Cladistics

Cladistics 32 (2016) 198–210 10.1111/cla.12125

A phylogenetic analysis of molecular and morphological characters of (, ): evolutionary relationships, , 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, ; bUniversity of Chinese Academy of Sciences, Beijing, 100049, China; cScience Directorate, Royal Botanical Gardens, Richmond, Surrey, TW9 3AB, UK; dKey Laboratory of Biodiversity and Biogeography of East , 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 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, , Frigidorchis and Porolabium. Apomorphic and plesiomorphic character states are identified for various recovered in this study. A few currently wrongly assigned to and 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, ; type, (King and Pantling, 1898; Lang, 1988; Pridgeon et al., (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 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 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. 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 characters were gathered by examining liv- ing in their natural habitats and herbarium Total DNA was extracted using a Plant Genomic specimens deposited in KATH, KUN, PE, and TUCH DNA Kit (Tiangen Biotech, Beijing, China) following and from digital images. For species for which only a the manufacturer’s protocol from leaf material that limited number of specimens were available, published had been dried with silica-gel during specimen collec- descriptions were also consulted (Hooker, 1890; King tion in the field. In total, seven markers including two and Pantling, 1898; Duthie, 1906; Tang and Wang, nuclear markers (ITS and Xdh) and five chloroplast 1937, 1940; Hunt, 1970; Seidenfaden, 1977; Hara markers (matK, psaB, psbA-trnH, rbcL, trnL-F) were et al., 1978; Lang, 1988; Pridgeon et al., 2001). A min- amplified using the primers listed in Table S2. imum of ten specimens were examined (when avail- PCR for nuclear and chloroplast regions was carried able) for each character for each species. Pollinia for out in 25-lL volumes containing 10–50 ng DNA, micromorphological examination were taken from 12.5 lL29 Taq PCR Master Mix (Biomed, Beijing, fully open flowers in dry specimens, coated with gold, China), 0.4 lM forward and reverse primers respec- and observed under an S-4800 scanning electron tively, and ddH2O to reach a total volume of 25 lL. microscope. Twenty-four species from the sampled The following cycling program was used: for ITS taxa were investigated (see Table S3). In a few cases region, 5 min (min) denaturation at 94 °C followed by where it was impossible to collect data for a particular 34 cycles of denaturation of 94 °C for 45 s, 45 s species, the relevant character state was treated as annealing at 55 °C and extension at 72 °C for 1 min, missing data. All voucher specimens examined to study followed by elongation for 10 min at 72 °C; for Xdh the pollinia were deposited in the Chinese National B. B. Raskoti et al. / Cladistics 32 (2016) 198–210 201

Herbarium (PE), Institute of Botany, Chinese Acad- Stamatakis et al. (2008) to assign support to branches emy of Sciences. in the ML tree. For the BI analysis, posterior probabilities (PP) for Matrix construction and phylogenetic analyses individual clades were inferred with MrBayes v.3.2.3 (Ronquist and Huelsenbeck, 2003) on the CIPRES The nucleotide sequences were edited independently Science Gateway. The best fitting DNA substitution and assembled using the ContigExpress program in model for each matrix was determined using MrModel Vector NTI Suite v. 6.0 (Informax, North Bethesda, test v.2.3 (Nylander et al., 2004) under the Akaike MD, USA). Clustal X v.2.1 (Larkin et al., 2007) was information criterion (AIC) in conjunction with PAUP used to align the DNA sequences of each region. v.4.0b10 (Swofford, 2002). The evolutionary model for Alignments were visually inspected and manually six markers, ITS, matK, psaB, psbA-trnH, rbcL and refined and adjusted to account for missing inserts by trnL-F, and combined nuclear, combined chloroplast sequential pairwise comparison using BioEdit v.7.1.3.0 and all combined markers was GTR+I+G (nst = 6, (Hall, 1999). All alignment gaps were treated as miss- rates = invagamma), and HKY+G (nst = 2, rates = ing data. gamma) for Xdh. Two separate four Markov chain Phylogenetic analyses were performed for each mar- Monte Carlo (MCMC) analyses were performed, start- ker individually and for the combined dataset. Maxi- ing with a random tree, proceeding for 5000 000 mum-parsimony (MP) analysis was performed in generations, and sampling every 1000 generations. PAUP v.4.0b10 (Swofford, 2002), and maximum-likeli- Majority rule (> 50%) consensus trees were recon- hood (ML) analysis was performed using the CIPRES structed after removing the ‘burn-in