Cladistics Cladistics 32 (2016) 198–210 10.1111/cla.12125 A phylogenetic analysis of molecular and morphological characters of Herminium (Orchidaceae, Orchideae): evolutionary relationships, taxonomy, and patterns of character evolution Bhakta Bahadur Raskotia,b,†, Wei-Tao Jina,†, Xiao-Guo Xianga,†, Andre Schuitemanc,†, De-Zhu Lid, Jian-Wu Lie, Wei-Chang Huangf, Xiao-Hua Jina,* and Lu-Qi Huangg,* aState Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China; bUniversity of Chinese Academy of Sciences, Beijing, 100049, China; cScience Directorate, Royal Botanical Gardens, Richmond, Surrey, TW9 3AB, UK; dKey Laboratory of Plant Biodiversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, 650201, China; eXishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Mengla County, Yunnan, 666303, China; fShanghai Chenshan Botanical Garden, Songjiang, Shanghai, 201602, China; gNational Resource Centre for Chinese Material Medica, China Academy of Chinese Medical Science, Beijing, 100700, China Accepted 12 May 2015 Abstract The first comprehensive phylogenetic study of the orchid genus Herminium and its allies is presented, based on seven molecu- lar markers (nuclear internal transcribed spacer, Xdh, chloroplast matK, psaB, psbA-trnH, rbcL and trnL-F) and 37 morphologi- cal characters. Phylogenetic analyses indicate that Herminium as currently delimited is paraphyletic and that several genera are deeply nested within it. Based on parsimony analysis of total evidence, the generic circumscription of Herminium is expanded to include Androcorys, Bhutanthera, Frigidorchis and Porolabium. Apomorphic and plesiomorphic character states are identified for various clades recovered in this study. A few species currently wrongly assigned to Peristylus and Platanthera are here included in Herminium. All necessary new combinations are made. © The Willi Hennig Society 2015. Introduction Pridgeon et al., 2001; Pearce and Cribb, 2002). Its gen- eric delimitation has been re-evaluated several times Herminium L. [Orchidaceae, Orchidoideae; type, (King and Pantling, 1898; Lang, 1988; Pridgeon et al., Herminium monorchis (L.) R.Br., Fig. 1a] is a genus 2001; Pearce and Cribb, 2002; Chen et al., 2009), but predominantly occurring in alpine grassland at eleva- no consensus has been reached. Govaerts et al. (2011) tions up to 5200 m. About 55 species are known, of listed 90 names under the genus Herminium, of which which 50 inhabit the Himalayan region, with a few about half were assigned to other genera. species extending into Europe and tropical Asia. Her- Some authors, such as Hooker (1890), King and minium is characterized by: globose tubers; more or Pantling (1898), Duthie (1906), and Tang and Wang less nutant, small, greenish flowers; spreading lateral (1937, 1940), recognized Herminium in a broad sense, sepals; curved ovary with a beaked apex; short col- while Hunt (1970), Seidenfaden (1977), Hara et al. umn; and two stigmas nearly protruding from beneath (1978), Lang (1988), and Pridgeon et al. (2001) took a the rostellum (Hooker, 1890; King and Pantling, 1898; more narrow view of Herminium and also transferred some species to Peristylus Blume and Platanthera Rich. Traditionally, Herminium, Peristylus and Platan- *Corresponding authors. E-mail addresses: [email protected]; [email protected] thera are distinguished by morphological characters of †These authors contributed equally to this work. the tuber, lip, stigma, and connective. Herminium © The Willi Hennig Society 2015 B. B. Raskoti et al. / Cladistics 32 (2016) 198–210 199 (a) (b) (c) (d) (e) (f) Fig. 1. Flowers of some representative species of Herminium and its alliance. (a) Herminium monorchis; (b) Peristylus fallax; (c) Platanthera lati- labris; (d) Androcorys pugioniformis; (e) Frigidorchis humidicola; (f) Hsenhsua chrysea. All photos taken by Xiaohua Jin. usually has globose tubers, a possibly saccate but not Renz is similar to Herminium but has pulvinate and slender-spurred lip, a narrow connective, and a stigma confluent stigma lobes. Frigidorchis Z.J. Liu & S.C. with two separate lobes that are free from the base of Chen is similar to Bhutanthera in having a pulvinate the lip; Peristylus has globose tubers, a variously stigma but differs in the subcorymbose inflorescence spurred lip (the spur ranging in shape from subglobose and flowers without bracts (Fig. 1e). to slender-tubular), stigma lobes that are separate and Several phylogenetic studies of the Orchideae that fused to the base of the lip and to the staminodes, and include Herminium have been conducted using molecu- a narrow connective; Platanthera has thick, fleshy lar data, but no species-level phylogenetic study of the roots (sometimes partly