Molecular Phylogenetics 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 (Orchideae, Orchidaceae) 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, China
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
b
a r t i c l e
i n f o
Article history:
Received 24 October 2013
Revised 30 March 2014
Accepted 3 April 2014
Available online 16 April 2014
Keywords:
Asia
Generic delimitation
Habenariinae
Molecular phylogenetics
Orchidinae
a b s t r a c t
The subtribe Orchidinae, distributed predominantly in Eastern Asia and the Mediterranean, presents
some of the most intricate taxonomic problems in the family Orchidaceae with respect to generic delimitation. Based on three DNA markers (plastid matK, rbcL, and nuclear ITS), morphological characters, and a
broad sampling of Orchidinae and selected Habenariinae mainly from Asia (a total of 153 accessions of
145 species in 31 genera), generic delimitation and phylogenetic relationships within the subtribe Orchidinae and Habenariinae from Asia were assessed. Orchidinae and Asian Habenariinae are monophyletic,
and Orchidinae is divided into distinct superclades. Many genera, such as Amitostigma, Habenaria, Hemipilia, Herminium, Platanthera, Peristylus and Ponerorchis, are not monophyletic. Habenaria is subdivided into
two distantly related groups, while Platanthera is subdivided into three even more disparate groups.
Many previously undetected phylogenetic relationships, such as clades formed by the Amitostigma–Neottianthe–Ponerorchis complex, Platanthera latilabris group, Ponerorchis chrysea, Sirindhornia, and Tsaiorchis,
are well supported by both molecular and morphological evidence. We propose to combine Hemipiliopsis
with Hemipilia, Amitostigma and Neottianthe with Ponerorchis, Smithorchis with Platanthera, and Aceratorchis and Neolindleya with Galearis, and to establish a new genus to accommodate Ponerorchis chrysea. Tsaiorchis 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
The subtribe Orchidinae consist of about 35 genera and
350–400 species, distributed mainly in Eastern Asia, the Mediterranean Region with a few species extending in Northern America,
Southern America and Africa (Chen et al., 2009; Dressler, 1993;
Kraenzlin, 1901; Lang, 1999; Pearce and Cribb, 2002; Pridgeon
et al., 2001). Orchidinae is similar to Habenariinae in habitat
preferences and many vegetative and floral characters, but these
⇑ Corresponding authors.
E-mail addresses: jinweitao@ibcas.ac.cn (W.-T. Jin), orchid@ibcas.ac.cn, xiaohuajin@ibcas.ac.cn (X.-H. Jin), A.Schuiteman@kew.org (A. Schuiteman), dzl@mail.kib.ac.
cn (D.-Z. Li), xiangxg2010@ibcas.ac.cn (X.-G. Xiang), hwc_zx@126.com (W.-C.
Huang), ljw@xtbg.org.cn (J.-W. Li), huangluqi01@126.com (L.-Q. Huang).
http://dx.doi.org/10.1016/j.ympev.2014.04.004
1055-7903/Ó 2014 Elsevier Inc. All rights reserved.
two subtribes can be distinguished on the basis of the structures
of stigma: Orchidinae usually has a concave and sessile stigma
often with confluent lobes, whereas Habenariinae usually has
stalked, convex and distinct stigma lobes (Dressler, 1993;
Pridgeon et al., 2001). This morphological distinction has been supported by molecular evidence (Douzery et al., 1999). However, Inda
et al. (2010, 2012) indicated that Habenariinae s.l. is paraphyletic,
and several genera from Africa, such as Stenoglottis, Cynorkis, and
Holothrix, were resolved as successive sister to Orchidinae + Habenaria and its alliance. Batista et al. (2013) showed that the Habenariinae clade (formed by Habenaria s.l. + Cynorkis + Stenoglottis)
is sister to the Orchidinae clade formed by (Orchis + Platanthera).
In practice, it can be difficult to distinguish between concave sessile stigma lobes and stalked convex ones in some alpine taxa, such
as Androcorys, Herminium, Peristylus, Ponerorchis, and Smithorchis
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
Platanthera species (Orchidinae) clearly have stalked stigma lobes.
Kurzweil and Weber (1992), Pridgeon et al. (2001) and Chase et al.
(2003) even suggested abandoning the recognition of Orchidinae
and Habenariinae as distinct clades.