phase’ samples Science Gateway web server (RAxML-HPC2 on (the first 25% of the sample trees). XSEDE 8.1.11) (Miller et al., 2010). Bayesian infer- ence (BI) was performed using MrBayes v.3.2.3 (Ron- Morphological dataset and ancestral state reconstruction quist and Huelsenbeck, 2003) and also on the CIPRES Science Gateway web server (MrBayes 3.2.3 on The morphological datasets with 37 characters of 56 XSEDE) (Miller et al., 2010). taxa (Table S3) were analysed by MP using PAUP Homogeneity between the nuclear (ITS, Xdh) and with the same options as described in the analysis of plastid datasets (matK, psaB, psbA-trnH, rbcL, and molecular data. For the BI analysis, two independent trnL-F) was tested using the incongruence length dif- runs of 3000 000 generations were performed using ference (ILD) (Farris et al., 1995) applied in PAUP four chains and sampling every 100 generations. v.4.0b10 (Swofford, 2002). The P-value for the ILD Majority rule (> 50%) consensus trees were recon- test of the combined dataset was below the threshold structed after removing the ‘burn-in phase’ samples value of 0.01. However, visual inspection of the indi- (the first 25% of the sample trees). vidual tree topologies of each DNA marker did not Ancestral character states were reconstructed with indicate hard conflicts. In such cases, nuclear and plas- the Trace Character History module of Mesquite tid datasets can be combined (Bull et al., 1993; v.3.02 (Maddison and Maddison, 2015) using a likeli- Mason-Gamer and Kellogg, 1996; Quicke et al., 2007). hood approach with Markov k-state 1 parameter This soft incongruence may disappear with additional (Mk1) model (Lewis, 2001). To perform this analysis, data (Wendel and Doyle, 1998). Therefore, nuclear the most parsimonious tree on the combined molecular (ITS, Xdh) and plastid datasets (matK, psaB, psbA- dataset was employed. trnH, rbcL, and trnL-F) were combined using Sequence Matrix v.1.7.8 (Vaidya et al., 2011) for phy- Total-evidence datasets logenetic analysis. For the MP analysis, all characters were equally The total-evidence datasets comprised all taxa from weighted, and gaps were coded as missing data. Heu- both the entire molecular and the morphological data- ristic searches of 1000 random addition replicates with sets (56 samples from 49 species). MP and Bayesian tree bisection reconnection (TBR) branch swapping analyses were carried out following the procedures as were conducted to obtain the most parsimonious trees; mentioned above for molecular analyses. For Bayesian ten trees from each random sequence were saved. To analysis, a total of 5000 000 generations were run, evaluate node support, bootstrap analyses (Felsenstein, with the chains sampled every 1000 generations. Burn- 1988) were performed using 1000 replicates with ten in was set at 5500 trees after MCMC convergence. random taxon additions and heuristic search options. Nexus files for MP analysis of the combined nuclear For the ML analyses, the default GTR + CAT model dataset, chloroplast dataset, combined molecular data- was used. Bootstrap resampling with 1000 replicates set, morphological character dataset and total-evidence was conducted using the rapid bootstrap procedure of dataset are given in Appendix S1. 202 B. B. Raskoti et al. / Cladistics 32 (2016) 198–210

Results the formed by the genus Hsenhsua: (i) the MON clade, which is subdivided into two strongly supported Molecular data analyses subclades, one the core Herminium species group, and the other formed by two species previously treated as For various reasons, a few sequences could not be Peristylus: P. mannii and P. forceps; (ii) the LAN clade obtained, such as Xdh for Androcorys pusillus, Andro- of nine species of Herminium; (iii) the BHU clade of corys sp., dentata, Habenaria rhodocheila Androcorys, Bhutanthera, Frigidorchis and Porolabium is and Herminium coiloglossum, psaB for Androcorys pus- divided into two well-supported sister subclades, one illus, psbA-trnH for Androcorys pusillus and Herminium formed by Androcorys and Porolabium, and the other by gramineum,andtrnL-F for Androcorys pusillus. The Bhutanthera and Frigidorchis; and (iv) the LAT clade, statistics for the sequence analysis for the nuclear and which is subdivided into two groups, one formed by chloroplast markers of each gene, and combined three species previously treated as Platanthera, and the molecular datasets are summarized in Table S4. MP other by one species formerly treated as Peristylus. The (Appendix S2), BI and ML analyses of the individual topology of the strict consensus tree is highly similar to regions revealed topologies with low support. that from the combined nuclear data, but presents strong support for each clade. The MON clade is imme- Phylogeny inferred from nuclear data diately sister to the polytomy formed