Comparative Anatomy of the Genus Pyrrosia Mirbel (Polypodiaceae) in Thailand

KANOKORN KOTRNON, ACHRA THAMMATHAWORN* AND PRANOM CHANTARANOTHAI

Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.

ABSTRACT.– Lamina epidermal peels and transections of leaves, stipes, rhizomes and roots of 17 Pyrrosia species in Thailand were investigated. The characteristics found to be of most use in distinguishing the species were: presence/absence of hydathodes, trichome type, stomatal type, shape and wall structure of the epidermal cells, presence/absence of hypodermis, presence/absence of sclerenchyma in the midrib and leaf margins, arrangement of the mesophyll, stipe shape in transection, distribution of sclerenchyma strands and presence/absence of sclerenchyma sheaths in the parenchyma ground tissue of the rhizomes.

KEY WORDS: Pyrrosia; Polypodiaceae; Plant anatomy; Thailand

Ogura (1972); Lin & Devol (1977); Sen & INTRODUCTION Hennipman (1981); Hovenkamp (1986) and

Schneider (1996). Hovenkamp (1986) The genus Pyrrosia Mirbel (Polypo- recognized 51 species worldwide and noted diaceae) comprises c. 100 species, widely the importance of anatomical characteristics distributed from tropical and subtropical for the delimitation of the genus and Asia to Africa and Australia (Holttum, species. These included the 1968). Tagawa & Iwatsuki (1989) recorded presence/absence of hydathodes, the wall 18 species of the genus in Thailand. The structure of the epidermal cells, the type genus is well circumscribed by the and level of stomata, the presence/absence presence of stellate hairs. Most species are of hypodermis, the structure of the epiphytic in lowland and montane forests; mesophyll, the presence/absence of others are terrestrial at low to high sclerenchyma sheaths, and the scattering elevations. Holttum (1968) noted that pattern of sclerenchyma strands in Pyrrosia is closely related to Drymoglos- rhizomes. Nayar & Chandra (1966) studied sum but differs in sori shape. the morphology and anatomy of 13 species Anatomical studies of this genus has of Indian Pyrrosia and divided the genus been carried out by many authors: Nayar & into six morphological groups namely: Chandra (1966); van Cotthem (1970); Flocculosa, Heteractis, Mannii, Nayariana, * Corresponding author: Obovata and Varia. The purpose of the Tel: (6643)-342-908 present paper is to investigate the leaf, Fax: (6643)-364-169 E-mail: achara @kku.ac.th 76 NAT. HIST. J. CHULALONGKORN UNIV. 7(1), MAY 2007 stipe, rhizome and root anatomy in Thai longifolia, P. mollis, P. nummularifolia, P. Pyrrosia. Our study will serve to fill in penangiana and P. piloselloides have gaps on the anatomy of the organs where irregularly-shaped sinuous walls (Fig. 1) published information on the genus is with straight, anticlinal walls in P. lacking. Once our data are combined with adnascens, P. lanceolata, P. nuda and P. other information on the comparative stigmosa. In transverse section, the outer anatomy of Polypodiaceae, they will add to walls of the epidermis are flat or convex a comprehensive analysis of this species- and project outwards to varying degrees. rich family. This report, then, represents a The epidermal cells on the midrib are partial effort towards that goal. smaller than those on the blade except in P. piloselloides and P. tonkinensis where their

