µmol m-2 sec-1 PPFD, with a 16:8 hr light:dark cycle. Tissue banking took place between 6/21/1995 and 7/26/1995. Fragments of gametophyte tissue were isolated by cutting the gametophyte with a scalpel into small pieces from throughout the gametophyte thallus. Pieces ranged in size from 1-3 mm in length and width. Gametophyte fragments were prepared for cryopreservation by culturing for seven days on ½ MS medium with the addition of filter-sterilized 10 µM abscisic acid (ABA). They were then cryopreserved using the encapsulation dehydration procedure (Fabre & Dereuddre, 1990), in which the fragments were encapsulated in alginate beads before an18 hr incubation in a 0.75 M sucrose solution, with more than one fragment being encapsulated per bead. The encapsulated tissues were then dried for 3 or 5 hrs under the airflow of a laminar flow hood. The dried beads were stored in 2 ml polypropylene cryovials (Corning®, Corning, NY), 6 to 19 beads per vial, the number of beads varying depending on the amount of tissue available. The samples were stored in a MVE XC 47/11, 900 vial capacity LN storage tank (Chart MVE, Ball Ground, GA), submerged in LN (-196oC). As a control at the time of banking, one vial of each species from each time of banking was rewarmed after a one-day exposure to LN and cultured on ½ MS medium for recovery. Recovery growth was scored as the number of beads showing green growing tissues, and data for the samples were taken between nine and 28 days after rewarming. In the case of three species, survival was also measured after 4.5 months of growth, with data taken after four weeks of growth. Samples from each species were also removed after 3.5 yrs of storage in LN, and recovery data was taken after two and seven weeks of growth. The data taken seven weeks after rewarming were reported previously (Pence, 2000). Samples were removed from LN between 7/16/2015 and 8/14/2015 to evaluate 20-yr survival. Recovery conditions in the present study replicated as much as possible the original recovery conditions, using ½ MS medium, only with modification in the source of the prepared MS salt mixture (Phytotechnology Laboratories, Overland Park, KS) and the source and amount of the gellan gum (Gelzan, Caisson Labs, Smithfield, UT), added at 0.33%. One vial representing each species and each banking time for that species was removed (9 vials total) and rewarmed at ambient temperature (22oC) for 15 min. The PENCE: FERN GAMETOPHYTES AFTER 20 YEARS IN LIQUID NITROGEN 341 beads were then plated on 1 – 4 plates of ½ MS medium, depending on the number of beads, 4-6 beads/plate. Cultures were maintained under the same temperature and lighting conditions originally used for growth. At the time of banking and at 3.5 yrs, survival was scored as the number of beads showing some recovery growth of encapsulated tissues, regardless of the number of fragments per bead. When tissues were evaluated after 20 years, survival was scored both as the percent of fragments and the percent of beads showing green, growing tissue at 3 – 4 weeks.

RESULTS Growth from cryopreserved gametophyte tissue occurred from all of the species of ferns tested in this study (Table 2). The values for the original survival data reported here are those recorded for the specific samples that were banked and are different from those reported previously (Pence, 2000), which were collected from experiments done in preparation for the long-term banking of samples. In all but two species, the percent of tissues showing growth after storage was greater than that observed before long-term storage. In Drynaria quercifolia and both samples of A. trapeziforme, post-storage growth was less than that observed originally, and in one sample of A. trapeziforme, there was no post-storage growth. However, that sample produced fungal growth on a few beads within the first few days of culture, and the remaining beads were transferred to fresh plates. The transferred beads did not show any contamination, but they also did not show any growth. Specific moisture levels of the banked samples used in this study are not available, except for two of the three samples, D. quercifolia and A. trapeziforme, which were dried for 5 hrs to 17.5% and 18.9% moisture, respectively. Samples recovered at 20 yrs were scored at 3 – 4 weeks. When these were compared with samples recovered at 3.5 yrs, the percent survivals were, in most cases, more similar to those recorded at two weeks from the 3.5 yr samples than with survivals recorded at seven weeks for those samples, which in all cases were higher than two week survival values (Table 2). Since pre-storage viability was based on growth per bead, post-storage measurements were calculated both on growth per bead and on growth per fragment for seven of the nine samples. The percent survival was very similar between the two measurements (Table 2). Most fragments showed some damaged areas, but ultimately, those with any surviving areas went on to regenerate gametophyte tissues (Figure 1).

