Orchids in the torrent: on the circumscription, conservation and rheophytic habit of Epipactis flava

bs_bs_banner Botanical Journal of the Linnean Society, 2013, 172, 358 370. With 4 figures Orchids in the torrent: on the circumscription, conservation and rheophytic habit of Epipactis flava HENRIK Æ. PEDERSEN 1 *, SANTI WATTHANA 2 and KANOK-ORN SRIMUANG 3 1 Herbarium, Botanical Garden, Natural History Museum of Denmark, University of Copenhagen, Øster Farimagsgade 2C, DK-1353 Copenhagen K, Denmark 2 Queen Sirikit Botanic Garden, PO Box 7, Mae Rim, Chiang Mai 50180, Thailand 3 School of Agriculture and Natural Resources, University of Phayao, 19 M. 2 T. Mae-ka, Muang District, Phayao 56000, Thailand Received 9 July 2012; revised 29 November 2012; accepted for publication 26 December 2012 The systematically difficult genus Epipactis (Orchidaceae) is often subdivided into two sections: Epipactis and Arthrochilium. Until now, the latter has attracted much less taxonomic attention than the former, but here we reassess the alleged distinction of two rare and nationally endangered taxa from tropical Asia (E. flava, E. atromarginata), based on morphological examination of live plants in situ in northern Thailand and of herbarium specimens from the entire range of the complex (Thailand, Laos and Vietnam). As the variation in vegetative and floral dimensions, flower colour and labellum morphology broadly overlaps between the two taxa, we merge them under the oldest valid name at species level, E. flava. Geomorphological features of our three study sites in Thailand and high shared occurrence of previously confirmed rheophytes among the accompanying species indicate that E. flava is itself rheophytic (a very rare feature in the Orchidaceae). Possible adaptations of E. flava to its rheophytic lifestyle are discussed. Among these, the formation of pure stands through dense clonal growth involving a creeping rhizome places E. flava as a mat-rooted rheophytic landplant (sensu van Steenis). Combining taxonomic, distributional and ecological data, we discuss the occurrence of this stream-inhabiting Epipactis in a conservation context. 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172, 358 370. ADDITIONAL KEYWORDS: adaptation distribution ecology Epipactis atromarginata rarity taxonomy typification. INTRODUCTION The systematically difficult genus Epipactis Zinn, encompassing 15 65 species (Delforge, 2005; Pridgeon et al., 2005; Chen et al., 2009), has a mainly Eurasian distribution, east to Vietnam, Japan and Sakhalin, but is also represented by two indigenous species in tropical Africa and one in western North America (Pridgeon et al., 2005: fig. 387.2). Furthermore, E. helleborine (L.) Crantz from Eurasia is naturalized and widespread in North America. The genus is often subdivided into section Epipactis (hypochile bowl-shaped without lateral lobes, epichile firmly *Corresponding author. E-mail: henrikp@snm.ku.dk attached) and section Arthrochilium Irmisch (hypochile shallowly boat-shaped with lateral lobes, epichile elastically attached). Taxonomic studies have focused on section Epipactis, which now seems to be overly split, mainly owing to over-emphasis on reversible floral traits (cf. Claessens & Kleynen, 1999), negligence of pronounced ecophenotypy (cf. Ehlers, Olesen & Ågren, 2002) and reliance on inadequate morphometric analyses (cf. Pedersen, 2010). Section Arthrochilium has attracted much less attention, but we recently found two rare and nationally endangered taxa from tropial Asia, E. flava Seidenf. and E. atromarginata Seidenf., to be so morphologically similar that a closer taxonomic study was warranted. At the same time, we noted that both taxa might be 358

TAXONOMY AND ECOLOGY OF EPIPACTIS FLAVA 359 rheophytic, a lifestyle that appears to be rare in Orchidaceae (van Steenis, 1981; Vermeulen & Tsukaya, 2011) and where it has never been described in detail. The original description of E. flava was based entirely on plants collected in south-western Thailand in 1926 (Seidenfaden, 1978). On the label of the type collection, the collector (Kerr) indicated: Forming thick patches on low rocks in streams. This growth habit was further emphasized by Seidenfaden & Pedersen (2003: 526) who, referring to C. Thorut (in litt.), indicated that:... this orchid must be growing submerged for months, because its habitat at [a newly discovered site in] Umphang (limestone rocks in streams) is flooded in the rainy season. Apart from the clearly distinct E. alata Aver. & Efimov, which grows in limestone coniferous forests in Vietnam (Averyanov, 2011), E. atromarginata is the only Epipactis species known from the area consisting of Thailand and the former Indo-China. The original description was based on recent collections and colour photos from Vietnam. According to the protologue (Seidenfaden, 1992: 22), the plants were found