sizes are similar. The cells of the lower MATERIALS AND METHODS epidermis are similar to those of the upper, in having sinuous walls. P. adnascens, P. Mature and fresh leaves, stipes, lanceolata and P. longifolia have straight rhizomes and roots of 17 taxa of Pyrrosia epidermal cell walls. The outer wall of the were obtained from the field in Thailand lower surface is thicker than that of the (Table 1), sectioned for anatomical studies upper surface except in P. mollis, P. using the sliding microtome, and stained. penangiana, P. stigmosa and P. varia Transverse sections were cut from the where it is thinner. The cutinized epidermis middle part of the midrib, leaf margin, on both surfaces of the leaf are distinct in stipe, rhizome and root. Two to five P. adnascens, P. angustata, P. heteractis, P. specimens were used to represent each heteractis var. minor, P. nummularifolia, P. species. For epidermal studies, samples stigmosa and P. varia. were prepared by mechanically scraping Hydathodes: Hydathodes are scattered midway between the base and the apex of all over the upper leaf surface (Fig. 2) in the lamina and stained. Permanent slides all species but absent in P. adnascens, P. were mounted in DePeX artificial mounting angustata, P. floccigera, P. lanceolata, P. medium. Photographs were taken with the longifolia, P. nuda and P. nummularifolia. aid of an Olympus BH2 light microscope. Stomata: Stomata are restricted to the Voucher specimens are kept at Khon Kaen lower leaf surface. Most species have University Herbarium (KKU). pericytic stomata (with vary narrow guard cells encircling the stomata) (Figs. 3, 5, 6) and polocytic stomata (with stomata RESULTS attached to the wall of the guard cells at the General anatomical description anterior end) occur only in P. penangiana (Fig. 4). Deeply sunken stomata occur in Lamina P. adnascens, P. angustata, P. eberhardtii, P. floccigera, P. heteractis, P. heteractis Epidermis: In surface view, the cells of the upper epidermis of most species are 5- var. minor, P. lanceolata, P. longifolia, P. 6-sided and orientated longitudinally (Fig. nuda and P. varia. Superficial stomata are 2). However, P. angustata, P. floccigera, present in P. flocculosa, P. mollis, P. P. flocculosa, P. heteractis var. minor, P. nummularifolia, P. penangiana, P. KOTRNON ET AL. – COMPARATIVE ANATOMY OF THE GENUS PYRROSIA MIRBEL 77

Sclerenchyma- - + + + + + + + + + + + + + + + + rib

Mid tous sheath traight, Sclerenchyma ------of margin + + + + + + , ST=s

mbination of

Leaf Mesophyll UN UN UN DIF DIF DIF DIF DIF DIF DIF DIF DIF DIF DIF DIF DIF DIF inuous entiated, +=presence, ------

Hypodermis + + + + + + + + + +

L L

Lower , G , G LA LA LA LA LA LA me AC, RI AC, RI LA LA AC, GL o AC, RI, GL AC, RI, GL AC, RI, GL AC, RI, GL LA, DI, GL ed, IR=irregular, SI=s LA, AC, RI, DI id

Trich C C C C

miate ray, RI=ribbon DI=co , SC , SC , SC , A , A , A Upper , A LA LA LA LA LA LA AC AC AC AC LA LA LA LA LA LA LA pongy parenchyma, UN=undiffer Level DE DE DE DE DE DE DE DE DE DE SUP SUP SUP SUP SUP SUP SUP and s Stomata ade Type PR PR PR PR PR PR PR PR PR PR PR PR PR PR PR PR PO ------

Hydathodes + + + + + + + + + + SI SI SI SI SI SI SI SI SI SI SI SI SI SI

Lower ST ST ST ata, LA=lanceolate ray, AC=acu wall om t Anticlinal SI SI SI SI SI SI SI SI SI SI SI SI SI

Upper ST ST ST ST DIF=differented into palis

Lower IR IR IR IR IR IR IR IR IR IR IR IR QU QU QU QU QU Epidermis uperficial s . (*=Group I, **=Group II, ***=Group III, QU=5-6-s Shape sia

Upper IR IR IR IR IR IR IR IR IR QU QU QU QU QU QU QU QU Pyrro unken, SUP=s

cale-like, GL=glandular hairs

***

, SC=s (C. Chr) Ching

t) Ching*** Species (Sw.) Ching minor (L.) M.G. Price *** (Hook.) Holtt. ** (H.Chris (Giesenh.) Ching (L.) Farwell (Sw.) Chin * (D.Don) Ching ** (Blume) Ching * (Mett. ex Kuhn) Ching *** var. (Sw.) Ching * (Burm.f.) C.V. Morton (Sw.) Ching *** sis

