Two Hybrids of Equisetum Sylvaticum (Equisetaceae) on the Island of Senja, Troms, Norway

FERN GAZ. 20(5):181-196. 2017 181

TWO HYBRIDS OF EQUISETUM SYLVATICUM (EQUISETACEAE) ON THE ISLAND OF SENJA, TROMS, NORWAY

MARCUS LUBIENSKI 1 & VEIT MARTIN DÖRKEN 2

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 Eurasia 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 Scotland (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 l r t s a a c a l m d g y

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t m m o e e e d h a l l l o o n i t i i i r r r s a r r r m c c e b e e e e i i t o t t t t u h n S m S s S s m s i 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 conifer-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 1 st order side branches of the fertile shoots of E. ×lofotense are in general longer than in E. sylvaticum , while having 2 nd 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 bryophyte 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.

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