on rocks in mountain streams. Until now, E. atromarginata has been considered endemic to Vietnam. A few years ago, various orchid enthusiasts showed us photos of E. flava from one of its localities in Thailand. We noted that some of the plants had floral coloration patterns similar to those of E. atromarginata. We therefore decided to look thoroughly into the taxonomy of the two taxa and to prepare an overall distribution survey. As this study involved several field trips, we also took the opportunity to assess the supposedly rheophytic habit of the plants. Combining taxonomic, distributional and ecological data, we assess the conservation needs of the streaminhabiting Epipactis of tropical Asia. MATERIAL AND METHODS PHENETIC VARIATION In Tak province of northern Thailand, field studies of phenetic variation were conducted at one locality in Mae Sot district (Ban Huai Tham Suea, 16 March 2010, 7 11 March 2011) and at three localities in Umphang district (Ko-Ta village, 27 February 2011; Se Pa La, 10 May 2009, 11 February and 18 March 2010; Thee Lo Su waterfall, 17 March 2010, 12 March 2011, 30 January 2 February 2012). All fieldwork was conducted towards the end of the dry season, partly to allow us to observe the plants in flower, and partly because transport along small dirt roads to the localities is highly problematic in the rainy season. Based on field examinations of numerous flowering clones, we prepared a morphological description of E. flava that is more detailed than those published by earlier authors. When previously published data on morphological variation in E. flava (from southwestern Thailand) were combined with our new original data from northern Thailand, the intraspecific range of variation turned out to be wider than previously realized (see Results). Against this background, we assessed the degree of morphological distinction of E. flava relative to E. atromarginata (as characterized in previously published descriptions, supplemented with unpublished material, including images of all herbarium specimens at LE). DISTRIBUTION The overall distribution of E. flava s.l. (including E. atromarginata) was studied through consultation of the literature and through examination of herbarium specimens, spirit samples and photographs deposited at BCU, BK, C, K, QBG and SING and images of herbarium specimens at LE (kindly provided by L. V. Averyanov) and P (via http://www. mnhn.fr/base/sonnerat.html). Supplementary distribution records for Thailand (without herbarium vouchers) were contributed by S. Sitthisajjadham (pers. comm.) and C. Thorut (pers. comm.). Special attention was paid to careful identification of Harmand 1324 (C, P), because Seidenfaden (1978, 1992) assigned this collection to E. flava as the only possible collection of this species from Laos. ECOLOGY During visits to Ban Huai Tham Suea, Se Pa La and Thee Lo Su waterfall, we examined the geomorphological reliefs and physical signs of erosion of stream beds with E. flava populations. This was done with the main purpose to assess the occurrence of the orchid in relation to the highest annual water level. Lists of vascular plants associated with E. flava were prepared for one plot (50 30 m) at Thee Lo Su waterfall (12 March 2011) and two plots (upstream plot: 50 10 m; downstream plot: 50 15 m) at Ban Huai Tham Suea (9 March 2011). The width of plots differed according to the individual widths of streams and stream beds, as we intended to list all species growing from below to slightly above the highest annual water level. The main purpose was to assess the occurrence of previously confirmed rheophytic species in habitats of E. flava. According to van Steenis (1981: 3): By definition rheophytes are plant species which are in nature confined to the beds of swift-running streams and rivers and grow there up to flood-level, but not beyond the reach of regularly occurring flash floods. When preparing our survey, we distinguished between obligate rheophytes and

360 H. Æ. PEDERSEN ET AL. facultative rheophytes (... species which are not true rheophytes, but occur regularly in streambeds ; cf. van Steenis, 1981: 11). Based on field observations at Ban Huai Tham Suea and Thee Lo Su waterfall, we assessed whether E. flava should be classified as terrestrial or lithophytic, we evaluated its morphological adaptations to rheophytic life and we searched for natural germination beds on various substrates. RESULTS PHENETIC VARIATION Based on comparative examination of numerous Epipactis clones at Ban Huai Tham Suea, Ko-Ta village, Se Pa La and Thee Lo Su waterfall, we are convinced that only one Epipactis sp. was represented at the study localities. Field data on variation in morphology and floral coloration patterns were compiled into one comprehensive description of E. flava that is presented in the Taxonomic treatment below (see also Fig. 1A D). In Table 