Anatomical characters of leaf Thai en (Kunze) Ching ** tata cens (Kaulf.) Farwell * (Giesenh.) Ching * in k 1. n nuda

ABLE P. adnas P. angus P. eberhardtii P. floccigera P. flocculosa P. heteractis P. heteractis P. lanceolata P. mollis P. P. to P. longifolia P. nummularifolia P. penangiana P. piloselloides P. stigmosa P. varia T PR=pericytic, PO=polocytic, DE=deeply s ribbon and lanceolate rays -=absence)

78 NAT. HIST. J. CHULALONGKORN UNIV. 7(1), MAY 2007

FIGURES 1-9. Upper epidermis (1-2) lower epidermis showing stomata (3-6) and surface trichomes (7-9): 1. P. angustata 2. P. tonkinensis 3. P. eberhardtii 4. P. penangiana 5. P. piloselloide 6. P. varia 7. P. angustata 8. P. mollis 9. P. adnascens.

KOTRNON ET AL. – COMPARATIVE ANATOMY OF THE GENUS PYRROSIA MIRBEL 79 piloselloides, P. stigmosa and P. thinner wall than the epidermal cells. tonkinensis. Hypodermal cells in P. eberhardtii are Trichomes: Stellate hairs develop on filled with tannin. both surfaces in all species, but are dense Mesophyll: Dorsiventral in most on the lower surface. Four forms of stellate species, but undifferentiated mesophyll rays are observed. Lanceolate rays (Fig. 9) only occur in P. flocculosa, P. longifolia with 1-7 stalk cells and 6-15 rays on both and P. piloselloides (Figs. 10-11) which surfaces occur in P. adnascens, P. are composed of parenchymatous cells, in angustata, P. eberhardtii, P. heteractis, P. which all the cells are large, thin-walled heteractis var. minor, P. lanceolata, P. and filled with tannin and chloroplasts. The nuda, P. piloselloides and P. varia. cells on each side are round and have 4-11 Acuminate rays (Fig. 7) (narrower and layers with large intercellular spaces. The longer than lanceolate rays) with 1-18 stalk palisade parenchyma has 1-5 layers and is cells and 6-10 rays on both surfaces occur composed of narrow, short cells in all in P. angustata, P. floccigera, P. species except P. nummularifolia, P. flocculosa, P. mollis, P. nummularifolia, floccigera and P. nuda in which the cells P. penangiana, P. stigmosa and P. are large and broad. Spongy parenchyma tonkinensis. Ribbon (or woolly) rays (Fig. has 2-7 layers and is irregularly shaped 8) (long, strongly and irregularly curled, with small cells and large intercellular closely appressed rays) with 1 stalk cell and spaces. In all species, the palisade and 5-15 rays occur only on the lower surface spongy cells contain chloroplasts. in P. angustata, P. floccigera, P. Leaf margin: Cells in the leaf margin flocculosa, P. mollis, P. nummularifolia, are slightly downwards in all species (Figs. P. stigmosa, and P. tonkinensis. Finally, 12-13), and sclerenchymatous in P. both ribbon rays and lanceolate rays occur adnascens, P. angustata, P. heteractis, P. on the lower surface in P. angustata and P. heteractis var. minor, P. lanceolata and P. eberhardtii. Glandular hairs are unicellular longifolia. and uniseriate and occur on the lower Midrib: The ground tissue is surface in P. eberhardtii, P. floccigera, P. parenchymatous and contains chloloplasts heteractis, P. heteratctis var. minor, P. in all species except P. flocculosa and P. mollis, P. nummularifolia, P. penangiana piloselloides in which it is densely filled and P. tonkinensis. Scale-like hairs occur with tannin. The sclerenchymatous sheath on the upper suface in P. lanceolata P. is below the lower and upper epidermis in longifolia and P. varia. the midrib of all species except P. Hypodermis: The hypodermis covers piloselloides (Figs. 14-15). the area of the leaf blade and develops on Vascular tissue: Vascular bundles the upper surface alone in all species except range in number from 1-5 through out the P. flocculosa, P. longifolia, P. mollis, P. width of the midrid, with concentric T- penangiana, P. piloselloides, P. stigmosa shaped xylem surrounded by phloem and and P. tonkinensis, where hypodermal cells pericyclic cells comprising 1-3 layers of are absent from the leaf margin and midrib. parenchyma. The vascular bundles are The hypodermis has 1-4 layers, and cells in surrounded by endodermis. (Fig. 17). each layer are larger with a distinctly 80 NAT. HIST. J. CHULALONGKORN UNIV. 7(1), MAY 2007