DISCUSSION These results demonstrate that in vitro-grown gametophytes of at least some fern species can survive up to 20 yrs in LN storage without appreciable deterioration using the encapsulation dehydration procedure. This method has been applied by others in the intervening yrs with good success (Table 1). There also have been reports of some survival of fern gametophytes using the encapsulation vitrification method (Makowski et al., 2016; Mikula et al., 2010), although when it was compared with encapsulation dehydration, the latter resulted in higher survival. Studies using vitrification without the protection of encapsulation have uniformly reported severe plasmolysis of the gametophyte tissues and no survival after exposure to LN (Makowski et al., 2016; Mikula et al., 2010). When the successful cryopreservation of fern gametophytes was reported originally, 3 – 4 hrs of drying brought samples to 19-27% moisture (wet weight basis) (Pence, 2000). 342 FERN GAZ. 20(8): 337-346. 2018 nd nd 0.0 79.5 82.3 83.5 91.7 66.7 40.5 Survival per Piece per 0.0 66.7 80.0 39.3 90.0 82.3 50.0 41.3 83.5 20 Yrs 20 Survival per Bead per 28 28 20 20 28 28 28 28 28 Day 50.0 75.0 67.0 90.0 88.0 100.0 Survival (at 7 wks) b 3.5 Yrs 3.5 33.0 60.0 80.0 50.0 67.0 63.0 Survival (at 2 wks) 40 80 73.3 TABLE 2 TABLE Survival 4.5 Mos a (at 4 wks) 6.7 25.0 60.0 84.6 46.2 46.2 91.7 19.3 41.7 Survival Pre-storage 9 4 8 6 12 16 12 16 27 Day 3 3 5 3 3 3 5 3 5 Hrs of Drying Species Seven week survival data from Pence, 2000. Data available for three species only. Davallia fejeensis Adiantum trapeziforme Drynaria quercifolia Phlebodium aureum Adiantum tenerum Cibotium glaucum a b Percent survival of six species of fern gametophytes, dried for 3 or 5 hrs, through LN exposure before storage and after 4.5 mos, 3.5 yrs, and 20 and yrs, 3.5 mos, 4.5 after and storage before exposure LN through hrs, 5 or 3 for dried gametophytes, fern of species six of survival Percent samples. all for same the if heading, the in or samples, different for different if column, adjacent in given is observation of Day storage. LN of yrs PENCE: FERN GAMETOPHYTES AFTER 20 YEARS IN LIQUID NITROGEN 343

The moisture contents available for two of the samples in this study that had been dried for five hrs were 18 – 19%. With seeds, a number of studies have been directed at defining optimum storage moisture levels, which are related to the seed oil content (e.g. Pritchard, 1995; Hor et al., 2005). Whether the moisture levels used is this study are optimum for fern gametophytes will require further research, but they were sufficient to allow regeneration of at least some of the tissues after cryostorage. The fact that survival after long-term storage was greater in most cases than pre- storage survival may have resulted from data being taken much earlier in the case of some of the pre-storage samples, as these were done by different operators, at different times, for various goals. The results of the 3.5 yr samples indicate that the initiation of growth continued for several weeks, with more fragments growing at seven weeks than at two weeks. Alternatively, the sample sizes in this study were small and may not be large enough to clearly track any small changes in viability over the time of storage. Similar increases in viability after LN exposure have been noted with small samples of other species, by others, as well as in this laboratory (Mix-Wagner, Schumacher & Cross, 2003; Vanhove, Philpott & Pence, 2016). The highly regenerative nature of fern gametophytes has been noted for several species (Maeda & Ito, 1981; Ong & Ng, 1998), and this ability makes these tissues well adapted to cryopreservation. Even if some tissues are damaged in the process of either cryoprotection or LN exposure, only a small amount of tissue is required (possibly only one cell) to regenerate the gametophyte tissue (Maeda & Ito, 1981). However, this ability makes it more difficult to evaluate survival quantitatively, because fragments are of different sizes and only portions of them may survive. Having more than one fragment encapsulated in an alginate bead can increase the number of fragments available for regrowth and minimize the number of beads needed, and survival per bead appears to