swollen into a fusiform tuber), group as a whole has been undertaken, and previous a slender-spurred lip, confluent or rarely separate and efforts to reconstruct the phylogeny of Herminium free stigma lobes, and a wide connective. However, have been only marginally successful. The first investi- morphological differentiation between these genera can gation of the monophyly of Herminium presented very be difficult in certain species in which characters of the preliminary conclusions on generic delimitation and column structure are more or less intermediate. interspecific relationships, as only four species and one This problem is compounded by the recognition of marker (internal transcribed spacer, ITS) were used several small satellite genera. Androcorys Schltr. is sim- (Bateman et al., 2003). A second study had an even ilar to Herminium but is characterized by the pulvinate more restricted taxon sampling, with only one species stigma lobes and a wide and hooded connective of Herminium being included in the analysis (Inda (Fig. 1d). Porolabium Tang et Wang is similar to And- et al., 2012). A more recent and more comprehensive rocorys in having pulvinate stigma lobes but is charac- investigation analysed chloroplast (rbcL, matK) and terized by the lip base with two pores. Bhutanthera nuclear (ITS) markers in 18 species of Herminium 200 B. B. Raskoti et al. / Cladistics 32 (2016) 198–210 sensu lato (s.l.) (Jin et al., 2014). It found that Andro- region, 2 min denaturation at 94 °C followed by 34 corys, Porolabium, the Platanthera latilabris group, cycles of denaturation of 94 °C for 45 s, 45 s anneal- and some species of Peristylus are nested within ing for 58 °C, and extension at 72 °C for 90 s, fol- Herminium, whereas some species of Herminium lowed by elongation for 5 min at 72 °C; for matK, belong to Platanthera. Generic delimitation, interspe- psaB and rbcL regions: 3 min denaturation at 95 °C cific interrelationships, and morphological character followed by 35 cycles of denaturation at 95 °C for evolution of Herminium remained unresolved. 30 s, 1 min annealing at 50 °C, and extension at 72 °C The aims of the present study can be summarized as for 90 s, followed by elongation for 10 min at 72 °C; follows: (i) to reconstruct the phylogeny of Herminium for psbA-trnH region, 3 min denaturation at 94 °C fol- based on extensive sampling, using multiple nuclear lowed by 34 cycles of denaturation of 94 °C for 45 s, and chloroplast sequence regions and morphological 1 min annealing from 52 to 55 °C, and extension at characters; (ii) to test the monophyly of Herminium; 72 °C for 2 min, followed by elongation for 5 min at and (iii) to understand the evolutionary pattern of 72 °C; for trnL-F region, 3 min denaturation at 94 °C morphological characters previously used in taxon followed by 35 cycles of denaturation of 94 °C for delimitation and to detect morphological synapomor- 30 s, 30 s annealing at 50 °C, and extension at 72 °C phies that support monophyletic groups. for 90 s, followed by elongation for 10 min at 72 °C. Sequencing was conducted in both directions using the same primers as in the PCR. Additional internal prim- Material and methods ers matK and trnL-F were also required for sequencing (Table S2). Sequencing reactions was carried out in Plant materials 10-lL volumes containing 1 lL template (approx. 10 ng), 4 lL ddH2O, 1 lL of sequencing primer (3.2 In total, 56 accessions were investigated (supporting pmol/lL) and 4 lL BigDye, with a cycler program of information Table S1). Forty-nine species across nine 25 cycles of 96 °C for 10 s, 50 °C for 5 s, and 60 °C genera (Androcorys, Bhutanthera, Frigidorchis, Habena- for 4 min after an initial denaturation of 96 °C for ria, Herminium, Hsenhsua, Peristylus, species currently 1 min. Sequencing was carried out on ABI Prism 154 misplaced in Platanthera,andPorolabium) were con- Bigdye Terminator Cycle Sequencing Kit (Applied sidered as the ingroup. This sampling includes about Biosystems, Foster City, CA, USA). 90% of the species of Herminium, and represents both the morphological and the geographical range of this Morphological character coding genus. The outgroup consists of three species of Orch- idinae, i.e. Galearis spathulata, Platanthera chlorantha A total of 37 morphological characters were scored and P. japonica, in accordance with previous phyloge- for 56 taxa based on traits traditionally used to cir- netic studies of the subtribes Orchidinae and Habe- cumscribe genera, including 21 binary and 16 multi- nariinae (Bateman et al., 2003; Jin et al., 2014). state morphological characters. The list of characters and their states is provided in the Appendix 1. Mor- DNA sequence data phological