by the LAN, LAT and BHU clades; the interrelationships among the last Maximum-parsimony analysis retrieved 81 most three clades are unknown. parsimonious trees (MPTs) with a score of 1156, The likelihood tree recovered for the combined consistency index (CI) of 0.678 and retention index molecular data has a very similar topology. The same (RI) of 0.800. The strict consensus tree (Fig. 2, left) four major clades have strong support with PP of 0.95 shows five major clades for which relationships with or higher from BI. each other are not well resolved: (i) a clade of 16 spe- cies of core Herminium and three species formerly trea- Phylogeny inferred from morphological data ted as part of Peristylus; (ii) a clade of eight species of Herminium; (iii) a clade of six species of Androcorys, The dataset for the MP analysis from 37 morpho- two species of Porolabium, one species of Bhutanthera, logical characters had 36 parsimony-informative char- and one species of Frigidorchis; (iv) a clade of three acters, CI = 0.347 and RI = 0.817. Most of the clades species previously treated as part of Platanthera; and found in the combined molecular analysis are col- (v) a clade of one species formerly treated as Peristy- lapsed in the strict consensus tree from morphological lus. The latter four clades form a moderately sup- data (Appendix S3). Several poorly supported clades ported clade which is sister to the clade of core form a polytomy. The likelihood tree and the BI tree Herminium. based on morphological data have a similar topology. The ML tree recovered for nuclear data has a very similar topology to the strict consensus tree. The same Phylogeny inferred from total-evidence data five major clades have strong support with PP of 0.92 or higher from BI. The total dataset (combined molecular and morpho- logical) has 6782 constant characters, 1036 parsimony- Phylogeny inferred from chloroplast data informative characters, with best tree score = 2982, CI = 0.677 and RI = 0.809. The strict consensus tree Maximum-parsimony analysis retrieved 530 MPTs from the total-evidence dataset revealed a topology with a score of 1587, CI = 0.709 and RI = 0.831. The highly congruent to that inferred from the combined strict consensus tree shows four moderately to highly molecular datasets, with higher support (Fig. 4), while supported major clades forming a polytomy with other the HSE clade is resolved as sister to four clades of ingroups (Fig. 2, right). The ML tree recovered for Herminium and its alliance. However, the LAN clade chloroplast data has a very similar topology. The four is resolved as the immediate sister to the BHU clade major clades have strong support with PP of 0.95 or plus LAT clade. The ML tree, recovered for the total- higher from BI. evidence dataset, has a very similar topology. The same four major clades have strong support with PP Phylogeny inferred from combined molecular data of 0.87 or higher from BI.

Maximum-parsimony analysis retrieved eight MPTs Character evolution and synapomorphies with a score of 2754, CI = 0.693 and RI = 0.816. The strict consensus tree (Fig. 3) shows that four major Phylogenetic analyses of 37 morphological char- clades form a strongly supported clade which is sister to acters show complicated evolutionary patterns in B. B. Raskoti et al. / Cladistics 32 (2016) 198–210 203

Fig. 2. Strict consensus of MPTs for combined analysis of sequence data from two nuclear genes (ITS, Xdh) (left) and five combined chloroplast genes (matK, psaB, psbA-trnH, rbcL, and trnL-F) (right). Numbers above branches are bootstrap values. Only support percentages above 50 are shown.

Herminium. All alpine species have (one or more) basal clade, once in the BHU clade and once in the LAT leaves, whereas most of the subalpine and temperate clade). Several species with a distinct spur are nested species in the LAN clade and LAT clade have cauline within the MON and LAT clades (such as Platanthera leaves. A subcorymbose inflorescence is restricted to latilabris, Fig. 1c). A callus on the lip has evolved Frigidorchis humidicola (BHU clade). An entire lip has independently several times (at least three times in the evolved at least five times (three times in the MON MON clade, once in the LAN clade and once in the 204 B. B. Raskoti et al. / Cladistics 32 (2016) 198–210

Fig. 3. Strict consensus of MPTs of Herminium and its alliance, based on the combined nuclear (ITS, Xdh) and chloroplast (matK, psaB, psbA- trnH, rbcL, and trnL-F) dataset. Numbers above branches are support values for MP, ML and BI (BSMP,BSML and PPBI) respectively. *Node support for BSMP,BSML = 100%, and PPBI = 1.00. A dash indicates nodes with < 50% support. Clade code: MON = monorchis, LAN = lance- um, BHU = Bhutanthera, LAT = latilabris, HES = Hsenhsua, HAB = Habenaria, PER = Peristylus.