Orchidinae is a medium-sized subtribe in Orchidaceae and one
of many well-studied groups in Orchidaceae, however, the generic
delimitation and classification within Orchidinae are still problematic and is complicated by the morphological diversity, wide distribution range, homoplasy of characters considered diagnostic at
generic level, and the intergrading and overlapping morphological
variation between genera (Aceto et al., 1999; Bateman et al., 2003,
2009; Box et al., 2008; Dressler, 1981, 1993; Hapeman and Inoue,
1997; Jin and Efimov, 2012; Luer, 1975; Pridgeon et al., 2001;
Soliva et al., 2001; Tyteca and Klein, 2008). Based on morphological
characters and/or analyses of molecular data, various generic
delimitations and taxonomies have been proposed, especially on
some systematically difficult genera, such as Dactylorhiza, Orchis
s.l., Platanthera, Ponerorchis, and Tsaiorchis (Aceto et al., 1999;
Bateman et al., 2003, 2009; Hapeman and Inoue, 1997; Hooker,
1890; King and Pantling, 1896, 1898; Lang, 1998, 1999; Soliva
et al., 2001; Luer, 1975; Pridgeon et al., 2001; Tyteca and Klein,
2008).
As previous molecular systematics of Orchidinae were largely
based on sampling from the Mediterranean area (Bateman et al.,
2003, 2009; Douzery et al., 1999; Soliva et al., 2001; Inda et al.,
2012), and/or mainly utilized a single DNA marker (ITS) (Bateman
et al., 2003, 2009; Douzery et al., 1999; Soliva et al., 2001), some
conclusions and results were weakly supported or even without
statistical support, and many taxonomic problems remain unresolved. Despite the high diversity of Orchidinae and Habenariinae
in Eastern Asia, many taxa, especially those from monotypic/oligotypic genera, were not represented in previous molecular studies.
Generic delimitation and systematic position of many genera in
Orchidinae and Habenariinae from Asia, such as Aceratorchis, Amitostigma, Hemipilia, Hemipiliopsis, Neolindleya, Neottianthe, Platanthera, Ponerorchis, Smithorchis, and Tsaiorchis, are not or little known,
and as a result many taxonomical suggestions and proposals
remain to be tested (Bateman et al., 2003, 2009; Chen et al., 2009;
Pridgeon et al., 2001). For a better understanding of the generic
delimitation within Orchidinae and of the interrelationships among
major clades within Orchidinae, it is desirable to base the analyses
on multiple DNA markers and a denser sampling across many systematically difficult genera and their allies from Asia.
In the present study, phylogenetic relationships were inferred
using three DNA markers (plastid matK, rbcL and nuclear ITS
sequence), with 153 samples representing 146 species of Orchidinae and selected Habenariinae, such as Androcorys, Habenaria, Herminium, and Peristylus, mainly from Asia, with the aims of (1)
increasing our understanding of the generic delimitation within
Orchidinae and Habenariinae; (2) reconstructing the phylogenetic
interrelationships within Orchidinae.
2. Materials and methods
including Bonatea, Gennaria, Habenaria, Ophrys, Pseudorchis, and
Serapias, were used to broaden the sampling of Orchideae in our
analyses. Since previous results indicated that Orchideae is closely
related to tribe Diseae (Bytebier et al., 2007; Douzery et al., 1999;
Pridgeon et al., 2001), two species of Disa, Disa tripetaloides and
Disa uniflora, were used as outgroups. The voucher information
and the GenBank accession numbers used in this study are listed
in Table A.1.
2.2. DNA extraction, amplification and sequencing
Total genomic DNA was isolated from silica-gel-dried materials
using a Plant Genomic DNA Kit (Beijing Biomed Co., LTD, Beijing,
China). For this study, two plastid markers (the coding gene matK,
rbcL) and the nuclear ribosomal DNA internal transcribed spacers
(ITS) were used. The PCR and sequencing primers for matK, rbcL,
and ITS are listed in Table A.2. The selected DNA regions were
amplified by using a standard polymerase chain reaction (PCR).
The sequencing reactions were performed by using the ABI Prism
Bigdye Terminator Cycle Sequencing Kit (Applied Biosystems, ABI).