1, our original data for selected quantitative characters are compared with corresponding data from previously published descriptions of E. flava and E. atromarginata. In Figure 2, a drawing of the labellum from one of the live plants studied by us (Fig. 2A) is shown alongside drawings of labella from specimens of E. flava from south-western Thailand (Fig. 2B, C) and E. atromarginata from Vietnam (Fig. 2D F). DISTRIBUTION The Epipactis collection Harmand 1324 from Laos was misidentified as Cephalanthera longibracteata Blume by Gagnepain & Guillaumin (1932 1934: 628) and Seidenfaden (1972: 112). This mistake was first realized by Seidenfaden (1978) who proposed that the plant might belong to E. flava (see also Seidenfaden, 1992). However, Seidenfaden later changed his mind and (contrary to Schuiteman et al., 2008) no longer considered the Thai and Laotian plants to be conspecific (Seidenfaden & Pedersen, 2003). Based on images of the P specimens of Harmand 1324 and on careful dissection of a flower from this collection at C, we confidently assign Harmand 1324 to E. flava. In its new, wider circumscription that also comprises E. atromarginata (see below), E. flava is known from Thailand (ten localities), Laos (one locality) and Vietnam (five localities). For a survey of all known populations, see Table 2. ECOLOGY At our three study sites, E. flava was consistently found to grow close to the level of the water surface in the dry season, either on the banks or among stones and rocks in the stream itself (Fig. 3A). At each site, the geomorphological relief of the stream bed (Fig. 3B) suggested that a considerably higher water level prevails during the rainy season, a scenario that was further supported by widespread signs of recent erosion of the sides of the stream bed. Thirty species of vascular plants were found in the three study plots, belonging to 25 genera and 18 families (Appendix). The highest number of species (18) was noted in the 750-m 2 downstream plot at Ban Huai Tham Suea, the second highest (14 species) in the 1500-m 2 plot at Thee Lo Su waterfall and the lowest (11 species) in the 500-m 2 upstream plot at Ban Huai Tham Suea. Details on the occurrence of (previously) confirmed rheophytes in the study plots are given in Table 3. Among the total of 30 species of vascular plants encountered, 14 are confirmed rheophytes, and this number includes at least nine obligately rheophytic species. The plot at Thee Lo Su waterfall contained nine confirmed rheophytic species (at least six obligate), constituting 64% of the species recorded in that plot, the upstream plot at Ban Huai Tham Suea contained eight confirmed rheophytic species (at least five obligate), constituting 73% of the species Table 1. Survey of vegetative and floral dimensions that have previously been recorded as (largely) non-overlapping between Epipactis flava in south-western Thailand (data from Seidenfaden, 1978) and E. atromarginata in Vietnam (data from Seidenfaden, 1992; Averyanov, 2011) compared with original data for our E. flava study populations in northern Thailand Measure E. flava (SW Thailand) E. flava (N Thailand) E. atromarginata (Vietnam) Height of plant (cm) 30 40 18 85 35 120 Length of leaves (cm) to 7 10 1.7 11.2 12 14 Length of sepals and petals (mm) 9 10 7 11 11 15 Length of epichile (mm) 5.5 5.1 6.2 6.5 Length of anther (mm) nearly 2 2.2 2.3 nearly 3

TAXONOMY AND ECOLOGY OF EPIPACTIS FLAVA 361 Figure 1. Morphology of Epipactis flava. A, large flowering clone growing on rock in a stream; B, small clone emerging from sandy substrate below c. 5 cm deep water; C, roots attaching directly to surface of limestone rock (note also the creeping rhizome); D, flower in front view; E, seedling with extant protocorm. A, B, D, Ban Huai Tham Suea, 16 March 2010; C, Ban Huai Tham Suea, 9 March 2011; E, Thee Lo Su waterfall, 12 March 2011. Photos by H. Æ. Pedersen.

362 H. Æ. PEDERSEN ET AL. Figure 2. Variation in labellum morphology among Epipactis flowers representing our study population of E. flava at Ban Huai Tham Suea in northern Thailand (A), E. flava from the locus classicus in south-western Thailand (B, C) and E. atromarginata from Vietnam (D F). Redrawn from sketches originally prepared by H. Æ. Pedersen (A, from Watthana 3746), P. Taylor (B, from Kerr 0261), G. Seidenfaden (C, from Kerr 0261; D and E, from LX-VN 1975) and L. V. Averyanov (F, from LX-VN 1975). Line drawing prepared by H. Æ. Pedersen. Table 2. Survey of all known populations of Epipactis flava, each with the year of the first record indicated; whenever available, a herbarium collection documenting the first record is cited, but otherwise another primary source is cited Country Province District Locality First record Herbarium voucher [or other source] Laos Attapeu N/A Sine loco 1877 Harmand 1324 (C, P) Thailand Kanchanaburi Sangkhla Buri Takien Thong waterfall 2005 