LAT clade). A spur-like saccate base is restricted to Pollen exine of all examined members of the BHU, the MON clade, while a distinct spur but shorter than MON, LAN and LAT clades is baculate-pilate except the ovary has evolved at least six times in Herminium. in Peristylus forceps, P. mannii and H. alaschanicum, Stigmas are extended along the entrance of spur in the where it is reticulate-heterobrochate. three subalpine species of the LAT clade (such as Pla- Phylogenetic analyses indicate that some charac- tanthera latilabris, Fig. 1c), and are very short projec- ters, such as tuber shape, beaked ovary, lip with tions protruding under the rostellum in alpine species concave base, and much abbreviated caudicles of the of the LAT clade, MON clade and LAN clade. They pollinia, are shared and uniform within all clades of are pulvinate in the BHU clade, which inhabits most Herminium and can be used for delimitation of this of the alpine regions in which Herminium occurs. genus. The following character states are recovered B. B. Raskoti et al. / Cladistics 32 (2016) 198–210 205

Fig. 4. Strict consensus of the MPTs of Herminium and its alliance, based on the total-evidence dataset. Numbers above branches are support values for MP and BI (BSMP, and PPBI). *Node support for BSMP, and PPBI = 1.00. Only support percentages above 50 are shown. Clade code: MON = monorchis, LAN = lanceum, BHU = Bhutanthera, LAT = latilabris, HES = Hsenhsua, HAB = Habenaria, PER = Peristylus. as synapomorphies for the MON clade: ovary Discussion beaked and strongly hooked, absence of pits on base of the lip, and horn-like viscidium. Ovary beaked The present study is based on the most comprehen- but not hooked and viscidium not horn like are syn- sive sampling achieved to date and is the first to spe- apomorphies of the LAN, BHU and LAT clades. A cifically address the generic limits of Herminium and horn-like viscidium is restricted to the MON clade, allied genera using an integrative phylogenetic whereas a globose viscidium is restricted to the approach. Results based on chloroplast, nuclear and LAN, BHU and LAT clades (Fig. 5). morphological data provide low to high resolution 206 B. B. Raskoti et al. / Cladistics 32 (2016) 198–210

Fig. 5. Character state distribution on the strict consensus tree from the total-evidence analysis. Open and closed squares are synapomorphic characters. Character number corresponds to those in Appendix 1. individually. However, they are largely consistent with or not, and the presence or absence of a spur, show each other in overall tree topology. The phylogeny complicated evolutionary patterns. Some stigma traits, reconstructed from the combined molecular and total- for example a projecting or a pulvinate stigma, are cor- evidence data is well supported and largely congruent related with the elevations at which members of a clade with the topologies of trees found using the partial occur. Characters such as phyllotaxy, leaf shape, length datasets mentioned above. of floral bracts, flower colour, length of ovary and pedi- cel, and petal shape show repeated losses and gains. The Character evolution associated character states are mostly uniform and pre- sumably apomorphic within clades or subclades and Characters of the lip and stigma have been widely although they are less informative for the generic delimi- used for generic or intraspecific classification in the Her- tation of Herminium as a whole, they are evidently use- minium alliance (King and Pantling, 1898; Pridgeon ful for recognizing the clades and subclades. Globose et al., 2001; Pearce and Cribb, 2002; Chen et al., 2009; tubers, which represent one of the few consistent Agrawala et al., 2010). Our phylogenetic analyses morphological differences between Herminium sensu indicate that lip characters, such as the lip being lobed lato and Platanthera, are probably a plesiomorphic or entire, deflexed or not deflexed, its base being dilated character state in the Herminium alliance, as it also B. B. Raskoti et al. / Cladistics 32 (2016) 198–210 207 occurs in other closely related genera, such as Hsenhsua, and articulated, lip three-lobed near the middle (five Peristylus and Habenaria. lobes in H. quinquelobum), without callus or spur (only H. longilobatum occasionally has