Figure 1. Encapsulated gametophtyes from two fern species resuming growth after 20 yrs of storage in liquid nitrogen. A. Adiantum tenerum gametophytes, as well as fragments that did not grow (arrow); B. Phlebodium aureum gametophytes. Scale bars = 0.5 cm. 344 FERN GAZ. 20(8): 337-346. 2018 correlate well with fragment survival. When survival of the 20-yr old samples was measured by both methods, the results were very similar. Cryopreservation of fern gametophytes has thus far been applied to in vitro cultures of gametophytes. This requires the initiation of gametophyte cultures by the aseptic germination of spores or gemmae, and in vitro fern gametophyte cultures initiated in this way have been established and reported for a number of species (Table 1). However, for species known only from the gametophyte in nature, in vitro cultures would need to be established directly from gametophytic tissue. Methods for this have been reported for gametophytes of bryophytes (Rowntree & Ramsay, 2005; Segreto et al., 2010) but not yet for fern gametophytes. Gametophytes of bryophytes are more complex structures than those of ferns, and further research is needed to determine whether fern gametophytes can survive the surface sterilization required for initiating in vitro cultures. The results of this study indicate gametophyte cryopreservation is an option for long- term conservation of in vitro collections of fern gametophytes that have been developed for horticultural or experimental purposes, as well as an option for the ex situ conservation of fern biodiversity. Thus, it is a tool that can contribute to the ex situ conservation goals (Target 8) of the Global Strategy for Plant Conservation (Convention on Biological Diversity, 2010). Further research is needed to test the applicability of encapsulation dehydration to more species with different habits and adaptations in order to more clearly define the breadth of its potential use. In addition, more long-term studies should be initiated to evaluate the effectiveness of cryopreservation in preserving tissues over the course of decades. However, these results with six species clearly demonstrate the potential of this method for fern conservation in the future.

ACKNOWLEDGEMENTS The helpful technical assistance of Kirsten Stewart in banking the original samples is acknowledged. Removal and evaluation of samples after 20 yrs of storage was supported by Institute of Museum and Library Services grant no. LG-25-12-0595-12.

REFERENCES ASHMORE, S.E., HAMILTON, K.N. & OFFORD, C.A. 2011. Conservation technologies for safeguarding and restoring threatened flora: Case studies from Eastern Australia. In Vitro Cell. Develop. Biol.- Plant 47: 99-109. BALLESTEROS, D. 2011. Conservation of Fern Spores. In: FERNÁNDEZ, H. & AL, E. (Eds) Working with ferns: Issues and applications, pp. 165-172. Springer Science+Business Media, LLC, New York. BARNICOAT, H., CRIPPS, R., KENDON, J. & SARASAN, V. 2011. Conservation in vitro of rare and threatened ferns—case studies of biodiversity hotspot and island species. In Vitro Cell. Develop. Biol.-Plant 47: 37-45. BERTRAND, A.M., ALBUERNE, M.A., FERNÁNDEZ, H., GONZÁLEZ, A. & SÁNCHEZ-TAMÉS, R. 1999. In vitro organogenesis of Polypodium cambricum. , Tiss. Org. Cult. 57: 65-69. CONVENTION ON BIOLOGICAL DIVERSITY (CBD). 2010. Consolidated update of the Global Strategy for Plant Conservation 2011-2020. Available online at http://www.cbd.int/gspc/strategy.shtml. (Accessed September 15, 2017) DESOTO, L., QUINTANILLA, L.G. & MÉNDEZ, M. 2008. Environmental sex determination in ferns: Effects of nutrient availability and individual density in Woodwardia radicans. J. Ecol. 96: 1319-1327. PENCE: FERN GAMETOPHYTES AFTER 20 YEARS IN LIQUID NITROGEN 345