Thaithong 2053 (BCU) Thailand Kanchanaburi Sangkhla Buri Huay Mae Kasat 1973 Phengklai et al. 3060 (BKF) Thailand Kanchanaburi Sangkhla Buri Kasat river 2007 S. Sitthisajjadham, pers. comm. Thailand Kanchanaburi Sangkhla Buri Suriya river 2007 S. Sitthisajjadham, pers. comm. Thailand Kanchanaburi Thong Pha Phum Ta Kanum 1926 Kerr 0261 (BK, C, K) Thailand Nan Mae Charim Lamnam Wah 2001 C. Thorut, pers. comm. Thailand Tak Mae Sot Ban Huai Tham Suea 2011 Watthana 3746 (QBG) Thailand Tak Umphang Ko-Ta village 2011 K. Srimuang, pers. obs. Thailand Tak Umphang Se Pa La 2010 Watthana 3387 (QBG) Thailand Tak Umphang Thee Lo Su waterfall 1998 Thorut 261 (BCU) Vietnam Gia Lai K Bang Kon Hannung 1985 Sine coll./lxvn 1975 (LE) Vietnam Kon Tum Kon Plong Mang Den 1985 Sine coll./lx-vn 2275 (LE) Vietnam Quang Tri Da Krong Cop village in Huc Nghi 2006 Averyanov et al. HLF 6181 (LE) Vietnam Quang Tri Da Krong La To village in Huc Nghi 2006 Loc et al. HLF 6139 (LE) Vietnam Quang Tri Da Krong Ta Long 2006 Trai & Quynh HLF 5757 (LE)

TAXONOMY AND ECOLOGY OF EPIPACTIS FLAVA 363 Figure 3. Habitats of Epipactis flava in the dry season. A, larger and smaller clones growing on and among rocks that appear to be largely submerged for long periods during the rainy season (Ko-Ta village, 27 February 2011); B, stream bed with E. flava flowering along the water margin apart from the elevated ground to the left front and right back, the entire area appears to be flooded in the rainy season (Se Pa La, 18 March 2010); C, natural germination bed among buttresses of Elaeocarpus varunua (Thee Lo Su, 12 March 2011). Photos by K. Srimuang (A) and H. Æ. Pedersen (B and C).

364 H. Æ. PEDERSEN ET AL. Table 3. Survey of confirmed rheophytic species of vascular plants found in study plots at Epipactis flava sites in Thailand Family Species Growth form Site 1 Site 2a Site 2b Araceae *Colocasia esculenta (L.) Schott Perennial herb - + - Athyriaceae *Diplazium esculentum (Retz.) Sw. 1 Perennial herb + - - Dipterocarpaceae Dipterocarpus alatus Roxb. ex G.Don Tree - + - Hopea odorata Roxb. Tree + + + Elaeocarpaceae *Elaeocarpus varunua Buch.-Ham. ex Mast. 2 Tree + + + Euphorbiaceae Bischofia javanica Blume 3 Tree + - - *Homonoia riparia Lour. 4,5,6 Shrub + - - Iteaceae *Itea riparia Collett & Hemsl. 4,7 Shrub + - - Moraceae *Ficus pyriformis Hook. & Arn. 4 Shrub + + + *Ficus saemocarpa Miq. 4 Shrub - + + Myrtaceae *Syzygium megacarpum (Craib) Ranthakr. & Tree - - + N.C.Nair 8 Oleaceae Chionanthus ramiflorus Roxb. Tree + - - Rhizophoraceae Carallia brachiata (Lour.) Merr. 9 Tree - + + Salicaceae *Salix tetrasperma Roxb. 6,10 Tree + + + Site 1: Thee Lo Su waterfall; Site 2: Ban Huai Tham Suea (a, upstream plot; b, downstream plot). A numbered footnote indicates a published source in which the species is described as rheophytic. Species without numbered footnotes are classified as rheophytes based on our own direct field observations from other localities. At least the asterisked species can be regarded as obligate rheophytes. 1 Tagawa & Iwatsuki (1988); 2 Phengklai (1981); 3 van Welzen (2005); 4 van Steenis (1981); 5 van Welzen (2007); 6 Puff & Chayamarit (2011); 7 Esser (2005); 8 Parnell & Chantaranothai (2002); 9 Hou (1970); 10 Larsen (1987). recorded, and the downstream plot at Ban Huai Tham Suea contained seven confirmed rheophytic species (at least five obligate), constituting 39% of the recorded species; all these figures exclude the study species, E. flava. The obligately rheophytic Elaeocarpus varunua Buch.-Ham. ex Mast., Ficus pyriformis Hook. & Arn. and Salix tetrasperma Roxb. and the facultatively rheophytic Hopea odorata Roxb. occurred in every plot. Among the ten remaining (previously confirmed) rheophytes, only Carallia brachiata (Lour.) Merr. was common to two plots (i.e. the two plots at Ban Huai Tham Suea). Most clones of E. flava were terrestrial, growing with their roots loosely embedded in sediments along the banks and among rocks in the streams. At Thee Lo Su waterfall and Ban Huai Tham Suea, however, we also observed a number of E. flava plants growing as true lithophytes, with their roots attaching directly to the surface of limestone rocks (Fig. 1C). Possible morphological adaptions to flowing water included dense clonal growth (Figs 1A, 3A) and slender flexible aerial shoots with narrow leaves (Fig. 1A, B). At Thee Lo Su waterfall, a natural germination bed was found in wet soil immediately adjoining the water surface in the dry season (Fig. 3C); the smallest seedlings maintained a distinct protocorm, but were devoid of distinctly developed roots (Fig. 1E). DISCUSSION VARIATION AND TAXONOMIC CIRCUMSCRIPTION When describing E. atromarginata, Seidenfaden compared his new species with E. flava, but found it to differ in some floral characters summarized in his key to the species (Seidenfaden, 1992: 21). First, he characterized the labellum