2.3. Phylogenetic analyses
Sequences were aligned using the program Clustal X 1.83
(Thompson et al., 1997) and manually adjusted using BioEdit
(Hall, 1999). The homogeneity between the ITS data and the combined plastid dataset (matK, rbcL) was tested using the incongruence length difference (ILD) (Farris et al., 1995), implemented in
PAUP v4.0b10 (Swofford, 2002). Following Cunningham (1997),
no cases of strongly supported incongruence were detected
(P = 0.17), therefore, we combined ITS data and the plastid dataset
(matK, rbcL) in SequenceMatrix v1.7.8 (Vaidya et al., 2011) to perform further phylogenetic analyses.
The phylogenetic analyses for each matrix were performed
using the maximum parsimony (MP) in PAUP v4.0b10 (Swofford,
2002) and Bayesian inference (BI) in MrBayes v3.1.2 (Ronquist
and Huelsenbeck, 2003) on CIPRES Science Gateway Web server
(Old MrBayes on XSEDE 3.1.2) (Miller et al., 2010).
For the MP analyses, heuristic searches were conducted with
1000 replicates of random addition, one tree held at each step during the stepwise addition, tree-bisection–reconnection (TBR)
branch swapping, MulTrees in effect, and steepest descent off. All
of the characters were unordered and equally weighted, and the
gaps were coded as missing data. To evaluate the node support,
bootstrap analyses (Felsenstein, 1988) were performed using
1000 replicates, with 10 random taxon additions and heuristic
search options.
Prior to the Bayesian analysis, a model for sequence evolution
for each matrix was determined by using ModelTest v3.7 (Posada
and Crandall, 1998) under the Akaike information criterion. For
the BI analyses, two separate four Markov chain Monte Carlo
(MCMC) analyses were run, with 10,000,000 generations and sampling every 1000 generation. Majority rule (>50%) consensus trees
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
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).
In order to represent the taxonomic diversity of Orchideae in Asia,
153 accessions of 145 species in 31 genera, including 27 genera
and 103 species from Asia (about 84% of Asian genera and 25% of
Asian species), were included in this study. Additionally, several
genera and some species from Africa, Europe, and South America,
3. Results
3.1. Sequences and alignment
In this study, 215 new sequences were obtained. Sequence
lengths were as follows: 825 bp for ITS region, 1254 bp for
rbcL, 1870 bp for matK. The combined alignment of ITS and
plastid regions comprised 3949 bp, 24% of which were
43
W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53
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
Aligned length
No. variable characters
No. parsimony-informative characters
Tree length (steps)
Consistency index (CI)
Retention index (RI)
Model
144
825
507(61%)
445(54%)
3052
0.33
0.77
GTR + I + G
136
1870
642(34%)
419(22%)
1428
0.58
0.82
GTR + I + G
119
1254
181(14%)
103(8%)
334
0.62
0.83
GTR + I + G
153
3949
1330(34%)
967(24%)
4868
0.42
0.78
GTR + I + G
parsimony-informative. Table 1 summarizes the properties of each
aligned data partition.
3.2. Phylogenetic analyses
The partition homogeneity test for plastid DNA + ITS shows
there were no strongly supported incongruent results in the datasets (P = 0.17), therefore, we combined the datasets for simultaneous analyses.
Phylogenetic relationships based on the ITS data had a better
resolution than the two combined plastid DNA data (results not
shown here). Based on the combined ITS and plastid DNA data,
our findings are consistent in the overall topology of the trees produced with maximum parsimony (MP) and Bayesian inference (BI)
methods, except for a few of the collapsed nodes. Bootstrap values
(BS) were often lower than the Posterior Probability (PP) from the
Bayesian analysis.
The BI topology from the combined dataset is chosen as the primary tree for discussion of phylogenetic relationships (Figs. 1 and
2; the MP strict consensus tree is not shown). Our results indicate
that the tribe Orchideae can be divided into two well supported
sister clades, Habenariinae (PP = 100, BS = 67) (Fig. 1) and Orchidinae (PP = 100, BS = 97) (Fig. 2). Within the Orchidinae, six of nine
major clades (Clade I to IX) are well supported along the backbone
of the Orchidinae, and many interrelationships among them are
well-resolved (Fig. 1). Within the Habenariinae, nine major clades,
Clade X to XVIII, can be recognized (Fig. 2). Our results show that
many morphological delimited genera of the Asian Orchideae in
Pridgeon et al. (2001) and Chen et al. (2009) are not monophyletic,
such as Hemipilia, Herminium, Platanthera, and Ponerorchis.
