sign in sign up
<<
Home , Gymnadenia, Habenaria, Hemipilia, Herminium, Orchideae, Ponerorchis

Molecular and Evolution 77 (2014) 41–53

Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

journal homepage: www.elsevier.com/locate/ympev

Molecular systematics of subtribe Orchidinae and Asian taxa of Habenariinae (, ) based on plastid matK, rbcL and nuclear ITS ⇑ Wei-Tao Jin a, Xiao-Hua Jin a, , André Schuiteman b, De-Zhu Li c, Xiao-Guo Xiang a, Wei-Chang Huang d, ⇑ Jian-Wu Li e, Lu-Qi Huang f, a State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 10093, b Herbarium, Library, Art and Archives Directorate, Royal Botanical Gardens, Kew, Richmond, Surrey TW9 3AB, UK c Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204, China d Shanghai Chenshan Botanical Garden, Chenhua Road 3888, Songjiang, Shanghai 201602, China e Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun Township, Mengla County, Yunnan 666303, China f National Resource Centre for Chinese Material Medica, China Academy of Chinese Medical Science, Beijing 100700, China article info abstract

Article history: The subtribe Orchidinae, distributed predominantly in Eastern and the Mediterranean, presents Received 24 October 2013 some of the most intricate taxonomic problems in the family Orchidaceae with respect to generic delim- Revised 30 March 2014 itation. Based on three DNA markers (plastid matK, rbcL, and nuclear ITS), morphological characters, and a Accepted 3 April 2014 broad sampling of Orchidinae and selected Habenariinae mainly from Asia (a total of 153 accessions of Available online 16 April 2014 145 in 31 genera), generic delimitation and phylogenetic relationships within the subtribe Orch- idinae and Habenariinae from Asia were assessed. Orchidinae and Asian Habenariinae are monophyletic, Keywords: and Orchidinae is divided into distinct superclades. Many genera, such as Amitostigma, , Hemip- Asia ilia, , , and , are not monophyletic. Habenaria is subdivided into Generic delimitation Habenariinae two distantly related groups, while Platanthera is subdivided into three even more disparate groups. Many previously undetected phylogenetic relationships, such as formed by the Amitostigma–Neot- Orchidinae tianthe–Ponerorchis complex, Platanthera latilabris group, Ponerorchis chrysea, , and Tsaiorchis, are well supported by both molecular and morphological evidence. We propose to combine Hemipiliopsis with , Amitostigma and Neottianthe with Ponerorchis, Smithorchis with Platanthera, and Acerator- chis and Neolindleya with , and to establish a new to accommodate Ponerorchis chrysea. Tsa- iorchis and Sirindhornia are two distinctive genera supported by both molecular data and morphological characters. A new genus, Hsenhsua, and 41 new combinations are proposed based on these findings. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction two subtribes can be distinguished on the basis of the structures of : Orchidinae usually has a concave and sessile stigma The subtribe Orchidinae consist of about 35 genera and often with confluent lobes, whereas Habenariinae usually has 350–400 species, distributed mainly in Eastern Asia, the Mediterra- stalked, convex and distinct stigma lobes (Dressler, 1993; nean Region with a few species extending in Northern America, Pridgeon et al., 2001). This morphological distinction has been sup- Southern America and (Chen et al., 2009; Dressler, 1993; ported by molecular evidence (Douzery et al., 1999). However, Inda Kraenzlin, 1901; Lang, 1999; Pearce and Cribb, 2002; Pridgeon et al. (2010, 2012) indicated that Habenariinae s.l. is paraphyletic, et al., 2001). Orchidinae is similar to Habenariinae in habitat and several genera from Africa, such as , , and preferences and many vegetative and floral characters, but these , were resolved as successive sister to Orchidinae + Habe- naria and its alliance. Batista et al. (2013) showed that the Habe- nariinae (formed by Habenaria s.l. + Cynorkis + Stenoglottis) ⇑ Corresponding authors. is sister to the Orchidinae clade formed by ( + Platanthera). E-mail addresses: [email protected] (W.-T. Jin), [email protected], xiaohua- In practice, it can be difficult to distinguish between concave ses- [email protected] (X.-H. Jin), [email protected] (A. Schuiteman), [email protected]. sile stigma lobes and stalked convex ones in some alpine taxa, such cn (D.-Z. Li), [email protected] (X.-G. Xiang), [email protected] (W.-C. Huang), [email protected] (J.-W. Li), [email protected] (L.-Q. Huang). as Androcorys, Herminium, Peristylus, Ponerorchis, and Smithorchis http://dx.doi.org/10.1016/j.ympev.2014.04.004 1055-7903/Ó 2014 Elsevier Inc. All rights reserved. 42 W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53

(Dressler, 1993; Lang, 1999; Pridgeon et al., 2001), while some true including , , Habenaria, , , and Platanthera species (Orchidinae) clearly have stalked stigma lobes. , were used to broaden the sampling of Orchideae in our Kurzweil and Weber (1992), Pridgeon et al. (2001) and Chase et al. analyses. Since previous results indicated that Orchideae is closely (2003) even suggested abandoning the recognition of Orchidinae related to tribe (Bytebier et al., 2007; Douzery et al., 1999; and Habenariinae as distinct clades. Pridgeon et al., 2001), two species of Disa, Disa tripetaloides and Orchidinae is a medium-sized subtribe in Orchidaceae and one Disa uniflora, were used as outgroups. The voucher information of many well-studied groups in Orchidaceae, however, the generic and the GenBank accession numbers used in this study are listed delimitation and classification within Orchidinae are still problem- in Table A.1. atic and is complicated by the morphological diversity, wide distri- bution range, homoplasy of characters considered diagnostic at 2.2. DNA extraction, amplification and sequencing generic level, and the intergrading and overlapping morphological variation between genera (Aceto et al., 1999; Bateman et al., 2003, Total genomic DNA was isolated from silica-gel-dried materials 2009; Box et al., 2008; Dressler, 1981, 1993; Hapeman and Inoue, using a Genomic DNA Kit (Beijing Biomed Co., LTD, Beijing, 1997; Jin and Efimov, 2012; Luer, 1975; Pridgeon et al., 2001; China). For this study, two plastid markers (the coding gene matK, Soliva et al., 2001; Tyteca and Klein, 2008). Based on morphological rbcL) and the nuclear ribosomal DNA internal transcribed spacers characters and/or analyses of molecular data, various generic (ITS) were used. The PCR and sequencing primers for matK, rbcL, delimitations and taxonomies have been proposed, especially on and ITS are listed in Table A.2. The selected DNA regions were some systematically difficult genera, such as , Orchis amplified by using a standard polymerase chain reaction (PCR). s.l., Platanthera, Ponerorchis, and Tsaiorchis (Aceto et al., 1999; The sequencing reactions were performed by using the ABI Prism Bateman et al., 2003, 2009; Hapeman and Inoue, 1997; Hooker, Bigdye Terminator Cycle Sequencing Kit (Applied Biosystems, ABI). 1890; King and Pantling, 1896, 1898; Lang, 1998, 1999; Soliva et al., 2001; Luer, 1975; Pridgeon et al., 2001; Tyteca and Klein, 2008). 2.3. Phylogenetic analyses As previous molecular systematics of Orchidinae were largely based on sampling from the Mediterranean area (Bateman et al., Sequences were aligned using the program Clustal X 1.83 2003, 2009; Douzery et al., 1999; Soliva et al., 2001; Inda et al., (Thompson et al., 1997) and manually adjusted using BioEdit 2012), and/or mainly utilized a single DNA marker (ITS) (Bateman (Hall, 1999). The homogeneity between the ITS data and the com- et al., 2003, 2009; Douzery et al., 1999; Soliva et al., 2001), some bined plastid dataset (matK, rbcL) was tested using the incongru- conclusions and results were weakly supported or even without ence length difference (ILD) (Farris et al., 1995), implemented in statistical support, and many taxonomic problems remain unre- PAUP v4.0b10 (Swofford, 2002). Following Cunningham (1997), solved. Despite the high diversity of Orchidinae and Habenariinae no cases of strongly supported incongruence were detected in Eastern Asia, many taxa, especially those from monotypic/oligo- (P = 0.17), therefore, we combined ITS data and the plastid dataset typic genera, were not represented in previous molecular studies. (matK, rbcL) in SequenceMatrix v1.7.8 (Vaidya et al., 2011) to per- Generic delimitation and systematic position of many genera in form further phylogenetic analyses. Orchidinae and Habenariinae from Asia, such as Aceratorchis, Amito- The phylogenetic analyses for each matrix were performed stigma, Hemipilia, Hemipiliopsis, Neolindleya, Neottianthe, Platanther- using the maximum parsimony (MP) in PAUP v4.0b10 (Swofford, a, Ponerorchis, Smithorchis, and Tsaiorchis, are not or little known, 2002) and Bayesian inference (BI) in MrBayes v3.1.2 (Ronquist and as a result many taxonomical suggestions and proposals and Huelsenbeck, 2003) on CIPRES Science Gateway Web server remain to be tested (Bateman et al., 2003, 2009; Chen et al., 2009; (Old MrBayes on XSEDE 3.1.2) (Miller et al., 2010). Pridgeon et al., 2001). For a better understanding of the generic For the MP analyses, heuristic searches were conducted with delimitation within Orchidinae and of the interrelationships among 1000 replicates of random addition, one tree held at each step dur- major clades within Orchidinae, it is desirable to base the analyses ing the stepwise addition, tree-bisection–reconnection (TBR) on multiple DNA markers and a denser sampling across many sys- branch swapping, MulTrees in effect, and steepest descent off. All tematically difficult genera and their allies from Asia. of the characters were unordered and equally weighted, and the In the present study, phylogenetic relationships were inferred gaps were coded as missing data. To evaluate the node support, using three DNA markers (plastid matK, rbcL and nuclear ITS bootstrap analyses (Felsenstein, 1988) were performed using sequence), with 153 samples representing 146 species of Orchidi- 1000 replicates, with 10 random taxon additions and heuristic nae and selected Habenariinae, such as Androcorys, Habenaria, Her- search options. minium, and Peristylus, mainly from Asia, with the aims of (1) Prior to the Bayesian analysis, a model for sequence evolution increasing our understanding of the generic delimitation within for each matrix was determined by using ModelTest v3.7 (Posada Orchidinae and Habenariinae; (2) reconstructing the phylogenetic and Crandall, 1998) under the Akaike information criterion. For interrelationships within Orchidinae. the BI analyses, two separate four Markov chain Monte Carlo (MCMC) analyses were run, with 10,000,000 generations and sam- pling every 1000 generation. Majority rule (>50%) consensus trees 2. Materials and methods were constructed after removing the ‘‘burn-in period’’ samples (the first 25% of the sampled trees). 2.1. Taxon sampling