callus), rostel- Phylogenetic relationships lum well developed, stigma lobes transversely oblong, beneath the rostellum, and viscidium suborbicular. Phylogenetic analyses based on the combined molec- The BHU clade (Figs 3 and 4), a group of some 19 ular datasets indicate that the MON clade is immedi- species, is mainly distributed in alpine regions and is ate sister to the polytomy formed by the BHU, LAN characterized by small plants (usually no more than and LAT clades (Fig. 3). Although seven molecular 15 cm tall), basal leaf or leaves, very small floral markers have been used in the study, the interrelation- bracts, and a pulvinate (cushion-like) stigma. Porolabi- ships of the latter three clades are not resolved. How- um was established based on the presence of a pair of ever, phylogenetic analyses based on total-evidence pits in the lip (Tang and Wang, 1940); however, Poro- data indicate that the BHU clade is immediately sister labium is closely related to Androcorys by sharing a to the LAT clade with moderate support, and the basal leaf, very small floral bracts, a lingulate entire lip LAN clade is sister to the group formed by these two that is dilated at the base, the absence of a spur, and a clades with strong support. wide connective that is more or less saccate. Our phy- Our total-evidence analyses indicate that Herminium logenetic analyses indicate that Porolabium is nested sensu stricto as currently understood is paraphyletic. within Androcorys. Frigidorchis humidicola (Peristylus Several genera, such as Androcorys, Bhutanthera, Frigi- humidicola) is characterized by subcorymbose inflores- dorchis and Porolabium, and a number of misplaced cence and flowers without bracts. Ormerod (2003) members of Peristylus and Platanthera are deeply treated it as a member of Bhutanthera. Chen et al. nested within Herminium with strong support (Figs 3 (2009) established a monotypic genus for it, Frigidor- and 4). Herminium s.l. is subdivided into two clades, chis. Our phylogenetic analyses indicate that Frigidor- one immediately sister to the clade formed by Andro- chis is the immediate sister to Bhutanthera. corys, Bhutanthera, Frigidorchis and Porolabium, and The LAT clade (Figs 3 and 4) comprises two sister the other sister to these two clades. Hsenhsua and an groups. The first group includes three subalpine species undescribed species were resolved as the immediate sis- and is well characterized by subglobose tubers, cauline ter group of Herminium plus Androcorys, Bhutanthera, leaves, an entire lip with a spur longer than the ovary Frigidorchis and Porolabium. The two other ingroup (except in Platanthera clavigera), a wide connective, a genera, Habenaria and Peristylus, used in the recon- well-developed rostellum, and two stigma lobes extend- struction of the phylogeny of Herminium have moder- ing far along the entrance of the spur. These three spe- ate support (Figs 3 and 4). cies have consistently been mistaken for members of The members of the core Herminium (MON) clade Platanthera due to their having an entire lip with a are mostly alpine species and are subdivided into two long spur, a wide anther connective and free stigma clades: the H. monorchis clade, and the Peristylus man- lobes. However, this subclade has an entire, globose nii and P. forceps clade. The former is characterized tuber instead of thick fleshy roots, an important char- by a three-lobed or sometimes entire lip, the mid-lobe acteristic of Platanthera. This subclade is readily dis- usually longer than the lateral lobes, lip base concave tinguished from others in the Herminium alliance by to saccate, and with horn-like viscidia. Pearce et al. its morphology, particularly its wide connective, long (2001) transferred Herminium alaschanicum to the spur, and two stigmas extending along the entrance of genus Peristylus based on the presence of a scrotiform the spur. The second group includes one species, Peri- spur; our analyses show that this species belongs in stylus fallax (Fig. 1b). Peristylus fallax differs from the MON clade of Herminium. The Peristylus mannii typical members of Peristylus (such as Peristylus gran- and P. forceps clade is characterized by horizontally dis) by its parallel locules and a stalked stigma that positioned flowers, a lip with a