side lobes as triangular obtuse and pointing forwards in E. flava, whereas he found them to be broad orbicular and sidewards and upwards pointing in E. atromarginata. When comparing labella of E. flava from south-western Thailand (Fig. 2B, C) and E. atromarginata from Vietnam (Fig. 2D, E), the side lobes appear to differ between the two taxa; but a typical labellum from one of our study populations in northern Thailand (Fig. 2A) is almost perfectly intermediate in shape. Second, Seidenfaden (1992: 21) characterized the epichile of E. flava as obpyriform with the sides upwards bent, forming a narrow V in section, whereas he found the epichile of E. atromarginata to be narrow ovate with the sides only slightly upwards bent, forming a broad open V in section. In reality, the outline of the epichile appears to be almost identical among the two taxa (Fig. 2). Admittedly, unpublished photographs of live material (accompanying Averyanov et al. HLF 6181 LE) do show relatively broader epichiles than in any Thai material examined by us. If the same

TAXONOMY AND ECOLOGY OF EPIPACTIS FLAVA 365 photographs and those published by Averyanov (2011: fig. 5B) and Seidenfaden (1992: pl. Ic), showing Vietnamese E. atromarginata, are compared with photos of E. flava flowers from northern Thailand (e.g. Fig. 1D; Sitthisajjadham, 2006: 243; Nanakorn & Watthana, 2008: 62 63), the flowers from Vietnam tend to show epichiles with less raised sides. However, we have observed moderate variation in this character in our study populations and we think that the apparent difference is partly an artefact caused by the epichile being photographed from different angles. Seidenfaden (1992: 21), who never saw live material of E. flava, described its flowers as yellow, orange at centre, in all probability representing a simplified rephrasing of Kerr s note on the label of the type collection: flowers pale yellow with deeper yellow markings on lip. Based on a colour photograph received from L. V. Averyanov, Seidenfaden (1992: 21) gave a more detailed description of E. atromarginata: Sepals cream to greenish with dark bluish-purple margins, midlobe brownish orange, sidelobes with warm brown stripes. To our knowledge, no colour photographs exist from the locus classicus of E. flava in south-western Thailand. On the other hand, Averyanov provided us with a few unpublished colour photographs of Vietnamese E. atromarginata and, against this background, we consider the flowers in Seidenfaden (1992: pl. Ic) and Averyanov (2011: fig. 5b) to represent opposite extremes of the colour variation in E. atromarginata (showing low and high anthocyanin levels, respectively). In our study populations of E. flava, the anthocyanin level varied among clones. Those with the highest level matched the flower depicted in Seidenfaden (1992: pl. Ic), whereas most clones had slightly paler flowers (see Fig. 1D for a typical colour form), and a few were even apochromic (matching Kerr s rough description). A few additional examples of colour variation in E. flava from northern Thailand can be found in Seidenfaden & Pedersen (2003: fig. 1), Sitthisajjadham (2006: 243; 2007: 50) and Nanakorn & Watthana (2008: 62 63). A few vegetative and floral dimensions have previously been recorded as (largely) non-overlapping between E. flava in south-western Thailand and E. atromarginata in Vietnam, the latter generally appearing larger than the former (Seidenfaden, 1978, 1992; Averyanov, 2011). However, if viewed in conjunction with our original data from E. flava populations in northern Thailand (Table 1), the variation in height of plant and length of sepals and petals appears to be continuous in the group. With regard to length of leaves, length of epichile and length of anther, our data similarly reduce the apparent gap between E. flava and E. atromarginata, but they do leave a tiny gap for the last. However, comparing with the size variation encountered in the same organs in most other Epipactis spp. (e.g. Delforge, 2005; Chen et al., 2009), these minor differences should hardly be attributed taxonomic significance. Furthermore, images of herbarium specimens from LE clearly demonstrate the lower leaves in some flowering individuals of E. atromarginata to be as short as 4 cm. Summing up, variation in vegetative and floral dimensions, flower colour and labellum morphology is broadly overlapping between Thai E. flava and Vietnamese E. atromarginata. Although the range of variation for each character does not always include the same extremes in the two areas (possibly an artefact resulting from undercollecting), we therefore conclude that the two taxa should be considered conspecific and recognized under the oldest valid name at species level, E. flava Seidenf. THE RHEOPHYTIC HABIT OF EPIPACTIS FLAVA Judging from geomorphological features alone (Fig. 3B), it is our impression that most (if