Clade I (Fig. 1) comprises 42 species (PP = 89, BS < 50), most of
which belong to Platanthera s.l., the remainders are from genera
Herminium, Peristylus, and Smithorchis. The interrelationships
within Clade I are not well resolved. Clade II comprises two species
of Galearis, two monotypic genera, Aceratorchis and Neolindleya,
and one species of Aorchis with weak support (PP = 86) (Fig. 1).
Clade III includes one species of Pseudorchis (Fig. 1). Clade IV consists of four species from Dactylorhiza and Gymnadenia with strong
support (PP = 100, BS = 85) (Fig. 1). Clade V includes three species
of Orchis s.s. with robust support (PP = 100, BS = 99) (Fig. 1). Clade
VI (PP = 100, BS = 93) consists of two species of Ophrys and two
species of Serapias with strong support (PP = 100, BS = 99) (Fig. 1).
Clade VII (Fig. 1) includes about 15 species from Amitostigma,
Neottianthe, and Ponerorchis, and is subdivided into three well supported subclades: one subclade contains five species from Amitostigma and Ponerorchis; another subclade includes four species
from Amitostigma and Neottianthe, including types of these two
genera; and the third subclade includes four to six species of Amitostigma. Clade VIII (Fig. 1) comprises nine species, six from Hemipilia, two from two monotypic genera, Hemipiliopsis and Tsaiorchis,
and one from Ponerorchis, with weak support (PP = 81, BS = 56).
Clade VIII can be divided into two subclades, one includes Tsaiorchis, and the other includes the remainder with strong support
(PP = 100, BS = 97). Clade IX includes a species from the oligotypic
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
XI consists of three species from Platanthera, i.e. Platanthera latilabris group, with strong support (PP = 100, BS = 100) (Fig. 2). Clade
XII includes one species from Herminium, H. lanceum. Clade XIII
(PP = 100, BS = 100) includes six species, two from Peristylus, and
four from Herminium (Fig. 2). An unidentified species of Herminium
is sister to clades X to XIII, with weak support for the sister group
(Fig. 2). Clade XIV (PP = 100, BS = 100) includes two terminals from
one species, Ponerorchis chrysea (Fig. 2). Clade XV includes nine
species, eight from Asian Habenaria and one from Pecteilis, with
strong support (PP = 100, BS = 99) (Fig. 2). Clade XVI includes seven
species, six from Peristylus, and one from Platanthera, with substantial support (PP = 100, BS = 84) (Fig. 2). Clade XVII includes 24 species of Habenaria from Africa, Asia and Southern America, and two
species of Botanea from Africa with some support (PP = 100,
BS = 59) (Fig. 2). Clade XVIII includes two monotypic genera from
Europe and Asia, Gennaria and Nujiangia, with strong support
(PP = 100, BS = 100) (Fig. 2).
4. Discussion
4.1. An overview of phylogenetics of Orchideae
With broader sampling and more DNA markers, our results confirmed the earlier findings that Orchidinae (PP = 100, BS = 97)
(Fig. 1) and Asian Habenariinae (PP = 100, BS = 67) (Fig. 2) are
two well resolved sister groups. However, our results indicated
that the morphological distinctions, such as the types of stigma
lobes, between these two subtribes are problematic (see discussion
of each clade). Inda et al. (2012) indicated that Habenariinae is
paraphyletic, and several genera from Africa, such as Stenoglottis,
Satyrium, Cynorkis, and Holothrix, were resolved as successive sister
to Orchidinae + Asian Habenariinae. Batista et al. (2013) showed
that the Habenariinae clade (formed by Habenaria s.l. + Cynorkis + Stenoglottis) is sister to the Orchidinae clade formed by
(Orchis + Platanthera). Given the complex taxonomy of Orchideae,
it would be premature to make any firm conclusion about these
two subtribes. More importantly, our results showed that many
previous findings of interrelationships within Orchideae based on
molecular phylogenetics were strongly supported, and many
unknown or overlooked phylogenetic relationships have been
detected.
Asian Orchidinae is subdivided into two superclades (Fig. 1).
Superclade A includes Dactylorhiza, Galearis, Gymnadenia, Orchis
s.s., and Platanthera s.l. (PP = 87, BS = 58). Superclade B includes
Amitostigma, Hemipilia, Neottianthe and Sirindhornia (PP = 100).