There are about 32 genera of Orchideae distributed in Asia, out 3. Results of which about ten genera are endemic (Chen et al., 2009; Dressler, 1993; Jin et al., 2012; Pedersen et al., 2002; Pridgeon et al., 2001). 3.1. Sequences and alignment In order to represent the taxonomic diversity of Orchideae in Asia, 153 accessions of 145 species in 31 genera, including 27 genera In this study, 215 new sequences were obtained. Sequence and 103 species from Asia (about 84% of Asian genera and 25% of lengths were as follows: 825 bp for ITS region, 1254 bp for Asian species), were included in this study. Additionally, several rbcL, 1870 bp for matK. The combined alignment of ITS and genera and some species from Africa, , and South America, plastid regions comprised 3949 bp, 24% of which were W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53 43

Table 1 The statistics from the analyses of the chloroplast and nuclear data sets from the parsimony analysis.

Information ITS matK rbcL Combined No. taxa 144 136 119 153 Aligned length 825 1870 1254 3949 No. variable characters 507(61%) 642(34%) 181(14%) 1330(34%) No. parsimony-informative characters 445(54%) 419(22%) 103(8%) 967(24%) Tree length (steps) 3052 1428 334 4868 Consistency index (CI) 0.33 0.58 0.62 0.42 Retention index (RI) 0.77 0.82 0.83 0.78 Model GTR + I + G GTR + I + G GTR + I + G GTR + I + G

parsimony-informative. Table 1 summarizes the properties of each (PP = 100, BS = 97). Clade IX includes a species from the oligotypic aligned data partition. genus Sirindhornia (Fig. 1). Clade X includes four species, two of Androcorys and two of Porolabium with strong support (PP = 100, BS = 100) (Fig. 2). Clade 3.2. Phylogenetic analyses XI consists of three species from Platanthera, i.e. Platanthera latila- bris group, with strong support (PP = 100, BS = 100) (Fig. 2). Clade The partition homogeneity test for plastid DNA + ITS shows XII includes one species from Herminium, H. lanceum. Clade XIII there were no strongly supported incongruent results in the data- (PP = 100, BS = 100) includes six species, two from Peristylus, and sets (P = 0.17), therefore, we combined the datasets for simulta- four from Herminium (Fig. 2). An unidentified species of Herminium neous analyses. is sister to clades X to XIII, with weak support for the sister group Phylogenetic relationships based on the ITS data had a better (Fig. 2). Clade XIV (PP = 100, BS = 100) includes two terminals from resolution than the two combined plastid DNA data (results not one species, Ponerorchis chrysea (Fig. 2). Clade XV includes nine shown here). Based on the combined ITS and plastid DNA data, species, eight from Asian Habenaria and one from , with our findings are consistent in the overall topology of the trees pro- strong support (PP = 100, BS = 99) (Fig. 2). Clade XVI includes seven duced with maximum parsimony (MP) and Bayesian inference (BI) species, six from Peristylus, and one from Platanthera, with substan- methods, except for a few of the collapsed nodes. Bootstrap values tial support (PP = 100, BS = 84) (Fig. 2). Clade XVII includes 24 spe- (BS) were often lower than the Posterior Probability (PP) from the cies of Habenaria from Africa, Asia and Southern America, and two Bayesian analysis. species of Botanea from Africa with some support (PP = 100, The BI topology from the combined dataset is chosen as the pri- BS = 59) (Fig. 2). Clade XVIII includes two monotypic genera from mary tree for discussion of phylogenetic relationships (Figs. 1 and Europe and Asia, Gennaria and Nujiangia, with strong support 2; the MP strict consensus tree is not shown). Our results indicate (PP = 100, BS = 100) (Fig. 2). that the tribe Orchideae can be divided into two well supported sister clades, Habenariinae (PP = 100, BS = 67) (Fig. 1) and Orchidi- nae (PP = 100, BS = 97) (Fig. 2). Within the Orchidinae, six of nine 4. Discussion major clades (Clade I to IX) are well supported along the backbone of the Orchidinae, and many interrelationships among them are 4.1. An overview of phylogenetics of Orchideae well-resolved (Fig. 1). Within the Habenariinae, nine major clades, Clade X to XVIII, can be recognized (Fig. 2). Our results show that With broader sampling and more DNA markers, our results con- many morphological delimited genera of the Asian Orchideae in firmed the earlier findings that Orchidinae (PP = 100, BS = 97) Pridgeon et al. (2001) and Chen et al. (2009) are not monophyletic, (Fig. 1) and Asian Habenariinae (PP = 100, BS = 67) (Fig. 2) are such as Hemipilia, Herminium, Platanthera, and Ponerorchis. two well resolved sister groups. However, our results indicated Clade I (Fig. 1) comprises 42 species (PP = 89, BS < 50), most of that the morphological distinctions, such as the types of stigma which belong to Platanthera s.l., the remainders are from genera lobes, between these two subtribes are problematic (see discussion Herminium, Peristylus, and Smithorchis. The interrelationships of each clade). Inda et al. (2012) indicated that Habenariinae is within Clade I are not well resolved. Clade II comprises two species paraphyletic, and several genera from Africa, such as Stenoglottis, of Galearis, two monotypic genera, Aceratorchis and Neolindleya, , Cynorkis, and Holothrix, were resolved as successive sister and one species of Aorchis with weak support (PP = 86) (Fig. 1). to Orchidinae + Asian Habenariinae. Batista et al. (2013) showed Clade III includes one species of Pseudorchis (Fig. 1). Clade IV con- that the Habenariinae clade (formed by Habenaria s.l. + Cynor- sists of four species from Dactylorhiza and with strong kis + Stenoglottis) is sister to the Orchidinae clade formed by support (PP = 100, BS = 85) (Fig. 1). Clade V includes three species (Orchis + Platanthera). Given the complex of Orchideae, of Orchis s.s. with robust support (PP = 100, BS = 99) (Fig. 1). Clade it would be premature to make any firm conclusion about these VI (PP = 100, BS = 93) consists of two species of Ophrys and two two subtribes. More importantly, our results showed that many species of Serapias with strong support (PP = 100, BS = 99) (Fig. 1). previous findings of interrelationships within Orchideae based on Clade VII (Fig. 1) includes about 15 species from Amitostigma, molecular phylogenetics were strongly supported, and many Neottianthe, and Ponerorchis, and is subdivided into three well sup- unknown or overlooked phylogenetic relationships have been ported subclades: one subclade contains five species from Amito- detected. stigma and Ponerorchis; another subclade includes four species Asian Orchidinae is subdivided into two superclades (Fig. 1). from Amitostigma and Neottianthe, including types of these two Superclade A includes Dactylorhiza, Galearis, Gymnadenia, Orchis genera; and the third subclade includes four to six species of Ami- s.s., and Platanthera s.l. (PP = 87, BS = 58). Superclade B includes tostigma. Clade VIII (Fig. 1) comprises nine species, six from Hemip- Amitostigma, Hemipilia, Neottianthe and Sirindhornia (PP = 100). ilia, two from two monotypic genera, Hemipiliopsis and Tsaiorchis, Clade VI, formed by Ophrys and Serapias, is suggested as a sister and one from Ponerorchis, with weak support (PP = 81, BS = 56). to Superclade A with strong support, and Sirindhornia is resolved Clade VIII can be divided into two subclades, one includes Tsaior- as sister to the rest of Superclade B with weak support (PP = 100) chis, and the other includes the remainder with strong support (Fig. 2). 44 W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53