not all) clones of E. flava at our study sites are submerged for several months each year and are often covered by at least 0.5 m of water in the rainy season. Further circumstantial evidence of a rheophytic habit comes from the high proportion of previously confirmed rheophytic species in each study plot (64% at Thee Lo Su waterfall, 39 73% at Ban Huai Tham Suea). Figure 4 shows a flooded E. flava site in the rainy season. Previous records of E. flava as growing in thick patches on low rocks in a stream in southwestern Thailand (Seidenfaden, 1978: 116) and on wet alluvial banks and riparian rocks along streams and small rivers in Vietnam (Averyanov, 2011: 19; under the synonym E. atromarginata) suggest that the rheophytic habit of E. flava may be universal and not confined to our study populations. Among the categories of rheophytes proposed by van Steenis (1981), we classify E. flava as a mat-rooted rheophytic landplant. As E. flava, the only known rheophytic species of Epipactis, is not an early branching species in the genus in phylogenetic trees based on internal transcribed spacer (ITS) sequences (Pedersen et al., 2009: fig. 1) or on those of ITS, trns-g and rbcl (Roy et al., 2009: fig. 2), the rheophytic life style seems to be a derived feature in Epipactis. The same is assumed to be the case in most other genera including rheophytes (van Steenis, 1981), and recurrent evolution of a rheophytic life style in Crinum L. (Amaryllidaceae) was convincingly documented by Bjorå et al. (2009). Rheophytes are rare in Orchidaceae. Apart from E. flava (based on the sparse information in Seidenfaden & Pedersen, 2003), Vermeulen & Tsukaya (2011) only listed the following orchid

366 H. Æ. PEDERSEN ET AL. Figure 4. Habitat of Epipactis flava in the rainy season (Thee Lo Su, 3 August 2012); all clones in the area are completely inundated. Photo by S. Janhomhuan. species from tropical Asia as (presumedly facultative) rheophytes: Agrostophyllum laterale J.J.Sm. (Borneo; see also Schuiteman, 1997), Arundina caespitosa Aver. (Vietnam, Laos, southern China; see also Averyanov, 2007), Bulbophyllum rheophyton J.J.Verm. & Tsukaya (Borneo), Eria spirodela Aver. (Vietnam, Laos), Eria Lindl. (section Conchidium Lindl.) sp. (Sulawesi), Porpax elwesii (Rchb.f.) Rolfe (Nepal to Vietnam and Peninsular Malaysia) and Poaephyllum selebicum J.J.Sm. (Sulawesi). To these might be added Appendicula rupestris Ridl. from Peninsular Malaysia (Holttum, 1957). Flowering of E. flava in Thailand occurs between January and March, depending on the locality, and Averyanov (2011) indicated the flowering time in Vietnam as March May. Thus, it seems that E. flava consistently flowers towards the end of the dry season, probably leaving just enough time for seed dispersal before being flooded. All known occurrences of E. flava in the Thai provinces of Tak and Kanchanaburi are located in limestone areas; at Lamnam Wah in Nan province, the bedrock is mixed with widespread occurrence of limestone (P. Suksathan, pers. comm.). In the Vietnamese province of Quang Tri, E. flava grows on shale (according to herbarium labels at LE; under the synonym E. atromarginata), whereas the edaphic conditions of the remaining sites are unknown. Our observation that E. flava does not only grow terrestrially, but also as a true lithophyte with its roots attached directly to the surface of limestone rocks (Fig. 1C), appears to be unique in the genus and might be seen as an adaptation to its rheophytic habit. Thus, the ability to attach firmly to rocks probably reduces the risk of being washed away by swiftly running water. The habit of forming pure stands (Fig. 3A) is a common feature of rheophytes (van Steenis, 1987). At Thee Lo Su waterfall, in particular, E. flava formed large clones, some of which were larger than we have encountered in any other orchid species. Incidentally, it might be more appropriate to refer to these aggregations as clumps rather than clones. Thus, each of them may represent two or more genets with profusely branched rhizomes growing densely interwoven [cf. Kull s, (1988) molecular identification of clones in the terrestrial Cypripedium calceolus L.]. The dense, clonal growth of E. flava is more pronounced and extensive than in any other species