short spur, and a ros- extends beyond the concave rostellum. Hooker (1890) tellum with a short arm. The pollen exine of Peristylus treated Peristylus fallax as a member of Herminium. forceps and P. mannii is reticulate-heterobrochate, which is different from all other members of four Taxonomic considerations clades of Herminium s.l. Tang and Wang (1940) trans- ferred these species to Herminium. Phylogenetic analyses indicate that Hsenhsua is The LAN clade (Fig. 4), including nine sampled immediately sister to a group formed by four clades species of Herminium, i.e. H. coiloglossum, H. kamen- (MON, LAN, BHU and LAT) with moderate to high gense, H. jaffreyanum, H. lanceum, H. longilobatum, support (Fig. 4), and is characterized by foliaceous flo- H. mackinonii, H. quinquelobum, H. souliei and H. yun- ral bracts enclosing the spur, pedicel longer than ovary nanense, is characterized by leaf or leaves usually basal (Fig. 1f), three slightly convex stigma lobes confluent or spirally arranged cauline, lip base nearly semicircular under the rostellum, and laevigate-scabrate pollen 208 B. B. Raskoti et al. / Cladistics 32 (2016) 198–210 exine. This evidence supports recognition of Hsenhsua 31107176, 31470299, J1310002) and the Chinese Spe- as a distinct genus. 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We propose to widen the circum- Bioinformatics 23, 2947–2948. scription of Herminium to include Androcorys, Bhutan- Lewis, P.O., 2001. A likelihood approach to estimating phylogeny thera, Frigidorchis and Porolabium as well as certain from discrete morphological character data. Syst. Biol. 50, 913– misplaced species of Platanthera and Peristylus. Hermin- 925. Maddison, W.P., Maddison, D., 2015. Mesquite, version 3.02: a ium can be distinguished from Platanthera by the modular system for evolutionary analysis. http//mosquite rounded tubers (tubers absent or drawn out in a root- project.org. like tip in Platanthera) and from Peristylus by the Mason-Gamer, R.J., Kellogg, E.A., 1996. Testing for phylogenetic conflict among molecular data sets in the tribe Triticeae stigma lobes, which are not adnate to the base of the lip. (Gramineae). Syst. Biol. 45, 524–545. Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway for Inference of Large Phylogenetic Trees. Acknowledgements Gateway Computing Environments Workshop (GCE), IEEE, pp. 1–8. Nylander, J.A., Ronquist, F., Huelsenbeck, J.P., Nieves-Aldrey, J., Funds were provided by grants from the National 2004. Bayesian phylogenetic analysis of combined data. Syst. Natural Science Foundation of China (Grant Nos. Biol. 53, 47–67. B. B. Raskoti et al. / Cladistics 32 (2016) 198–210 209

Ormerod, P., 2003. Orchidaceous additions to the floras of China Table S3. Morphological data matrix for the phylo- and Vietnam. Taiwania 48, 139–146. genetic analysis. Pearce, N.R. and Cribb, P.J., 2002. Flora of Bhutan. Vol. 3. Royal Botanic Garden, Edinburgh, Edinburgh and Royal Government Table S4. The statistics of MP analysis for nuclear of Bhutan, Thimphu. and chloroplast markers. Pearce, N., Cribb, P.J., Renz, J., 2001. Notes relating to the Flora of Bhutan: XLIV. Taxonomic notes, new taxa and additions to the Orchidaceae of Bhutan and Sikkim (India). Edinburgh J. Bot. 58, 99–122. Appendix 1 Pridgeon, A.M., Cribb, P.J., Chase, M.W., 2001. Genera Orchidacearum: Volume 2. Orchidoideae. Oxford University Press, Oxford. Morphological characters coded in phylogenetic analysis Quicke, D.L., Jones, O.R., Epstein, D.R., 2007. Correcting the problem of false incongruence due to noise imbalance in the 1 Tuber shape: tuber branched (0), unbranched (1) incongruence length difference (ILD) test. Syst. Biol. 56, 496–503. 2 Phyllotaxy: distichous (0), spiral (1), single leaf (2) Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian 3 Leaves: cauline (0), basal (1) phylogenetic inference under mixed models. Bioinformatics 19, 4 Leaf base: petiolate (0), sessile (1) – 1572 1574. 5 Leaf shape: elliptic to ovate-orbicular (0), linear-lanceolate to Seidenfaden, G., 1977. Orchid genera in Thailand: 5. Orchidoideae. oblong (1) Dansk. Bot. Ark. 31, 3. 