Fig. 1. Phylogenetic tree obtained by Bayesian analysis of the combination of ITS and plastid regions, showing the detailed relationships of subtribe Orchidinae. Numbers above the branches indicate posterior probabilities (PP) and bootstrap percentages (BS). ‘‘–’’ indicates node is not supported in the analysis. ‘‘Ã’’ indicates node is with support value 100%. W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53 45

Fig. 2. Phylogenetic tree obtained by Bayesian analysis of the combination of ITS and plastid regions, showing the detailed relationships of subtribe Habenariinae. Numbers above the branches indicate posterior probabilities (PP) and bootstrap percentages (BS). ‘‘–’’ indicates node is not supported in the analysis. ‘‘Ã’’ indicates node is with support value 100%.

Asian Habenariinae is subdivided into three moderately to genera from Europe and Asia, Gennaria and Nujiangia, mainly strongly supported superclades (Fig. 2). Superclade C (PP = 98), occurs in tropical regions with a few species extending into sub- consisting of Androcorys, Porolabium, Herminium, Ponerorchis chry- tropical mountains. The clade formed by Gennaria and Nujiangia sea, Platanthera latilabris group, and some species of Habenaria, is resolved as sister to the remainder in Superclade E. occur mostly in montane to alpine regions in Asia. Superclade D As Orchidinae is well-represented in Europe, the generic delim- (PP = 100, BS = 84), comprising Peristylus, occur mainly in tropical itation and classification of many genera around the Mediterra- and subtropical Asia with a few species extending into alpine nean have been thoroughly studied, for example, Orchis and its regions. Superclade E (PP = 100), including Habenaria from Asia, alliance by Aceto et al. (1999), Ophrys by Soliva et al. (2001), Sera- Africa and the Neotropics, African Bonatea, and two monotypic pias by Bellusci et al. (2008), Dactylorhiza by Devos et al. (2006) and 46 W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53

Box et al. (2008). Recently, molecular phylogenetics of Habenaria comparison established that these species have oblong to fusiform from Neotropics has been investigated based on comprehensive rootstock (which are near horizontal in S. calceoliformis), entire and sampling from Neotropics (Batista et al., 2013). Hence, in our anal- spurred lip, naked viscidium, and a relative obvious anther connec- yses, sampling of genera or species from the Africa, Europe, and tive, which supports the transfer of these species into Platanthera. Neotropics is mainly used as placeholders for these taxa. Their Dozens of Platanthera species from the , such as P. phylogeny will not be discussed here because our focus is on phy- bakeriana, P. leptocaulon, P. roseotincta, and others, have two pro- logenetic analyses on Asian taxa. jecting stigmatic lobes (stalked stigma lobes) and an entire lip (Fig. 3b). Duthie (1906), Hooker (1890), King and Pantling (1896, 4.2. Phylogeny and generic delimitation 1898), and Tuyama (1966, 1971, 1975) treated these species as Habenaria, while Kraenzlin (1901), Lindley (1835), Lang (1998, 4.2.1. Platanthera (Clade I, and XVI) 1999), and Soó (1929) placed them in Platanthera. Lang (1998) Platanthera consists of about 100–150 species and is among the even proposed a subgenus, subgen. Stigmatosae, to contain these larger genera in Orchideae, however, generic delimitation of Pla- species. Our results indicate that stalked stigma lobes have evolved tanthera is unclear. Luer (1975) suggested that several previous at least twice in Platanthera, one in P. yadongensis (Jin et al., 2013), recognized genera, such as Blephariglottis, Lacera, Limnorchis, and the other in P. exelliana and its relative species. This renders the Tulotis, should be treated as sections within Platanthera, and morphological distinction between Habenariinae and Orchidinae, Dressler (1993) suggested that Platanthera is close to Dactylorh- which is mainly based on the morphology of the stigma, iza–Galearis alliance instead of Habenaria. These suggestions have problematic. been supported by recent results of molecular studies (Bateman et al., 2003, 2009; Hapeman and Inoue, 1997). Bateman et al. 4.2.2. Aceratorchis, Aorchis, Galearis, and Neolindleya (Clade II) (2009) broadened Platanthera to include more genera, such as Both Aceratorchis (Fig. 3c) and Neolindleya are little-known Diphylax, , and Tsaiorchis. Based on seed micromorphology, monotypic genera. Bateman et al. (2009) considered Aceratorchis however, Gamarra et al. (2008) stated that Limnorchis should be to be a peloric form of Galearis. Neolindleya was previously consid- separated from Platanthera as a distinct genus. ered as Gymnadenia (Bateman et al., 2003), then it was separated as Our analyses do not only support most of these conclusions, but a distinctive genus and sister to Galearis (Bateman et al., 2009; also indicate that Platanthera s.l. is polyphyletic and subdivided Efimov et al., 2009). Pridgeon et al. (2001) considered Aorchis as into three distant related groups that belong to two subtribes, Pla- congeneric with Galearis. In our phylogenetic analyses, Acerator- tanthera clade (Clade I, Orchidinae), Platanthera latilabris group chis, Aorchis, Galearis and Neolindleya form a weak supported clade (Clade XI, Habenariinae), and Platanthera biermanniana (included (PP = 86) (Fig. 1), while Aceratorchis and Neolindleya were resolved in Clade XVI, Habenariinae). as successive sister to Galearis and Aorchis. These four genera typ- The Platanthera clade (Clade I, Orchidinae) is not strongly sup- ically share a stolon-like rhizome, anther loculi parallel with dis- ported and the interrelationships within the genus are not well tinct rostellum between them, concave stigma, and viscidium resolved. However, the Platanthera clade is well characterized by enclosed in bursicle. Considering the weak support of the clade, morphological characters, such as fleshy rootstock (no tuber), we tentatively proposed to subsume all other three genera into basal to cauline and grading into foliaceous bracts, sheath- Galearis. However, this needs to be tested by further studies. ing at base, lip spurred, short and truncate, anther broad, loculi more or less separated by connective, stigmas two and more or less divergent at the base of the entrance of spur. 4.2.3. Amitostigma, Neottianthe, and Ponerorchis (Clade VII) Platanthera latilabris group, consisting of three closely related The generic borders between Ponerorchis and Amitostigma are species, P. clavigera, P. edgeworthii, P. latilabris (Fig. 3a), form an unclear due to the difficulty of distinguishing the supposed diag- unambiguous clade deeply nested within Habenariinae. Species nostic characters, such as appendages of column and the bursicle, of this clade are characterized by ovoid to globose tubers, densely and several species have been transferred back and forth between flowered inflorescence, linear and fleshy lip with a callus at base, these genera. However, it is nevertheless somewhat unexpected stigma lobes more or less stalked, extending at base of lip that Amitostigma, Neottianthe and Ponerorchis are nested together (Fig. 3a). Hooker (1890) treated these species as members of Hab- and form a moderately supported group (Fig. 1). This clade mainly enaria, Szlachetko and Kras (2006) transferred these species into occurs in Eastern Asia, with one or two species extending into the the Neotropical genus, Habenella. Our results indicated that Platan- northern temperate zone. Members of this clade share an ovoid thera latilabris group and Habenella (included in Clade XVII) are two tuber, slender with 1–3 slightly fleshy leaves, flowers quite distantly related groups. Instead, Platanthera latilabris group secund along rachis, dorsal and forming a hood, the are resolved as sister to Androcorys plus Porolabium with weak sup- base of lip more or less connecting the column, column short, lat- port (PP = 92, BS = 56). eral appendages obvious, parallel anther cells, stigma lobes situ- Platanthera biermanniana resembles species of Peristylus in gross ated and confluent under rostellum. The subclade formed by morphology. Our analyses show that P. biermanniana is deeply three species of Neottianthe (including the type, Neottianthe cucul- nested within Peristylus with robust support, which is congruent lata) and Amitostigma gracile (type of Amitostigma) is strongly sup- with morphological characters, such as cylindric-ovoid tuber, 3- ported and well characterized by morphological characters, such as lobed lip, short spur, anther with very narrow connective. leaves basal, elliptic to ovate, petals and three forming a Several species of Herminium (H. carnosilabre and H. angustilab- hood, lip papillose. Ponerorchis is said to differ from Amitostigma re), Peristylus (P. nematocaulon), and the monotypic Smithorchis (S. and Neottianthe by having a viscidium enveloped in a bursicle calceoliformis) are deeply nested within Platanthera s.l. These spe- (see Pridgeon et al., 2001). Our results indicate that a bursicle cies are restricted in alpine region with elevation ranging from has independently evolved four times in Orchideae (Clade II, VII, 3500 to 4300 m. The taxonomy has been complicated by these spe- VIII, XIV). Moreover, our fieldwork established that the presence cies having minute flower (the smallest in Orchidinae) and a of a bursicle is difficult to determine even in living specimens, greatly reduced column which is occupied almost entirely by the and some species of Amitostigma, such as A. monanthum (Fig. 3g), anther. Most genera of Asian Orchideae were distinguished on A. yuanum (Fig. 3h), do have bursicles. It is almost impossible to the basis of floral characters, especially of the column (Lang, assign some species, such as Amitostigma yuanum (Fig. 3h), Pone- 1999; Pridgeon et al., 2001). Our observations and morphological rorchis nana (Fig. 3i), and A. farreri,toAmitostigma or Ponerorchis, W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53 47