TAXONOMY AND ECOLOGY OF EPIPACTIS FLAVA 367 of Epipactis and probably increases the chance of the plants to maintain a foothold in swiftly running water, as similarly proposed by Schuiteman (1997) for Agrostophyllum laterale. Indeed, dense clonal growth with the root system forming a netting or mat is the main unifying feature of mat-rooted rheophytes (van Steenis, 1981). The aerial shoots of E. flava are more slender and flexible and have narrower leaves than most other species of Epipactis (Fig. 1A, B). These features must be expected to minimize the physical impact of the current see also Schuiteman (1997) on A. laterale and Vermeulen & Tsukaya (2011) on B. rheophyton. Indeed, flexible stems and narrow leaves are among the most universal features of rheophytes (van Steenis, 1981, 1987). The natural germination bed at Thee Lo Su waterfall was characterized by wet soil with virtually no cover of herbs or mosses (Fig. 3C). The youngest seedlings observed were devoid of well-developed roots, despite already having produced two or three leaves (Fig. 1E). These in situ observations are contrary to observations from asymbiotic in vitro germination experiments, in which a few roots are usually formed prior to the first leaf (A. Kongbungkead, pers. comm.). In the few other Epipactis spp. for which young seedlings have been examined, several roots are likewise produced before the first leaf (Rasmussen, 1995). This is also evident from the drawings illustrating the in situ observations published by Fuchs & Ziegenspeck (1924: fig. 6) on young seedlings of E. helleborine (L.) Crantz, and by Fuchs & Ziegenspeck (1926: fig. 7) on those of E. atrorubens (Hoffm. ex Bernh.) Besser. We suspect that the virtually rootless condition of young E. flava seedlings in situ is a result of the wet environment in which they grow. It also seems likely that, due to the lack of well-developed roots, young seedlings may easily be washed away and dispersed downstream. Our preliminary observations on the rheophytic habit of E. flava raise several new questions, for example: (1) are the seeds mainly wind- or waterdispersed; (2) in which season do the seeds germinate, and how do the young seedlings cope with the rainy season; (3) what are the mycorrhizal associates of juvenile and adult plants in the periodically aqueous environment; and (4) what characterizes the pollination ecology, and how important is sexual reproduction relative to clonal growth? We are currently studying the last topic, and we are also planning to subsequently address the others. CONSERVATION Epipactis flava is considered endangered (EN) in Thailand (Santisuk et al., 2006) and critically endangered (CR) in Vietnam (Averyanov, 2011, as E. atromarginata) and it is unknown whether the only collection from Laos (from 1877) represents an extant population. As demonstrated by our findings concerning the distribution (Thailand to Vietnam), habitat requirements (calcareous ground in periodically flooded streambeds in lowland forests) and local abundancy (partly caused by extensive clonal growth), the rare E. flava should be classified as predictable in the typological framework of rarity proposed by Rabinowitz (1981: 208 209). This category accommodates species that are: locally abundant over a large range in a specific habitat. The worst threats to such species are not collection, but destruction of the specialized habitats. As evident from Table 2, no fewer than 14 of the 16 known populations of E. flava were only discovered in the last five decades, and most (if not all) of them still exist. This does not immediately signal a species on the decline. However, the dominance of recently discovered populations in all probability reflects the intensified botanical exploration of two previously underexplored countries (cf. Smitinand, 1989; Averyanov et al., 2003). Against this background, it seems highly likely that a considerable number of undiscovered E. flava populations have been lost. In the Thai province of Kanchanaburi, for example, several valleys with small streams were permanently flooded in the 1980s. Thus, a large valley system situated between the type locality of E. flava (Ta Kanum) in the southern district of Thong Pha Phum and its four known localities in the northern district of Sangkhla Buri (Table 2) is now covered by the Khao Laem reservoir. This artificial lake was formed when in 1984 the construction of the Vajriralongkorn dam in the Khwae Noi river was completed. Judging from the geographical positions of known E. flava populations, and from the general topography and vegetation cover of the area, it seems likely that the now permanently flooded area previously held a number of undiscovered E. flava populations. The case of E. flava highlights the need for detailed natural history inventories being conducted prior to the (potential) implementation of major construction works in natural environments. Due to the highly specific habitat requirements of predictable rare species such as E. flava, it would even be feasible to perform special searches for these in areas where the specialized type of habitat is known (or expected) to exist. Ex situ conservation of E. flava, as in vitro cultures, is being developed in Anupan Kongbungkead s laboratory at Naresuan University in Phitsanulok, Thailand. In the near future, in vitro cultures will also be established in the Botanical Garden, University of Copenhagen, where they will be combined with seed banking and cryopreservation.