FIGURES 10-17. TS of leaf (10-11) TS of leaf margins (12-13) and TS of leaf midribs (14-15): 10. P. flocculosa 11. P. longifolia 12. P. angustata 13. P. stigmosa 14. P. eberhardtii 15. P. nummularifolia 16. P. floccigera, showing stomata level 17. P. mollis, showing vascular bundle.

KOTRNON ET AL. – COMPARATIVE ANATOMY OF THE GENUS PYRROSIA MIRBEL 81

FIGURES 18-25. TS of stipes (18-19) TS of rhizomes (20-23) TS of roots (24-25): 18. P. nuda 19. P. piloselloides 20. P. angustata 21. P. nummularifolia 22. P. floccigera 23. P. mollis 24. P. penangiana 25. P. tonkinensis.

82 NAT. HIST. J. CHULALONGKORN UNIV. 7(1), MAY 2007

Stipe penangiana and P. tonkinensis. In P. The outlines of the stipe in transverse angustata, sclerenchyma strands are sections (TS) are mostly rounded, while restricted to the peripheral zone and occur heart-shaped (Fig. 18) in P. adnascens, P. in between the vascular bundles (Fig. 20). lanceolata, P. nuda and P. varia, elliptic in Sclerenchyma strands are situated centrally P. piloselloides (Fig. 19) and spindle- in the inner parenchyma (Fig. 21) in P. shaped in P. tonkinensis. The epidermal adnascens, P. eberhardtii, P. heteractis, P. cells of stipes contain stomata. Below the heteractis var. minor, P. lanceolata, P. epidermis is the sclerenchymatous sheath, nuda, P. nummularifolia and P. varia. formed by 3-10 layers. Dense starch grains Sclerenchyma strands are absent (Fig. 22) in many species are present in the in P. floccigera, P. flocculosa, P. parenchymatous ground tissue. piloselloides and P. stigmosa. The structure Vascular tissue: Three shapes of of the cell walls in sclerenchyma strands is vascular bundle arrangement in the stipes similar to that of the walls of are recognized: U-shaped in P. adnascens, sclerenchymatous sheaths, but is often P. angustata, P. eberhardtii, P. floccigera, distinctly darker. P. flocculosa, P. heteractis, P. lanceolata, Vascular tissue: In all species, P. longifolia, P. penangiana, P. stigmosa, rhizomes are dictyostelic, with meristeles P. tonkinensis and P. varia; V-shaped (Fig. arranged in a ring. The number of 18) in P. mollis, P. heteractis var. minor meristeles ranges from 4-10. Each and P. nuda, and Linear-shaped in P. meristele is oval, with plate-like xylem nummularifolia and P. piloselloides. The surrounded by phloem and endodermis, and vascular tissue system consists of 2-13 1-3 layers of parenchymatous pericycle. vascular bundles with extended ends. Vascular bundles are round or oval in TS, Root with plate-like xylem and phloem on both The root consists of a single layer of sides. The vascular bundle has 1 - 3 layers epidermis. Bristle-like root hairs are of pericycle and is enclosed by the present in all species. The cells of the outer endodermis. cortex are thin-walled and parenchymatous, consisting of 2-5 layers, while those of the Rhizome inner cortex form sclerenchymatous The ground tissue of rhizomes is sheaths, consisting of 5-10 layers (Figs. 24- parenchymatous and the cells contain dense 25). starch grains. In most species, the Vascular tissue: The stele is a diarch sclerenchymatous sheaths are distinct and surrounded by endodermis and a layer of situated peripherally to the vascular tissue. parenchymatous pericycle. They have 2-8 layers and are absent from the sheath in P. flocculosa, P. penangiana DISCUSSION AND CONCLUSIONS and P. stigmosa. The sclerenchyma strands may be divided into 4 groups. The results show that a combination of Sclerenchyma strands scattered irregularly some characteristics is useful in the through the inner parenchyma (Fig. 23) identification of Thai Pyrrosia (Tables 1 & occur in P. longifolia, P. mollis, P. 2). It is impossible to identify the KOTRNON ET AL. – COMPARATIVE ANATOMY OF THE GENUS PYRROSIA MIRBEL 83