Fig. 3. Diversity of Orchideae from Asia. (a) flowers of Platanthera latilabris, front view; (b) flowers of Platanthera roseotincta, front view; (c) Plants of Aceratorchis tschiliensis, showing the stolon-like rhizome; (d) plant of Hemipilia purpureopunctata; (e) flowers of Hemipilia purpureopunctata; (f) plants of Tsaiorchis neottianthoides; (g) flower of Ponerorchis monantha; (h) flowers of Amitostigma yuana; (i) flower of Ponerorchis nana; (j) flowers of Sirindhornia pulchella; (k) Hsenhsua chrysea. (Photos taken by Xiaohua Jin.)

based on morphological characters. On these grounds, we propose cylindrical to ovoid-globose tubers, solitary, basal, prostrate to unite Amitostigma and Neottianthe into Ponerorchis. fleshy, more or less distinctly spotted, inflorescence with purple spots (Fig. 3d, e), stigma lobes confluent. In contrast to these, Tsa- iorchis (Fig. 3f) is characterized by hairy and horizontally extending 4.2.4. Hemipilia, Hemipiliopsis,and Tsaiorchis (Clade VIII) rhizomes, one to two chartaceous and green leaves, inflorescence Both Hemipiliopsis (Fig. 3d, e) and Tsaiorchis (Fig. 3f) are little- green, two lateral appendages longer than anther, rostellum elon- known and narrowly distributed monotypic genera. Garay and gate, stigma lobes separate, linear and extending at the base of lip. Kettredge (1985) placed Tsaiorchis neottianthoides (= Habenaria Although Luo and Chen (2003) established Hemipiliopsis on the keiskeoides)inAmitostigma, Pridgeon et al. (2001) treated Tsaiorchis basis of its less developed rostellum and the shape of spur, Hemip- as Diphylax. Bateman et al. (2009) positioned Tsaiorchis neottian- iliopsis is characterized by the well-developed and erect rostellum thoides in Platanthera. Bateman et al. (2003) stated that the mono- as long as half of anther locus (Fig. 3e). The sister taxon of Hemip- typic Hemipiliopsis is closely related to Ponerorchis brevicalcarata, iliopsis, Ponerorchis brevicalcarata, fits well in Hemipilia. In addition, and suggested these two species should be assigned to Hemipilia. Hemipiliopsis is sympatric with Hemipilia. Based on these findings, In our analyses, Hemipiliopsis is nested within Hemipilia with we propose to broaden Hemipilia to include Hemipiliopsis, and robust support (PP = 100, BS = 97), while Tsaiorchis is resolved as maintain Tsaiorchis as a distinctive genus. sister to Hemipiliopsis plus Hemipilia with weak support. These results agree with morphological characters. On the one hand, members of this clade (Clade VIII) typically share a distinctly elon- 4.2.5. Sirindhornia (Clade IX) gate and erect rostellum. On the other hand, Tsaiorchis differs Sirindhornia is a recently established and morphologically dis- greatly from Hemipiliopsis and Hemipilia both in vegetative and flo- tinctive genus. There are three species and they are mainly distrib- ral characters. Hemipiliopsis and Hemipilia are characterized by uted in border regions in , , and China. However, 48 W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53 the systematic position of Sirindhornia has been disputed. Pedersen 4.2.7. Ponerorchis chrysea (Habenaria chrysea, Orchis chrysea, Clade et al. (2002) stated that Sirindhornia is close to Hemipilia, Poneror- XIV) chis and reminiscent of Orchis. Instead, Chen et al. (2009) included Ponerorchis chrysea (Fig. 3h) may have the largest flowers in Sirindhornia monophylla (type of Sirindhornia)inPonerorchis. Orchidinae, however, its systematic position is poorly understood. In our phylogenetic analyses, Sirindhornia is unambiguously Schlechter (1924) treated Habenaria chrysea as a member of Orchis resolved as sister to the remaining genera, including Hemipilia, Tsa- s.l., Soó (1966) transferred it to Ponerorchis, Hunt (1971) included it iorchis, in Superclade B, which is supported by morphological char- in Chusua. In our analyses, Ponerorchis chrysea is resolved as sister acters of Sirindhornia. Sirindhornia is characterized by the elongate to the entity comprised of Clades X–XIII, with moderate support tuber, solitary and fleshy leaf, leaf convolute and not spreading, (PP = 100, BS = 69) (Fig. 2). Most members of these clades have stem and ovary papillose–pubescent, ciliate bract, lip spurred, basal and green leaf (or leaves), yellow, yellowish or green flowers, stigma lobes concave, and viscidium enveloped in a bursicle which strikingly differ from Ponerorchis. Our detailed morphologi- (Fig. 3j). In addition, species of Sirindhornia typically grow in lime- cal examination indicate that Ponerochis chrysea is a quite out of stone regions, and flower from April to June, which is one to two place in Ponerorchis on account of several morphological charac- months earlier than other Orchidinae from Eastern Asia. Based on ters, such as leaves basal, sheaths long and clasping peduncle, floral these findings, we recognize Sirindhornia as a distinct genus. bracts similar to leaf and enclosing the spur, pedicel longer than ovary, anther connective elongate and drawn out, three slightly 4.2.6. Androcorys, Herminium, and Porolabium (Clade X, XI, XII, and convex stigma lobes confluent under rostellum. Some of these XIII) characters, such as pedicel longer than ovary, connective elongate Morphologically, it is difficult to determine the systematic posi- and drawn out, are even unique in Orchideae in Eastern Asia. On tions of Androcorys, Herminium, and Porolabium in Orchideae due to these grounds, we propose to establish a new genus, Hsenhsua,to the relative small flowers, short column occupied almost by anther, accommodate this taxon. greatly reduced stigma and rostellum (Pridgeon et al., 2001). Hsenhsua X.H. Jin, Schuit. et W.T. Jin, gen. nov. Figs. 3k and 4. Pridgeon et al. (2001) proposed to broaden Androcorys to include Type species: Hsenhsua chrysea (W.W. Sm.) X.H. Jin, Schuit., W.T. some members of Herminium with spurred lip; Bateman et al. Jin et L.Q. Huang. (2003) suggested that some temperate members of Peristylus Included taxa: Hsenhsua chrysea (W.W. Sm.) X.H. Jin, Schuit.,W.T. should be transferred to Herminium. In our analyses, Androcorys, Jin et L.Q. Huang, comb. nov. (Basionym Habenaria chrysea W.W. Herminium, and Porolabium are deeply embedded within Habe- Sm., Notes from the Royal Botanic Garden, Edinburgh 13(63–64): nariinae, and Herminium is not monophyletic. Some misplaced 204–205. 1921.) members have to be excluded and assigned to other genera, while Diagnosis: Tuber subglobose. Stem 2 leaved. Leaves green, basal. some species currently residing in other genera need to be trans- Inflorescence 1-flowered. Floral bracts foliaceous, sheathing, ferred to it. enclosing the spur. Pedicel longer than ovary; anther connective Herminium lanceum is resolved as immediate sister to groups elongate and drawn out; rostellum protruding with two arms; formed by clade X plus XI with moderate support (PP = 99, three slightly convex stigma lobes confluent under rostellum. BS < 50). Our observations indicate that H. lanceum differs from Morphological evidence: Morphologically, Hsenhsua is similar to other species of Herminium by having parallel anther loculi, one Ponerorchis s.l. by sharing subglobose tuber, three-lobed lip with pulvinate stigma swelling at the base of the column, and transverse a distinct spur, rostellum protruding with two arms, but differs rostellum, while most Herminium (including the type, H. monor- from the latter by having foliaceous bracts sheathing and enclosing chis) have more or less divergent anther loculi, two stalked stigma the spur, pedicel longer than ovary, anther connective elongate and lobes extending along the base of column, and a cylindrical drawn out. Hsenhsua can be readily distinguished from Herminium rostellum. s.l. by its foliaceous bracts, elongate anther connective, three A little known species of Herminium is resolved as immediate slightly convex stigma lobes confluent under rostellum. sister to clade X, XI, XII and XIII with weak support. Our morpho- Molecular evidence: In the current study, the combined analyses logical studies show that this entity is similar to Herminium and of the datasets of three markers (two chloroplast, one nuclear) shares many morphological characters, such as ovoid tubers, visci- shows moderate support for a sister group relationships of Hsenh- dium involute and hornlike, two stalked stigma lobes extending at sua with Herminium s.l. (including Androcorys, Herminium, Porolabi- the base of column. This entity differs from Herminium by having um and other species) (PP = 100, BS = 69). parallel anther loculi with a long (as long as half of anther locus) Habit and Distribution: Growing in humid alpine grassland and and slender appendage between them. More molecular data and shrub between 3600 and 4200 m in the Eastern Himalayas, includ- a better understanding of the morphological characters are needed ing China (NW Yunnan, SE to S Xizang), and Bhutan. to elucidate the phylogeny of this little known entity. Etymology: Hsenhsua is named in honor of Prof. Hsen-Hsu Hu, Tang and Wang (1940) separated Porolabium from Herminium the renowned Chinese scientist who was a pioneer and founder on the basis of having a lip with two pores. Pridgeon et al. (2001) of modern botany in China. stated that the rostellum of Porolabium is identical to Herminium; Pearce and Cribb (2002) suggested that Androcorys ophioglossoides 4.2.8. Habenaria and its alliance (Clade XV, XVII, and XVIII) resembles Porolabium biporosum in morphological characters. Our Habenaria is one of the largest genera of orchid family and results indicate that Porolabium is nested within Androcorys with widespread across the tropical and subtropical regions of the world strong support, and these two genera are sympatric. We found that (Pridgeon et al., 2001). There is much debate about the generic Androcorys and Porolabium both have a pulvinate stigma at the delimitation and infrageneric systems (see Batista et al., 2013; base of the column. There are several divergent taxonomic options Pridgeon et al., 2001). Bateman et al. (2003) stated that Habenaria to classify Herminium and its alliance into one or more monophy- is polyphyletic and needs to be dismantled into many monophy- letic groups. One is to broaden Herminium to include Androcorys letic genera. On the other hand, Batista et al. (2013) indicated that and its alliance (Clade X, XI, XII, XIII). Another is to recognize each Neotropical Habenaria is monophyletic and closely related to some clade in this alliance (Clade X, XI, XII, XIII) as a distinctive genus. African congeners, and suggested that a revision of the infrageneric However, with the currently available sampling and weak sup- system rather than an extensive generic fragmentation is most ported interrelationships among these clades, it would be prema- appropriate. In our phylogenetic analyses with 36 sampled species ture to make any firm decision. from Asia, Africa and the Neotropics, Habenaria is resolved into two W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53 49