368 H. Æ. PEDERSEN ET AL. TAXONOMIC TREATMENT EPIPACTIS FLAVA SEIDENF. Dansk Bot. Ark. 32(2): 116, figure 71 (1978). Arthrochilium flavum (Seidenf.) Szlach., Orchidee (Hamburg) 54: 588 (2003). Type: Thailand, southwestern floristic region, Kanchanaburi province, Thong Pha Phum district, Ta Kanum, 19 January 1926, Kerr 0261 [lectotype C! (designated here), isotypes BK! K!]. Epipactis atromarginata Seidenf., Opera Bot. 114: 22, figure 5 (1992). Arthrochilium atromarginatum (Seidenf.) Szlach., Orchidee (Hamburg) 54: 588 (2003). Type: Vietnam, Gia Lai, Kon Hannung, 15 May 1985, sine coll./lx-vn 1975 [sphalm.: 2275 in the protologue] [holotype LE (photographs seen), isotype HN]. [Figures in square brackets indicate extreme values reported by Seidenfaden, 1978, 1992 or Averyanov, 2011]. GEOPHYTE OR LITHOPHYTE with fibrous roots arising from a branched creeping rhizome; aerial shoots vegetative or flowering, densely gregarious, sometimes forming large colonies. STEM slender, 18 85[ 120] cm tall, glabrous (below) to pubescent (above), covered by two or three cataphylls at base. LEAVES four to seven (or nine), spreading, somewhat recurved, more or less distichous along most of the stem, sheathing at base (the uppermost one or two leaves sometimes non-sheathing), (bluish-)green, ovate to elliptic or linear-lanceolate, acute to acuminate, 1.7 11.2[ 14.0] (0.3 )0.5 5.8 cm, decreasing in size towards the proximal part and (at least in large individuals) the distal part of the shoot. INFLORES- CENCE racemose, lax, more or less secund; rachis flexuose, 3.5 21.0 cm long; bracts spreading to suberect, ovate to lanceolate or elliptic, acute to acuminate, 0.5 4.7 0.3 1.4 cm, decreasing in size towards the distal part of the rachis. FLOWERS one to 14 (normally three or four), opening successively from below, long-pedicelled, wide-open, more or less spreading. SEPALS greenish-yellow with or without reddish-brown margins, spreading, shallowly boatshaped, pubescent on the dorsal side; dorsal sepal lanceolate (to lanceolate-oblong), (sub)acute, threeveined from the base, 8.0 10.1[ 15.0] 2.5 3.9 mm; lateral sepals obliquely ovate to falcately lanceolate, acute, often slightly keeled in their distal part, threeto four-veined from the base, 8.9 11.0[ 15.0] 2.7 4.6 mm. PETALS greenish-yellow with or without reddish-brown margins, recurved (apices often meeting behind the dorsal sepal), lanceolate to oblong with irregular margins, obtuse to rounded, threeto five-veined from the base, glabrous, 7.0 9.4[ 15.0] 2.3 3.8 mm. LABELLUM porrect, glabrous, 8.3 9.8 mm long, differentiated into hypochile and epichile; hypochile concave with two erect, obliquely oblong to obliquely subtriangular, rounded lateral lobes, cream to greenish-yellow, suffused with dull orange to reddish-brown (especially along the veins), 2.9 4.0 mm long, 6.3 8.2 mm wide when flattened (distance between lateral lobes in natural position 1.5 2.6 mm), the concave central part provided with a pair of adjoining keels in its proximal to middle part that are furnished with tiny orange warts and end in two orange oblongoid calli; epichile elastically attached, fleshy, broadly oblanceolate to oblongsubspathulate, obtuse to rounded, cream to pale yellow with a large (brownish-)orange patch 5.1 6.2[ 6.5] 2.3 2.8 mm, in its proximal half with two marginal ridges, just above the middle with a small median shiny drop-shaped callus. COLUMN slightly reflexed from the base (describing an obtuse angle to the ovary), slightly incurved in its distal part, subparallel to the labellum, 5.4 6.2 mm long; anther versatile, subapical, oblongoid, obtuse to rounded in front, [nearly 2.0 to] 2.2 2.3 [to nearly 3.0] mm long; pollinia two, oblongoid, fragile; fertile stigma subapical, hemielliptic with the margin elevated laterally and distally, and with a pair of low rounded swellings at base; rostellum well developed, presenting a barrier between anther and fertile stigma, producing a well-functioning diffuse viscidium at apex. OVARY slenderly fusiform, pubescent, 11 17 mm long (including the thread-like pedicel). CAPSULE spreading to nodding, fusiform to oblongoid, with six distinct longitudinal ridges, pubescent, 11 17 mm long (excluding perianth remnants and pedicel), 3.1 5.6 mm in diameter; fruit pedicel 9.1 10.5 mm long. Note. According to Averyanov (2011), the holotype of E. atromarginata is deposited at HN, whereas the specimen at LE is an isotype. In the protologue, however, Seidenfaden (1992) clearly indicated that he had only examined material of the specimen deposited at LE (which must therefore be regarded as the holotype). ACKNOWLEDGEMENTS Henrik Æ. Pedersen s field work and herbarium visits were financially supported by Queen Sirikit Botanic Garden (in 2010) and the Augustinus Foundation (in 2011). Additionally, we gratefully thank the Department of National Parks, Wildlife and Plant Conservation for permission to conduct field work in Umphang Wildlife Sanctuary, Suchart Janhomhuan for permission to use his photograph (Fig. 4), Hanne N. Rasmussen and Anupan Kongbungkead for fruitful discussions, the curators of BCU, BK, K and SING for help and hospitality during our visits, and Leonid V. Averyanov for kindly sending images of herbarium specimens deposited at LE.

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