6 Sterile bracts of stem: absent (0), present (1) Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap 7 Inflorescence type: subcorymbose (0), racemose (1), single- algorithm for the RAxML web servers. Syst. Biol. 57, 758–771. flowered (2) Swofford, D.L. 2002. PAUP*: Phylogenetic Analysis Using Parsimony (and Other Methods), version 4.0b10. Sinauer, 8 Fertile floral bracts: absent (0), truncate (1), short-triangular Sunderland, MA. (2), lanceolate to linear (3) Tang, T., Wang, F., 1937. Notes on Orchidaceae of China II. Bull. 9 Length of floral bracts: equal or longer than ovary (0), minute (1), Fan. Mem. Inst. Biol. Bot. Ser. 7, 127–144. well developed but less than half as long as the ovary (2), absent (3) Tang, T., Wang, F., 1940. Contributions to the knowledge of eastern 10 Ovary: straight (0), arcuate (1) Asiatic Orchidaceae. Bull. Fan. Mem. Inst. Biol. Bot. Ser. 10, 28– 11 Apex of ovary: not beaked (0), beaked not hooked (1), 31. beaked and strongly hooked (2) Vaidya, G., Lohman, D.J., Meier, R., 2011. SequenceMatrix: 12 Ovary: not twisted (0), twisted and cylindrical (1), ovary concatenation software for the fast assembly of multi-gene twisted and fusiform (2) datasets with character set and codon information. Cladistics 27, 13 Flowers: secund (0), resupinate (1), other (2) 171–180. 14 Flower colour: other colour (0), yellowish green or green (1), Wendel, J.F., Doyle, J.J., 1998. Phylogenetic incongruence: window green with white lip (2) into genome history and molecular evolution. In: Soltis, D.E., 15 Ovary pedicel: not distinct (0), distinct but shorter than ovary Soltis, P.S., Doyle, J.J. (Eds.), Molecular Systematics of Plants (1), nearly equal to or longer than ovary (2) – II. Springer, Berlin, pp. 265 296. 16 Petal: falcate (0), oblique (1), straight (2) 17 Petal: linear (0), lanceolate (1), ovate (2), oblong (3), 18 Petals: connivent with sepals (0), spreading (1) Supporting Information 19 Lateral sepal: not deflexed (0), deflexed (1) 20 Lip: lobed (0), entire (1) Additional Supporting Information may be found in 21 Lip: not deflexed (0), deflexed (1) the online version of this article: 22 Lip base: not dilated (0), dilated (1) 23 Lip base: not concave (0), concave (1) 24 Lip base: not spurred (0), spurred (1), saccate base (2) Appendix S1. Nexus files for MP analysis of the 25 Spur: globose (0), cylindrical and shorter than ovary (1), combined nuclear dataset, chloroplast dataset, com- cylindrical and longer than ovary (2), absent (3) bined molecular dataset, morphological character 26 Lip: without callus (0), with callus (1) 27 Lip base: without pits (0), with pits (1) dataset, and total-evidence dataset. 28 Anther connective: neither elongate nor drawn out (0), elon- Appendix S2. Phylogenetic hypothesis for Hermini- gate and drawn out (1) um, based on MP analysis of each molecular dataset, 29 Anther connective narrow (0), anther connective broad (1) including non-Herminium outgroups from Galearis and 30 Viscidium: not enrolled into horn-like (0), enrolled into horn- Platanthera. Values above branches represent MP sup- like (1) 31 Pollinium caudicles: absent or nearly indistinct (0), distinct (1) port. 32 Anther locules: divergent (0), parallel (1) Appendix S3. Phylogenetic hypothesis for Hermini- 33 Stigma: not pulvinate (0), pulvinate (1) um, based on MP analysis of the morphological data- 34 Rostellum: without arms (0), with erect arms, but not broad set, including non-Herminium outgroups from Galearis (1), with erect arms, broad (2) and Platanthera. Numbers above branches are support 35 Stigma: bilobed (0), trilobed (1) 36 Stigma: beneath the rostellum (0), extended along the values for MP and BI (BSMP and PPBI). entrance of spur (1), adnate to the lip base (2), free from ros- Table S1. Voucher information and GenBank acces- tellum, the entrance of spur and the lip base (Habenaria type) sion numbers for the sequences analysed in this study. (3) Table S2. Primers used for PCR and sequencing in 37 Pollen exine: baculate-pilate (0), reticulate-heterobrochate (1), this study. laevigate-scabrate (2) 210 B. B. Raskoti et al. / Cladistics 32 (2016) 198–210