Fig. 4. Plant of Hsenhsua chrysea. (a) habit of Hsenhsua chrysea; (b) front view of flower; (c) bract; (d) dorsal sepal, lateral sepal, and lip; (e) lateral view of flower (bract removed); (f) lateral view of longitudinal section of column and upper part of ovary. (Drawn by Yunxi Zhu.)

unambiguously supported groups: Clade XV and clade XVII (Fig. 2). genera share several morphological characters, such as two alter- Clade XV includes nine Asian species mainly distributed in sub- nate leaves, densely flowered inflorescence, 3-lobed lip with a tropical region, such as H. dentata, H. rhodocheila and Pecteilis short spur, auricles longer than anther, and a convex stigma (Jin gigantea, and is sister to the predominantly alpine Herminium s.l. et al., 2012). clade (Clades X–XIII) and Ponerorchis chrysea. Clade XVII, mainly Although with limited sampling of Habenaria and its alliance including African and South American Habenaria, and Bonatea,is from Africa and Neotropics, our phylogenetic analyses indicate that resolved as sister to two monotypic genera, Nujiangia and Gennaria. the taxonomy of Habenaria is even more complicated than previ- It is interesting to note that five Asian Habenaria are nested within ously thought. It is clear that as currently defined Habenaria is pat- Clade XVII. Asian tropical Habenaria stenopetala is resolved as sister ently polyphyletic. There have been several highly divergent to the African-Neotropical H. heyneanna plus H. foliosa with strong taxonomic proposals to delimit Habenaria to be monophyletic. support (PP = 100, BS = 100). Asian alpine H. intermedia is resolved One is the generic fragmentation of Habenaria into many genera, as sister to African montane H. praestans with strong support such as Bonatea, Habenella, and many others; another is to broaden (PP = 100, BS = 100); both belong to Habenaria sect. Mutipartitae. Habenaria to include nearly all genera, such as Androcorys, Hermin- Two monotypic and morphologically isolated genera in Habe- ium, and Peristylus, in Habenariinae. The third is to redefine clade nariinae, Gennaria and Nujiangia, were uncovered as sister to Afri- XVII as Habenaria s.s., with typification of Habenaria macroceratitis, can and American Habenaria with strong support. These two while clade XV is segregated as a distinct genus. With the currently 50 W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53 available sampling it is premature to make any firm decision. as Herminium and its alliance (including Clade X, XI, XII, XIII, and Future work with more comprehensive sampling and more molec- XIV, most endemic in Pan-Himalayas), are deeply nested within ular data will undoubtedly provide new insights into the taxonomy Habenariinae (Fig. 2). of Habenaria. Taken together, it seems that the tropical ancestral group of Orchideae radiated in the Mediterranean region in the last 15 mil- 4.2.9. Peristylus (Clade XVI) lion years and in the Pan-Himalayas in the last 8 million years Peristylus is a widespread genus in Asia with about 70 species (Geographical history of Pan-Himalayas see Shi et al., 1998; Bioge- (Pridgeon et al., 2001), however, it is almost neglected in previous ography of Pan-Himalayas see Sun, 2002a, 2002b). studies of Orchideae. There is some debate on its distinction from Habenaria. Seidenfaden (1977) proposed to define Peristylus on the 5. Main conclusions basis of a set of characters, such as size of floral parts, the position of stigma lobes and caudicles. Pridgeon et al. (2001) stated that Based on three DNA markers (plastid matK, rbcL, and nuclear Peristylus typically have pulvinate stigmatic swellings at the base ITS), morphological characters and comprehensive sampling, our of the column and adnate to the base of the lip. Comber (2001) also current study greatly advances our understanding of the phylog- suggested a set of morphological characters to define Peristylus.In eny of Orchidinae and Asian Habenariinae. Orchidinae are subdi- our phylogenetic analyses, Peristylus is resolved as an unambigu- vided into two sister groups: one is Superclade A + Clade VI ously supported clade within Habenariinae, and immediate sister (including Gymnadenia, Ophrys, Orchis and its alliance, and Platan- to the group formed by Clades X to XV (Fig. 2). Some misplaced thera and its alliance) with diversity centers in the Mediterranean species of Peristylus, such as P. nematocaulon and P. coeloceras, region and Pan-Himalayas, the other is Superclade B (Hemipilia, are nested within Platanthera or Herminium, whereas at least one Ponerorchis, Sirindhornia, and Tsaiorchis) and is mainly distributed misplaced Platanthera with trilobed lip and ovoid tubers, namely in Eastern Asia. Habenaria is subdivided into two distant related P. biermanniana, is nested within Peristylus. Geographically, Peristy- groups: one group mainly distributed in tropical and subtropical lus is mainly distributed in Asia in tropical to subtropical regions, Asia, the other group (including type of Habenaria) is widespread while Platanthera and Herminium mainly occur in alpine regions. in both Old and New Tropics. Elements of alpine regions are deeply In our phylogenetic analyses, the currently delimited Peristylus nested within each tropical/subtropical group. Many genera, such s.s. is strongly supported by molecular evidence and is more uni- as Herminium, Platanthera, Peristylus and Ponerorchis, are not form in morphological characters, characterized by having ovoid monophyletic. Many previously undetected phylogenetic relation- to globose tubers, flowers small, dorsal sepal and petals forming ships, such as clades formed by Ponerorchis chrysea, the Platanthera a hood, lip trilobed and spurred, spur shorter than ovary, and pul- latilabris group, Sirindhornia, and Tsaiorchis, are well supported by vinate stigmas adnate to the base of lip. Therefore, we suggest to both molecular and morphological evidence. We propose to com- make some intergeneric transfers to retain Peristylus as a mono- bine Hemipiliopsis with Hemipilia, to broaden Ponerorchis to include phyletic genus which mainly includes members from tropical Amitostigma and Neottianthe, to combine Aorchis, Aceratorchis, and and subtropical regions. Neolindleya with Galearis, and to establish a new genus to accom- modate Ponerorchis chrysea. Tsaiorchis and Sirindhornia are recog- 4.3. Biogeography nized as two distinctive genera. A nomenclatural revision is provided in the Appendix A. At the same time, the relationships Orchidinae are mostly distributed in northern temperate or of many groups, such as Herminium and its alliance (including And- alpine regions, however, recent studies indicate that the wide- rocorys, Herminium, Platanthera latilabris group, and Porolabium) spread tropical genus is sister to the remaining and Galearis and its alliance (including Aceratorchis, Aorchis, Gale- Orchidinae (Bateman et al., 2003; Inda et al., 2010). There are aris, and Neolindleya) remain unresolved. Further work based on two centers of biodiversity of Orchidinae. One is the Mediterranean broader sampling and more markers is needed. Region, the other is the Pan-Himalayas (including Himalayas and the Hengduan Mountains) (Pridgeon et al., 2001; Lang, 1998). Acknowledgements Inda et al. (2012) stated that Orchidinae have diversified in the Mediterranean Region in the last 15 Million years. Many taxa of Funds were provided by grants from the National Natural Sci- Superclade A, such as , Ophrys, Pseudorchis and Ser- ence Foundation of China (Grant Nos. 31107176, 31311120061) apias, are predominantly Mediterranean (Pridgeon et al., 2001). On and the Chinese Special Fund for Medicine Research in the Public the other hand, many genera in Superclade A, such as Platanthera Interest (201407003). and its alliance, have their biodiversity centers in Pan-Himalayas (Chen et al., 2009; Inda et al., 2012; King and Pantling, 1898; Lang, 1998; Pridgeon et al., 2001). Bateman et al. (2003) and Inda Appendix A et al. (2012) showed that Platanthera and its alliance are deeply nested within the Mediterranean Region group. Our results indi- A.1. Nomenclatural revision cate that Clade III, IV, V, and VI (the Mediterranean Region group) are resolved as successive sister to Platanthera and its alliance. 1. Galearis Raf. Members of Superclade B, including Hemipilia, Ponerochis, Sirind- (1) Galearis camtschatica (Cham.) X.H. Jin, Schuit. et W.T. Jin, hornia and Tsaiorchis, are mostly restricted to mountain regions comb. nov. in Eastern Asia, especially the Pan-Himalayas. Tropical/subtropical Basionym Orchis camtschatica Cham., Linnaea 3: 27 (1828). genera, such as Sirindhornia and Tsaiorchis, are resolved as succes- 2. Hemipilia Lindl. sive sister to alpine taxa (Fig. 1). Synonym. In contrast to Orchidinae, Habenariinae are widespread in trop- Hemipiliopsis Y.B. Luo et S.C. Chen, syn. nov. ical/subtropical regions around the world and only a few species (2) Hemipilia purpureopunctata (K.Y. Lang) X.H. Jin, Schuit. et occur in alpine regions. Recent studies indicate that tropical Afri- W.T. Jin, comb. nov. can genera, such as Cynorkis and, Stenoglottis, are basal groups in Basionym Habenaria purpureopunctata K.Y. Lang, Acta Phytotax. the subtribe (Bateman et al., 2003; Batista et al., 2013; Inda Sin. 16(4): 127 (1978). et al., 2010, 2012). Our results indicate that alpine groups, such 3. Peristylus Blume. W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53 51