) 1 2-4 2-5 2-3 3-6 2-6 2-3 2-3 2-6 5-6 2-3 2-3 2-8 2-3 2-3 2-8 5-10

Sclerenchyma (no. of layers Root f o. o 1 1 1 2 1 1 1 5 2-3 3-4 1-2 2-3 2-3 3-4 4-5 3-4 2-3 layers) Parenchy cular bundle) ma (n

8 6 4 6 4 7-8 6-7 5-6 7-8 4-5 5-6 5-6 3-4 2-3 9-10 7-10 7-10 (no.) , VB=vas bundle Vascular

f ections o . e s e vers - - - 3-4 5-7 5-8 2-4 5-8 4-6 4-6 6-8 3-5 4-6 3-5 2-4 3-4 5-6 ths (no layers) Rhizom shea Sclerenchymatous

nce, TS=trans e - - - - of central central central central central central central central vacular scattered scattered scattered scattered (-=abs tous strands Arrangement sclerenchyma

sia

f o Pyrro . 3-5 4-6 7-9 4-5 3-4 7-9 4-6 4-6 5-7 5-8 4-5 4-6 4-6 6-8 4-5 4-6 8-10 ths (no layers) shea Sclerenchymatous ection of Thai in s 8 3 6 6 6 5 2 3 7 10 6-7 6-7 7-8 7-9 8-9 (no.) 10-12 12-13 bundle Vascular Stipe and roots L L V V V U U U U U U U U U U U U VB omes Shape of

ed ed S ed ed ed p p p p p e e e e e e e e e a h tic-sha Round Round p Globos Globos Globos Globos Globos Globos Globos Globos Globos indle-s p Shape of T Heart-sha Heart-shaped Heart-sha Heart-sha Elli S

minor olia f

var. Anatomical characters of stipes, rhiz Species sis iana era 2. en g cens olia g f i in ustata mosa k g g g n enan iloselloides locci locculosa ABLE f f p p T P. sti P. P. eberhardtii P. P. P. to P. varia P. mollis P. nuda P. nummulari P. heteractis P. heteractis P. lanceolata P. lon P. P. adnas P. an