FABRE, J., & DEREUDDRE, J. 1990. Encapsulation-dehydration: A new approach to cryopreservation of Solanum shoot-tips. CryoLett. 11: 413-426. FIORI, C.C.L., SANTOS, M. & RANDI, Á.M. 2009. Aspects of gametophyte development of Dicksonia sellowiana Hook (Dicksoniaceae): an endangered tree fern indigenous to South and Central America. Amer. Fern J. 99: 207-216. HOLLINGSWORTH, S.N., ANDRES, E.A. & GREER, G.K. 2012. Pheromonal interactions among gametophytes of Osmundastrum cinnamomeum and the origins of antheridiogen systems in leptosporangiate ferns. Intern. J. Plant Sci. 173: 382-390. HOR, Y.L., KIM, Y.J., UGAP, A., CHABRILLANGE, N., SINNIAH, U.R., ENGELMANN, F. & DUSSERT, S. 2005. Optimal hydration status for cryopreservation of intermediate oily seeds: Citrus as a case study. Annals of Botany 95: 1153-1161. KAMACHI, H. & NOGUCHI. M. 2012. Negative gravitropism in dark-grown gametophytes of the fern Ceratopteris richardii. Amer. Fern J. 102: 147-153. KANG, M., HUANG, H., JIANG, M. & LOWE, A.J. 2008. Understanding population structure and historical demography in a conservation context: Population genetics of an endangered fern. Divers. Distrib. 14: 799-807. KAźMIERCZAK, A. 2003. Induction of cell division and cell expansion at the beginning of gibberellin A3-induced precocious antheridia formation in Anemia phyllitidis gametophytes. Plant Sci. 165: 933-939. KISHIDA, W. 2015. Asplenium dielmannii. The IUCN Red List of Threatened Species 2015: e.T80229074A80229092. http://dx.doi.org/10.2305/IUCN.UK.2015- 4.RLTS.T80229074A80229092.en. Downloaded on 16 April 2018. LINSMAIER, E.M. & SKOOG, F. 1965. Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant. 18: 100-127. MAEDA, M. & ITO, M. 1981. Isolation of protoplasts from fern prothallia and their regeneration to gametophytes. Bot. Mag. Tokyo 94: 35-40. MAKOWSKI, D., JRYBCZYńSKI, .J., MIKULA, A. & KLIMASZEWSKA, K. 2015. A simple way to overcome the recalcitrance of the water fern Ceratopteris thalictroides (L.) Brongn. to cryopreservation. Acta Soc. Bot. Pol. 84: 385-388. MAKOWSKI, D., TOMICZAK, K., RYBCZYńSKI, J.J. & MIKUłA, A. 2016. Integration of tissue culture and cryopreservation methods for propagation and conservation of the fern Osmunida regalis L. Acta Physiol. Plant. 38: 1-12. MARIMUTHU, J., & MANICKAM, V.S. 2011. Ex situ conservation of two threatened ferns of the Western Ghats through in vitro spore culture. J. Threat. Taxa 3: 1919- 1928. MIKUłA, A., JATA, K., & RYBCZYSKI, J.J. 2009. Cryopreservation strategies for Cyathea australis (R. Br.) Domin. CryoLett. 30: 429-439. MIKULA, A., MAKOWSKI, D., WALTERS, C. & RYBCZYNSKI, J.J. 2010. Exploration of cryo-methods to preserve tree and herbaceous fern gametophytes. In: FERNÁNDEZ, H., KUMAR, A. & REVILLA, M. A. (Eds), Working with ferns: Issues and applications, pp. 173-192. Springer Science+Business Media, LLC, New York. MIX-WAGNER, G., SCHUMACHER, H.M. & CROSS, R.J. 2003. Recovery of potato apices after several years of storage in liquid nitrogen. CryoLett. 24: 33-41. ONG, B.L., & NG, M.L. 1998. Regeneration of drought-stressed gametophytes of the epiphytic fern, Pyrrosia pilosellodes (L.) Price. Plant Cell Rep. 18: 225-228. PENCE, V.C. 2000. Cryopreservation of in vitro grown fern gametophytes. Amer. Fern 346 FERN GAZ. 20(8): 337-346. 2018