(3) Peristylus biermannianus (King et Pantl.) X.H. Jin, Schuit. & (18) Ponerorchis faberi (Rolfe) X.H. Jin, Schuit. et W.T. Jin, W.T. Jin, comb. nov. comb. nov. Basionym Habenaria biermanniana King et Pantl., J. Asiat. Soc. Basionym Habenaria faberi Rolfe, Kew Bull. 1896: 201 (1896). Bengal. 64: 343 (1895). (19) Ponerorchis farreri (Schltr.) X.H. Jin, Schuit. et W.T. Jin, 4. Platanthera Rich. comb. nov. Synonym. Basionym Amitostigma farreri Schltr., Repert. Spec. Nov. Regni Smithorchis Tang et F.T. Wang, syn. nov. Veg. 20: 378 (1924). (4) Platanthera angustilabris (King et Pantl.) X.H. Jin, Schuit. et (20) Ponerorchis gonggashanica (K.Y. Lang) X.H. Jin, Schuit. et W.T. Jin, comb. nov. W.T. Jin, comb. nov. Basionym Herminium angustilabre King et Pantl. J. Asiat. Soc. Basionym Amitostigma gonggashanicum K.Y. Lang, Acta Phyto- Bengal, 65(2): 131. 1895 (1896). tax. Sin. 22(4): 315 (1984). (5) Platanthera calceoliformis (W.W. Sm.) X.H. Jin, Schuit. et (21) Ponerorchis gracilis (Blume) X.H. Jin, Schuit. et W.T. Jin, W.T. Jin, comb.nov. comb. nov. Basionym Herminium calceoliforme W.W. Sm., Notes Roy. Bot. Basionym gracile Blume, Mus. Bot. Lugd.-Bat. 2: 190 Gard. Edinburgh. 13: 211 (1921). (1856). (6) Platanthera carnosilabris (Tang et F.T. Wang) X.H. Jin, (22) Ponerorchis keiskei (Finet) X.H. Jin, Schuit. et W.T. Jin, Schuit. et W.T. Jin, comb. nov. comb. nov. Basionym Herminium carnosilabre Tang et F.T. Wang, Bull. Fan Basionym Gymnadenia gracilis var. keiskei Finet, Bull. Soc. Bot. Mem. Inst. Biol. Bot. 10: 32 (1940). France 47: 280 (1900). 5. Ponerorchis Rchb. f. (23) Ponerorchis kinoshitae (Makino) X.H. Jin, Schuit. et W.T. Synonyms. Jin, comb. nov. Amitostigma Schltr., syn. nov. Basionym Gymnadenia kinoshitae Makino, Bot. Mag. (Tokyo) 23: Neottianthe (Rchb.) Schltr., syn. nov. 137 (1909). (7) Ponerorchis alpestris (Fukuy.) X.H. Jin, Schuit. et W.T. Jin, (24) Ponerorchis lepida (Rchb. f.) X.H. Jin, Schuit. et W.T. Jin, comb. nov. comb. nov. Basionym Amitostigma alpestre Fukuy., Bot. Mag. Tokyo 49: 664 Basionym Gymnadenia lepida Rchb. f., Otia Bot. Hamburg.: 51 (1935). (1878). (8) Ponerorchis amplexifolia (Tang et F.T. Wang) X.H. Jin, (25) Ponerorchis luteola (K.Y. Lang et S.C. Chen) X.H. Jin, Schuit. Schuit. et W.T. Jin, comb. nov.Basionym Amitostigma amplexifoli- et W.T. Jin, comb. nov. um Tang et F.T. Wang, Bull. Fan Mem. Inst. Biol. Bot. 7: 3 Basionym Neottianthe luteola K.Y. Lang et S.C. Chen, Acta Phyto- (1936). tax. Sin. 35(6): 545 (1996). (9) Ponerorchis basifoliata (Finet) X.H. Jin, Schuit. et W.T. Jin, (26) Ponerorchis monantha (Finet) X.H. Jin, Schuit. et W.T. Jin, comb. nov. comb. nov. Basionym Peristylus tetralobus f. basifoliatus Finet, Rev. Gén. Bot. Basionym Peristylus monanthus Finet, Rev. Gén. Bot. 13: 323 13: 525, pl. 13(C). (1901). (1901). (10) Ponerorchis bidupensis (Aver.) X.H. Jin, Schuit. et W.T. Jin, (27) Ponerorchis oblonga (K.Y. Lang) X.H. Jin, Schuit. et W.T. Jin, comb. nov. comb. nov. Basionym Hemipilia bidupensis Aver., Lindleyana 14: 222 (1999). Basionym Neottianthe oblonga K.Y. Lang, Acta Phytotax. Sin. (11) Ponerorchis bifoliata (Tang et F.T. Wang) X.H. Jin, Schuit. 35(6): 544 (1997). et W.T. Jin, comb. nov. (28) Ponerorchis ovata (K.Y. Lang) X.H. Jin, Schuit. et W.T. Jin, Basionym Amitostigma bifoliatum Tang et F.T. Wang, Bull. Fan comb. nov. Mem. Inst. Biol. Bot. 7: 127 (1936). Basionym Neottianthe ovata K.Y. Lang, Acta Phytotax. Sin. 35(6): (12) Ponerorchis capitata (Tang et F.T. Wang) X.H. Jin, Schuit. et 542 (1997). W.T. Jin, comb. nov. (29) Ponerochis papilionacea (Tang, F.T. Wang et K.Y. Lang) Basionym Amitostigma capitatum Tang et F.T. Wang, Bull. Fan X.H. Jin, Schuit. et W.T. Jin, comb. nov. Mem. Inst. Biol. Bot. 7: 4–5 (1936). Basionym Amitostigma papilionaceum Tang, F.T. Wang et K.Y. (13) Ponerorchis camptoceras (Rolfe) X.H. Jin, Schuit. et W.T. Lang, Acta Phytotax. Sin. 20(1): 83 (1982). Jin, comb. nov. (30) Ponerorchis parciflora (Finet) X.H. Jin, Schuit. et W.T. Jin, Basionym Habenaria camptoceras Rolfe, J. Linn. Soc., Bot., 29: comb. nov. 319 (1892). Basionym Peristylus tetralobus f. parciflorus Finet, Rev. Gén. Bot. (14) Ponerochis compacta (Schltr.) X.H. Jin, Schuit. et W.T. Jin, 13: 525 (1902) (as ‘parceflorus’). comb. nov. (31) Ponerorchis (Schltr.) X.H. Jin, Schuit. et W.T. Basionym Neottianthe compacta Schltr., Acta Horti Gothob. 1: Jin, comb. nov. 136 (1924). Basionym Amitostigma physoceras Schltr., Acta Horti Gothob. 1: (15) Ponerorchis cucullata (L.) X.H. Jin, Schuit. et W.T. Jin, 133 (1924). comb. nov. (32) Ponerorchis pinguicula (Rchb. f. et S. Moore) X.H. Jin, Basionym Orchis cucullata L., Sp. Pl.: 939 (1753). Schuit. et W.T. Jin, comb. nov. (16) Ponerorchis cucullata var. calcicola (W.W. Sm.) X.H. Jin, Basionym Gymnadenia pinguicula Rchb. f. et S. Moore., J. Bot. 16: Schuit. et W.T. Jin, comb. nov. 135 (1878). Basionym Gymnadenia calcicola W.W. Sm., Notes Roy. Bot. Gard. (33) Ponerochis simplex (Tang et F.T. Wang) X.H. Jin, Schuit. et Edinburgh. 8: 188 (1914). W.T. Jin, comb. nov. (17) Ponerorchis dolichocentra (Tang, F.T. Wang et K.Y. Lang) Basionym Amitostigma simplex Tang et F.T. Wang, Bull. Fan X.H. Jin, Schuit. et W.T. Jin, comb. nov. Mem. Inst. Biol. Bot. Ser. 10: 25 (1940). Basionym Amitostigma dolichocentrum Tang, F.T. Wang et K.Y. (34) Ponerorchis secundiflora (Hook. f.) X.H. Jin, Schuit. et W.T. Lang, Acta Phytotax. Sin. 20(1): 84 (1982). Jin, comb. nov. 52 W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53