84 NAT. HIST. J. CHULALONGKORN UNIV. 7(1), MAY 2007 sclerenchymatous sheaths in the mibrib, This group grows on tree-trunks in open stipe, rhizome and root of all species. The places or mixed deciduous forest and dry sclerenchymatous strands in rhizomes may evergreen forest, and has thick cuticles on be divided into four groups based on their both surfaces, deeply sunken stomata and position: (1) scattered irregularly through mesophyll with few intercellular spaces. the inner parenchyma; (2) restricted to the Group II: P. floccigera, P. flocculosa, peripheral zone; (3) situated centrally in the P. mollis, P. nummularifolia, P. penangina inner parenchyma; and (4) absent. and P. tonkinensis Hovenkamp (1986) suggested that the This group grows on mossy tree-trunks, arrangement of sclerenchymatous strands especially along stream in evergreen forest, and their position in parenchyma is of and has thin or very thin cuticles, taxonomic value in Pyrrosia. superficial stomata and mesophyll with According to Sen & Hennipman (1981); large intercellular spaces. P. floccigera Ogura (1972), pericytic stomata are differs by having sunken stomata. restricted to the genus Pyrrosia. Price Group III: P. adnascens, P. (1974, cited in Hovenkamp, 1986) notes eberhardtii, P. heteractis, P. heteractis that Drymoglossum pilioselliodes should var. minor P. lanceolata P. piloselloides therefore be transferred to Pyrrosia. and P. stigmosa Using a combination of anatomical This group grows on tree-trunks in dry characteristics, only two taxa (P. heteractis evergreen forest or on mossy tree-trunks in and P. heteractis var. minor) could not be moist evergreen, and has thick cuticles on separated because they are morphologically both surfaces and sunken stomata. P. similar (Tagawa & Iwatsuki, 1989). The piloselloides and P. stigmosa differ by latter differs from the typical variety in its having superficial stomata. smaller stipe, 1-3 cm in length, and its However, this study indicates that the fronds 3-6 cm by up to 2 cm. Both taxa level of stomata may be a genetically also grow at the same altitude. Further inherited characteristic and not a response study is needed to elucidate their status. to environmental conditions. Pyrrosia lanceolata and P. adnascens are similar in morphology. The former can be readily distinguished by the scale-like SPECIMES EXAMINED hairs on its upper lamina surface and the cortical parenchyma with dense deposits of All specimens examined are deposited at starch in the rhizomes. The latter lacks KKU. Pyrrosia adnascens, Kanokorn scale-like hairs on the upper surface, and 35/1999. – P. angustata, Kanokorn has cortical parenchyma with dense 67/1999. – P. eberhardtii, Kanokorn deposits of tannin in the rhizomes. 41/1999. – P. floccigera, Kanokorn Based on ecological and lamina 39/1999. – P. flocculosa, Kanokorn anatomical characteristics, the genus can be 31/1999. – P. heteractis, Kanokorn divided into three groups. 67/2000. – P. heteractis var. minor, Group I: P. angustata, P. longifolia, P. Kanokorn 80/2000. – P. lanceolata, nuda and P. varia Kanokorn 51/2000. – P longifolia, Kanokorn 36/1999. – P. mollis, Kanokorn 70/2000. – P. nummularifolia Kanokorn KOTRNON ET AL. – COMPARATIVE ANATOMY OF THE GENUS PYRROSIA MIRBEL 85

59/2000. – P. nuda, Kanokorn 54/2000. – Hovenkamp, P.H. 1986. A monograph of the fern P. penangiana, Kanokorn 64/2000. – P. genus Pyrrosia (Polypodiaceae). Leiden Netherlands. piloselloides, Kanokorn 10/1999. – P. Lin, B.L and Devol, C.E. 1977. The use of stipe stigmosa, Kanokorn 40/1999. – P. characters in fern taxonomy Part 1. Taiwania, tonkinensis, Kanokorn 52/2000. – P. varia, 22: 91-99. Kanokorn 37/1999. Nayar, B.K. and Chandra, S. 1965. Ferns of India- XV, Pyrrosia. Bulletin of the National Botanic Gardens, 117: 1-98. ACKNOWLEDGEMENTS Ogura, Y. 1972. Comparative anatomy of vegetative organs of the Pteridophytes. Berlin: Gebruder Barntraeger. We would like to thank Dr. David A. Schneider, H. 1996. The root anatomy of ferns: a Simpson for reviewing the manuscript. comparative study. In: Camus, J.M., Gibby, M This work was supported by the and Johns, R.J (Eds). Pteridology in Perspective. TRF/BIOTEC Special Program for Royal Botanic Gardens, Kew, 271-283 pp. Sen, U. and Hennipman, E. 1981. Structure and Biodiversity Research and Training grant ontogeny of stomata in Polypodiaceae. Blumea, (BRT 543001). 27: 175-201. Tagawa, M. and Iwatsuki, K. 1989. Polypodiaceae. In: Smitinand, T and Larsen, K (Eds). Flora of LITERATURE CITED Thailand (Pteridophytes) 3(4). Tistr, Bangkok. van Cotthem, W.R.J. 1970. A classification of Holttum, R.E. 1968. Flora of Malaya (ferns of stomatal type. Botanical Journal of the Linnean Malaya). Government printing office, Singapore. Society, 63: 235-246.

Received: 22 November 2006 Accepted: 10 May 2007