Appendix 2 Herminium humidicola (K.Y. Lang & D.S. Deng) X.H. Jin, Schu- it., Raskoti & L.Q. Huang, comb. nov. Basionym: Peristylus humidicola K.Y. Lang & D.S. Deng, Novon Taxonomic treatment of Herminium 6: 190 (1996). Herminium L., Opera Varia: 251 (1758). Herminium latilabre (Lindl.) X.H. Jin, Schuit., Raskoti & L.Q. Androcorys Schltr., Repert. Spec. Nov. Regni Veg. Beih.4:52 Huang, comb. nov. (1919), syn. nov. Basionym: Platanthera latilabris Lindl., Gen. Sp. Orchid. Pl.: 289 Bhutanthera Renz, Edinburgh J. Bot. 58: 99 (2001), syn. nov. (1835). Frigidorchis Z.J. Liu & S.C. Chen, J. Fairylake Bot. Gard. 6(3): 14 Herminium oxysepalum (K.Y.Lang) X.H. Jin, Schuit., Raskoti & (2007), syn. nov. L.Q. Huang, comb. nov. Monorchis Seg., Pl. Veron. 3: 251 (1754). Basionym: Androcorys oxysepalus K.Y. Lang, Guihaia 16: 106 Monorchis Ehrh., Beitr. Naturk.4: 147 (1789). (1996). Porolabium Tang & F.T. Wang, Bull. Fan Mem. Inst. Biol. 10: 36 Herminium wangianum X.H. Jin, Schuit., Raskoti & L.Q. Huang, (1940), syn. nov. nom. nov. Herminium albomarginatum (King) X.H. Jin, Schuit., Raskoti & Basionym: Androcorys spiralis Tang & F.T. Wang, Bull. Fan L.Q. Huang, comb. nov. Mem. Inst. Biol. 10: 38 (1940). Basionym: Habenaria albomarginata King, Ann. Roy. Bot. Gard. Not Herminium spirale (A.Rich.) Rchb.f., Bonplandia (Hannover) (Calcutta) 8: 322 (1898). 3: 213 (1855). Herminium albosanguineum (Renz) X.H. Jin, Schuit., Raskoti & Note: named after the Chinese botanist Fa Tsuan Wang. L.Q. Huang, comb. nov. The following five species, currently treated as Peristylus species, Basionym: Bhutanthera albosanguinea Renz, Edinburgh J. Bot. 58: belong in Herminium. 101 (2001). Herminium coeloceras (Finet) Schltr., Notes Roy. Bot. Gard. Herminium albovirens (Renz) X.H. Jin, Schuit., Raskoti & L.Q. Edinburgh 5: 97 (1912). Huang, comb. nov. Basionym: Peristylus coeloceras Finet, Rev. Gen.Bot. 13: 519 Basionym: Bhutanthera albovirens Renz, Edinburgh J. Bot. 58: (1902). 102 (2001). Herminium elisabethiae (Duthie) Tang & F.T.Wang, Bull. Fan Herminium clavigerum (Lindl.) X.H. Jin, Schuit., Raskoti & L.Q. Mem. Inst. Biol. 7: 129 (1936). Huang, comb. nov. Basionym: Habenaria elisabethiae Duthie, J. Asiat. Soc. Bengal, Basionym: Platanthera clavigera Lindl., Gen. Sp. Orchid. Pl.: 289 Pt. 2, Nat. Hist. 71: 44 (1902). (1835). Herminium fallax (Lindl.) Hook.f., Fl. Brit. India 6: 129 (1890). Herminium edgeworthii (Hook.f. ex Collett) X.H. Jin, Schuit., Basionym: Peristylus fallax Lindl., Gen. Sp. Orchid. Pl.: 298 Raskoti & L.Q. Huang, comb. nov. (1835). Basionym: Habenaria edgeworthii Hook. f. ex Collett, Flora Siml- Herminium forceps (Finet) Schltr., Notes Roy. Bot. Gard. Edin- ensis: 504, f. 166 (1907) burgh 5: 97 (1912). Herminium esquirolii X.H. Jin, Schuit., Raskoti & L.Q. Huang, Basionym: Peristylus forceps Finet, Rev. Gen.Bot. 13: 521 (1902). nom. nov. Herminium mannii (Rchb.f.) Tang & F.T. Wang, Bull. Fan Mem. Basionym: Androcorys ophioglossoides Schltr., Repert. Spec. Nov. Inst. Biol. 7: 128 (1936). Regni Veg. Beih. 4: 53 (1919). Basionym: Coeloglossum mannii Rchb.f., Linnaea 41: 54 (1876). Not Herminium ophioglossoides Schltr., Notes Roy. Bot. Gard. The following two new combinations are, respectively, a correc- Edinburgh 5: 96 (1912). tion and an addition to Jin et al. (2014). Note: named after the collector of the type, J. Esquirol. Platanthera stenochila X.H. Jin, Schuit., Raskoti & L.Q. Huang, Herminium fimbriatum (Raskoti) X.H. Jin, Schuit., Raskoti & nom. nov. L.Q. Huang, comb. nov. Basionym: Herminium angustilabre King & Pantl. J. Asiat. Soc. Basionym: Bhutanthera fimbriata Raskoti, Phytotaxa 62: 57 Bengal, 65(2): 131. 1895 (1896). (2012). Platanthera angustilabris (King & Pantl.) X.H. Jin, Schuit. & Herminium handelii X.H.Jin, Schuit., Raskoti & L.Q. Huang, W.T. Jin, Molec. Phylogen. Evol. 77: 51 (2014), nom. illeg. nom. nov. Not Platanthera angustilabris Seidenf., Opera Bot. 124: 11 (1995). Basionym: Habenaria alpina Hand.-Mazz., Symb. Sin.7: 1336 Platanthera superantha (J.J.Wood) X.H. Jin, Schuit., Raskoti & (1936). L.Q. Huang, comb. nov. Not Herminium alpinum (L.) Sweet, Hort. Brit.: 382 (1826). Peristylus superanthus J.J.Wood, Kew Bull.41: 811 (1986). Note: named after the Austrian botanist H. R. E. von Handel- Note: this species is closely related to Platanthera nematocaulon Mazzetti. (Hook.f.) Kraenzl., which was included as Peristylus nematocaulon Herminium himalayanum (Renz) X.H. Jin, Schuit., Raskoti & (Hook.f.) Banerji & P. Pradhan in the cladogram in Jin et al. (2014, L.Q. Huang, comb. nov. fig. 1). Basionym: Bhutanthera himalayana Renz, Edinburgh J. Bot. 58: 104 (2001).