Basionym Peristylus secundiflorus Kraenzl., Orchid. Gen. Sp. 1: Cunningham, C.W., 1997. Can three incongruence tests predict when data should be 518 (1898). combined? Mol. Biol. Evol. 14, 733–740. Devos, N., Raspé, O., Jacquemart, A., Tyteca, D., 2006. On the of (35) Ponerorchis tetraloba (Finet) X.H. Jin, Schuit. et W.T. Jin, Dactylorhiza Necker ex Nevski (Orchidaceae): is Coeloglossum viride (L.) comb. nov. Hartman a Dactylorhiza? Bot. J. Linn. Soc. 152, 261–269. Basionym Peristylus tetralobus Finet, Rev. Gén. Bot. 13: 524 Douzery, E.J.P., Pridgeon, A.M., Kores, P., Linder, H.P., Kurzweil, H., Chase, M.W., 1999. Molecular phylogenetics of Diseae (Orchidaceae): a contribution from (1901). nuclear ribosomal ITS sequences. Am. J. Bot. 86, 887–899. (36) Ponerorchis thailandica (Seidenf. et Thaithong) X.H. Jin, Dressler, R.L., 1981. The Orchids: Natural History and Classification. Harvard Schuit. et W.T. Jin, comb. nov. University Press, Cambridge. Dressler, R.L., 1993. Phylogeny and classification of the orchid family. Dioscorides Basionym Amitostigma thailandicum Seidenf. et Thaithong in G. Press, Portland. Seidenfaden, Contr. Orchid Fl. Thailand 13: 8 (1997). Duthie, J.F., 1906. The orchids of the North-Western Himalaya. Ann. Roy. Bot. Gard. (37) Ponerorchis tibetica (Schltr.) X.H. Jin, Schuit. et W.T. Jin, (Calcutta) 9, 85–211. Efimov, P.G., Lauri, R.K., Bateman, R.M., 2009. Neolindleya Kraenzl. (Orchidaceae), an comb. nov. enigmatic and largely overlooked autogamous genus from temperate East Asia. Basionym Amitostigma tibeticum Schltr., Repert. Spec. Nov. Reg- Kew. Bull. 64, 661–671. ni Veg. 20: 379 (1924). Farris, J.S., Kälersjö, M., Kluge, A.G., Bult, C., 1995. Constructing a significance test for (38) Ponerorchis trifurcata (Tang, F.T. Wang et K.Y. Lang) X.H. incongruence. Syst. Biol. 44, 570–572. Felsenstein, J., 1988. Phylogenies from molecular sequences: inference and Jin, Schuit. et W.T. Jin, comb. nov. reliability. Annu. Rev. Genet. 22, 521–565. Basionym Amitostigma trifurcatum Tang, F.T. Wang et K.Y. Lang, Gamarra, R., Galán, P., Herrera, I., Ortúnˇez, E., 2008. Seed micromophology supports Acta Phytotax. Sin. 20(1): 80, pl. 1(5–8) (1982). the splitting of Limnorchis from Platanthera (Orchidaceae). Nordic J. Bot. 26, 61– 65. (39) Ponerorchis wenshanensis (W. H. Chen, Y. M. Shui et K.Y. Garay, A., Kettredge, W., 1985. Notes from the Ames Orchid Herbarium. Bot. Mus. Lang) X.H. Jin, Schuit. et W.T. Jin, comb. nov. Leaf. Harv. Univ. 30, 47–59. Basionym Amitostigma wenshanense W. H. Chen, Y. M. Shui et Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, K.Y. Lang, Acta Bot. Yunnan. 25(5): 521 (2003). 95–98. (40) Ponerorchis yuana (Tang et F.T. Wang) X.H. Jin, Schuit. et Hapeman, J.R., Inoue, K., 1997. Plant-pollinator interactions and floral radiation in W.T. Jin, comb. nov. Platanthera (Orchidaceae). In: Givnish, T.J., Sytsma, K.J. (Eds.), Molecular Evolution and Adaptive Radiation. Cambridge University Press, Cambridge, pp. Basionym Amitostigma yuanum Tang et F.T. Wang, Bull. Fan 433–454. Mem. Inst. Biol. Bot. 10: 26 (1940). Hooker, J.D., 1890. The flora of British 6: Orchideae. Reeve, London. 6. Tsaiorchis Tang et F.T. Wang. Hunt, P.F., 1971. Notes on Asiatic Orchids: VI. Kew. Bull. 26, 171–185. Inda, L.A., Pimentel, M., Chase, M.W., 2010. Contribution of mitochondrial cox1 (41) Tsaiorchis keiskeoides (Gagnep.) X.H. Jin, Schuit. et W.T. intron sequences to the phylogenetics of tribe Orchideae (Orchidaceae): do the Jin, comb. nov. distribution and sequence of this intron tell us something about its evolution? Basionym Habenaria keiskeoides Gagnep., Bull. Soc. Bot. Fr. Taxon 59, 1053–1064. 78:71. (1931). Inda, L.A., Pimentel, M., Chase, M.W., 2012. Phylogenetics of tribe Orchideae (Orchidaceae: ) based on combined DNA matrices: inferences Synonym Tsaiorchis neottianthoides Tang et F.T. Wang, Bull. Fan regarding timing of diversification and evolution of pollination syndromes. Ann. Mem. Inst. Biol. Bot. 7: 133 (1936). Bot. London 110, 71–90. Jin, X.H., Efimov, P.G., 2012. Platanthera ovatilabris and P. dulongensis spp. nov. and new records of Platanthera (Orchidaceae, Orchidoideae) for Yunnan and Tibet, Appendix B. Supplementary material China. Nordic J. Bot. 30, 291–298. Jin, X.H., Li, D.Z., Xiang, X.G., Lai, Y.J., Shi, X.C., 2012. Nujiangia (Orchidaceae: Orchideae): a new genus from the Himalayas. J. Syst. Evol. 50, 64–71. Supplementary data associated with this article can be found, Jin, W.T., Zhou, H.L., Jin, X.H., 2013. Platanthera yadongensis (Orchidaceae, in the online version, at http://dx.doi.org/10.1016/j.ympev.2014. Orchideae), a new species from Tibet, China. Syst. Bot. 38 (4), 982–986. King, G., Pantling, R., 1896. A second series of new orchids from Sikkim. J. Asiat. Soc. 04.004. Bengal, Pt. 2, Nat. Hist., 118–134. King, G., Pantling, R., 1898. The orchids of Sikkim-Himalaya. Ann. Roy. Bot. Gard. (Calcutta) 8, 1–342. References Kraenzlin, F., 1901. Orchidacearum Genera et Species, vol. 1. Mayer and Müller, Berlin. Aceto, S., Caputo, P., Cozzolino, S., Gaudio, L., Moretti, A., 1999. Phylogeny and Kurzweil, H., Weber, A., 1992. Floral morphology of southern African Orchideae. II. evolution of Orchis and allied genera based on ITS DNA variation: morphological Habenariinae. Nordic J. Bot. 12, 39–61. gaps and molecular continuity. Mol. Phylogenet. Evol. 13, 67–76. Lang, K.Y., 1998. A new subgenus of Platanthera (Orchidaceae). Acta Phytotaxon. Sin. Bateman, R.M., Hollingsworth, P.M., Preston, J., Luo, Y.B., Pridgeon, A.M., Chase, 36, 449–458. M.W., 2003. Molecular phylogenetics and evolution of Orchidinae and selected Lang, K.Y., 1999. Flora Republicae Popularis Sinicae, vol. 17. Science Press, Beijing. Habenariinae (Orchidaceae). Bot. J. Linn. Soc. 142, 1–40. Lindley, J., 1835. The Genera and Species of Orchidaceous Plants. Ridgways Bateman, R.M., James, K.E., Luo, Y.B., Lauri, R.K., Fulcher, T., Cribb, P.J., Chase, M.W., Piccadilly, London. 2009. Molecular phylogenetics and morphological reappraisal of the Platanthera Luer, C.A., 1975. The Native Orchids of the and Canada Excluding clade (Orchidaceae: Orchidinae) prompts expansion of the generic limits of Florida. New York Botanical Garden, New York. Galearis and Platanthera. Ann. Bot.-London 104, 431–445. Luo, Y.B., Chen, S.C., 2003. Hemipiliopsis, a new genus of Orchidaceae. Novon 13, Batista, J.A.N., Borges, K.S., de Faria, M.W.F., Proite, K., Ramalho, A.J., Salazar, G.A., 450–453. van den Berg, C., 2013. Molecular phylogenetics of the species-rich genus Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway Habenaria (Orchidaceae) in the new world based on nuclear and plastid DNA for inference of large phylogenetic trees. In: Proceedings of the Gateway sequences. Mol. Phylogenet. Evol. 67, 95–109. Computing Environments Workshop (GCE), 14 November 2010, New Orleans, Bellusci, F., Pellegrino, G., Palermo, A.M., Musacchio, A., 2008. Phylogenetic LA, pp. 1–8. relationships in the orchid genus Serapias L. based on noncoding regions of Pearce, N.R., Cribb, P.J., 2002. Orchideae. In: Pearce, N.R., Cribb, P.J. (Eds.),The the chloroplast genome. Mol. Phylogenet. Evol. 47, 986–991. Orchids of Bhutan. Royal Botanic Garden Edinburgh, Edinburgh, Royal Box, M.S., Bateman, R.M., Glover, B.J., Rudall, P.J., 2008. Floral ontogenetic evidence Government of Bhutan, Bhutan, pp. 122–192. of repeated speciation via paedomorphosis in subtribe Orchidinae Pedersen, H.A., Suksathan, P., Indhamusika, S., 2002. Sirindhornia, a new orchid (Orchidaceae). Bot. J. Linn. Soc. 157, 429–454. genus from Southeast Asia. Nordic J. Bot. 22, 391–404. Bytebier, B., Bellstedt, D.U., Linder, H.P., 2007. A molecular phylogeny for the large Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA substitution. African orchid genus Disa. Mol. Phylogenet. Evol. 43, 75–90. Bioinformatics 14, 817–818. Chase, M.W., Cameron, K.M., Barret, R.L., Freudenstein, J.V., 2003. DNA data and Pridgeon, A.M., Cribb, P.J., Chase, M.W., Rasmussen, F.N., 2001. Genera Orchidaceae systematics: a new phylogenetic classification. In: Dixon, K.W., Orchidacearum, vol. 2. Oxford University Press, Oxford. Kell, S.P., Barret, R.L., Cribb, P.J. (Eds.), Orchid Conservation. Natural History Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference Publications, Kota Kinabalu, pp. 69–89. under mixed models. Bioinformatics 19, 1572–1574. Chen, S.C., Lang, K.Y., Stephan, W.G., Phillip, J.C., Paul, O., 2009. Subfam. Schlechter, R., 1924. Additamenta ad Orchideologiam chinensem. (Orchidaceae Orchidoideae. In: Wu, Z.Y., Raven, P.H., Hong D.Y. (Eds.), . vol. novae et criticae) Repert. Spec. Nov. Regni Veg. 19, 372–383. 25. Science Press, Beijing, Missouri Botanical Garden, St. Louis, pp. 45–166. Seidenfaden, G., 1977. Orchid genera in Thailand V: Orchidoideae. Dansk Bot. Ark. Comber, J.B., 2001. Orchids of Sumatra. The Royal Botanic Gardens Kew, London. 31, 1–149. W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53 53