J. 90: 16-23. PENCE, V.C. 2015. Propagation and cryopreservation of Asplenium scolopendrium var. americanum, the American hart’s-tongue fern. Amer. Fern J. 105: 211-225. PRITCHARD, H.W. 1995. Cryopreservation of seeds. In: DAY, J.G. & MCLELLAN, M.R. (Eds) Methods in molecular biology. Vol. 38: Cryopreservation and freeze- drying protocols, pp. 133-144. Humana Press Inc., Totowa, NJ. RAINE, A.C.A. & SHEFFIELD, E. 1997. Establishment and maintenance of aseptic culture of Trichomanes speciosum gametophytes from gemmae. Amer. Fern J. 87: 87-92. ROGERS, S.M.D. & BANISTER, S. 1992. Micropropagation of Notholaena. HortSci. 27: 1224-1225. ROWNTREE, J.K. & RAMSAY, M.M. 2005. Ex situ conservation of bryophytes : Progress and potential of a pilot project. Bol. Soc. Esp. Briol. 26-27: 17-22. SARA, S.C. & MANICKAM, V.S. 2007. In vitro developmental ontogeny and life cycle of a rare fern species - Thelypteris confluens (Thunb.) Morton. Ind. J. Biotech. 6: 372-380. SEGRETO, R., HASSEL, K., BARDAL, R. & STENøIEN, H.K. 2010. Desiccation tolerance and natural cold acclimation allow cryopreservation of bryophytes without pretreatment or use of cryoprotectants. The Bryol. 113: 760-769. SOMER, M., ARBESú, R., MENÉNDEZ, V., REVILLA, M.A. & FERNÁNDEZ, H. 2009. Sporophyte induction studies in ferns in vitro. Euphytica 171: 203-210. VANHOVE, A.-C., PHILPOTT, M. & PENCE, V.C. 2016. Evaluating survival in shoot tips of several species stored in liquid nitrogen for 4 to 16 years. In Vitro Cell. Develop. Biol.-Plant 52: S33. WHITTIER D.P. & STEEVES, T. 1960. The induction of apogamy in the bracken fern. Can. J. Bot. 38: 925-930. WILKINSON, T. 2002. In vitro techniques for the conservation of Hymenophyllum tunbrigense (L.) Sm. Fern Gaz. 16: 458. YU, R., ZHANG, G., LI, H., CAO, H., MO, X., GUI, M., ZHOU, X., JIANG, Y., LI, S. & WANG, J. (2017) In vitro propagation of the endangered tree fern Cibotium barometz through formation of green globular bodies. Plant Cell Tiss. Org. Cult. 128: 369-379. FERN GAZ. 20(8). 2018 347

Continued from page 336

CHRISTENHUSZ, M. 2018. Plant Gateway’s The Global Flora A practical flora to vascular plant species of the world special edition GLOVAP Nomenclature Part 1, Vol. 4: 1-155. Plant Gateway, Bradford. COPELAND, E. B. 1942. Filicum Novarum CXXXVIII Diagnoses. Univ. Calif. Publ. Bot. 18: 217-226. PARRIS, B. S. 2007. Five new genera and three new species of Grammitidaceae (Filicales) and the re-establishment of Oreogrammitis. Gard. Bull. Singapore 58(2): 233-274. THIERS, B. 2018. Index Herbariorum. New York Botanical Gardens, on-line.

Barbara S. Parris Fern Research Foundation, 21 James Kemp Place, Kerikeri, Bay of Islands, New Zealand 0230 Email: [email protected] 348 FERN GAZ. 20(8). 2018

INSTRUCTIONS FOR AUTHORS PAPERS should not usually exceed 20 printed pages and are generally expected to be considerably shorter. As well as reports of original research, review articles and short notes, e.g. new records, are encouraged. The senior author should supply an email address to facilitate correspondence. REVIEWS should cover recent developments in any aspect of research on ferns and lycopods. This can include unpublished research by the author, and/or the possible direction of future research. They should be aimed at the general reader who may not be an expert in the chosen topic. Proposals for a review should be discussed first with the review editors. MANUSCRIPTS should be submitted in English (British) in electronic format in 10-point Times New Roman font and single spaced. Electronic versions of text and tables should be compatible with WORD, with figures as TIFF files, and sent as email attachments. All manuscripts will be refereed. THE TITLE should reflect the content of the paper and be in BOLD CAPITALS (11-point) and centrally aligned. Generic and specific names should be in italics and any title containing a generic or specific name should be followed by the family name in brackets e.g. TRICHOMANES SPECIOSUM (HYMENOPHYLLACEAE) IN SOUTHERN SPAIN AUTHOR ABBREVIATIONS should follow Pichi Sermolli’s (1996) Authors of scientific names in Pteridophyta, Royal Botanic Gardens, Kew. MAIN HEADINGS: should be in BOLD CAPITALS (10-point) and centrally aligned. SUBSIDIARY HEADINGS: should be in bold, the first letter of each word in capitals, the rest in lower case and left-aligned. AUTHORS’ NAMES AND FULL ADDRESSES (including email address): follow the title and are centrally aligned. KEY WORDS: up to ten. ABSTRACT: should reflect the content of the paper. FIGURES: there is no distinction between photographs and line drawings in numbering. All should be supplied in mono or colour, TIFF format, 300 dpi and CMYK. (please contact the Production Editor with queries), with a scale bar where appropriate. Lettering or numbers (Arabic) should be in the bottom left using uppercase Times Roman. Figure captions should be on a separate sheet. TABLES: can be printed in either portrait or landscape format. Authors should consider this when preparing tables. Authors should ensure that tables fit the printed page size in a legible form. PAGESIZE: The Gazette has a useable page size of 123 × 204 mm. This can be used in portrait or landscape mode but any supplied artwork should not be larger than this. MEASUREMENTS: follow the metric system. CHECKLISTS: follow the format of Baksh-Comeau, Fern Gaz. 16(1, 2): 11- 122. REFERENCES: follow the style of a recent issue of The Fern Gazette, e.g.:- HOOKER, W.J. 1864. Species Filicum, 5. Dulau & Co., London. MORTON, C.V. 1947. The American species of Hymenophyllum, section Sphaeroconium. Contr. U.S. Natl. Herb. 29(3): 139-201. STEVENSON, D.W. & LOCONTE, H. 1996. Ordinal and familial relationships of pteridophyte genera. In: CAMUS, J.M., GIBBY, M. & JOHNS, R.J. (Eds) Pteridology in perspective, pp. 435-467. Royal Botanic Gardens, Kew. JOURNAL ABBREVIATIONS: should follow Botanico Periodicum Huntianum & Supplements. Alterations from the original text at proof stage will be charged for unless they are minor points of detail. A pdf will be provided free to the senior author.