Shi, Y.F., Li, J.J., Li, B.Y., 1998. Uplift and Environmental Changes of Qinghai-Tibetan Szlachetko, D.L., Kras, M., 2006. Notes sur le genre Habenella. Richardiana 6, 33–39. Plateau in the Late Cenozoic. Guangdong Science and Technology Press, Tang, T., Wang, F.T., 1940. Contributions to the knowledge of eastern Asiatic Guangzhou. Orchidaceae I. Bull. Fan Mem. Inst. Biol. Bot. Ser. 10, 23–46. Soliva, M., Kocyan, A., Widmer, A., 2001. Molecular phylogenetics of the sexually Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The deceptive orchid genus Ophrys (Orchidaceae) based on nuclear and chloroplast CLUSTAL_X windows interface: flexible strategies for multiple sequence DNA sequences. Mol. Phylogenet. Evol. 20, 78–88. alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882. Soó, R., 1929. Revision der Orchideae-Ophrydineae von Ostasien und dem Tuyama, T., 1966. Orchidaceae. In: Hara, H. (Ed.), The Flora of Eastern Himalaya. Himalaya. Annls Hist.-Nat. Mus. Nat. Hung. 26, 339–384. University of Tokyo, Tokyo, pp. 424–452. Soó, R., 1966. Die sog. Orchis Arten der Ostasiatisch-Nordamerikanischen Flora. Acta Tuyama, T., 1971. Orchidaceae. In: H. Hara (Ed.), The Flora of Eastern Himalaya, Bot. Acad. Sci. Hung. 12, 351–354. Second Report. University of Tokyo, Tokyo, pp. 176–196. Sun, H., 2002a. Tethys retreat and Himalayas-Hengduanshan Mountains uplift and Tuyama, T., 1975. Orchidaceae. In: H. Ohashi (Ed.), The Flora of Eastern Himalaya, their significance on the origin and development of the Sino-Himalayan Third Report. University of Tokyo Press, Tokyo, pp. 137–164. elements and alpine flora. Acta Bot. Yunnan. 24, 273–288. Tyteca, D., Klein, E., 2008. Genes, morphology and biology – the systematics of Sun, H., 2002b. Evolution of Arctic-Tertiary Flora in Himalayan-Hengduan Orchidinae revisited. J. Eur. Orch. 40, 501–544. Mountains. Acta Bot. Yunnan. 24, 671–688. Vaidya, G., Lohman, D.J., Meier, R., 2011. SequenceMatrix: concatenation software Swofford, D.L., 2002. PAUP⁄: phylogenetic analysis using parsimony (⁄ and Other for the fast assembly of multi-gene datasets with character set and codon Methods), version 4.0b10. Sinauer, Sunderland. information. Cladistics 27, 171–180. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

学霸图书馆（www.xuebalib.com）是一个“整合众多图书馆数据库资源，

提供一站式文献检索和下载服务”的24 小时在线不限IP 图书馆。 图书馆致力于便利、促进学习与科研，提供最强文献下载服务。

图书馆导航：

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具