Clade VI, formed by Ophrys and Serapias, is suggested as a sister
to Superclade A with strong support, and Sirindhornia is resolved
as sister to the rest of Superclade B with weak support (PP = 100)
(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. ‘‘’’ 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. ‘‘’’ indicates node is with support
value 100%.
Asian Habenariinae is subdivided into three moderately to
strongly supported superclades (Fig. 2). Superclade C (PP = 98),
consisting of Androcorys, Porolabium, Herminium, Ponerorchis chrysea, Platanthera latilabris group, and some species of Habenaria,
occur mostly in montane to alpine regions in Asia. Superclade D
(PP = 100, BS = 84), comprising Peristylus, occur mainly in tropical
and subtropical Asia with a few species extending into alpine
regions. Superclade E (PP = 100), including Habenaria from Asia,
Africa and the Neotropics, African Bonatea, and two monotypic
genera from Europe and Asia, Gennaria and Nujiangia, mainly
occurs in tropical regions with a few species extending into subtropical mountains. The clade formed by Gennaria and Nujiangia
is resolved as sister to the remainder in Superclade E.
As Orchidinae is well-represented in Europe, the generic delimitation and classification of many genera around the Mediterranean have been thoroughly studied, for example, Orchis and its
alliance by Aceto et al. (1999), Ophrys by Soliva et al. (2001), Serapias 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
from Neotropics has been investigated based on comprehensive
sampling from Neotropics (Batista et al., 2013). Hence, in our analyses, sampling of genera or species from the Africa, Europe, and
Neotropics is mainly used as placeholders for these taxa. Their
phylogeny will not be discussed here because our focus is on phylogenetic analyses on Asian taxa.
4.2. Phylogeny and generic delimitation
4.2.1. Platanthera (Clade I, and XVI)
Platanthera consists of about 100–150 species and is among the
larger genera in Orchideae, however, generic delimitation of Platanthera is unclear. Luer (1975) suggested that several previous
recognized genera, such as Blephariglottis, Lacera, Limnorchis, and
Tulotis, should be treated as sections within Platanthera, and
Dressler (1993) suggested that Platanthera is close to Dactylorhiza–Galearis alliance instead of Habenaria. These suggestions have
been supported by recent results of molecular studies (Bateman
et al., 2003, 2009; Hapeman and Inoue, 1997). Bateman et al.
(2009) broadened Platanthera to include more genera, such as
Diphylax, Piperia, and Tsaiorchis. Based on seed micromorphology,
however, Gamarra et al. (2008) stated that Limnorchis should be
separated from Platanthera as a distinct genus.
Our analyses do not only support most of these conclusions, but
also indicate that Platanthera s.l. is polyphyletic and subdivided
into three distant related groups that belong to two subtribes, Platanthera clade (Clade I, Orchidinae), Platanthera latilabris group
(Clade XI, Habenariinae), and Platanthera biermanniana (included
in Clade XVI, Habenariinae).
The Platanthera clade (Clade I, Orchidinae) is not strongly supported and the interrelationships within the genus are not well
resolved. However, the Platanthera clade is well characterized by
morphological characters, such as fleshy rootstock (no tuber),
leaves basal to cauline and grading into foliaceous bracts, sheathing at base, lip spurred, column 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.
Platanthera latilabris group, consisting of three closely related
species, P. clavigera, P. edgeworthii, P. latilabris (Fig. 3a), form an
unambiguous clade deeply nested within Habenariinae. Species
of this clade are characterized by ovoid to globose tubers, densely
flowered inflorescence, linear and fleshy lip with a callus at base,
stigma lobes more or less stalked, extending at base of lip
(Fig. 3a). Hooker (1890) treated these species as members of Habenaria, Szlachetko and Kras (2006) transferred these species into
the Neotropical genus, Habenella. Our results indicated that Platanthera latilabris group and Habenella (included in Clade XVII) are two
quite distantly related groups. Instead, Platanthera latilabris group
are resolved as sister to Androcorys plus Porolabium with weak support (PP = 92, BS = 56).
Platanthera biermanniana resembles species of Peristylus in gross
morphology. Our analyses show that P. biermanniana is deeply
nested within Peristylus with robust support, which is congruent
with morphological characters, such as cylindric-ovoid tuber, 3lobed lip, short spur, anther with very narrow connective.
Several species of Herminium (H. carnosilabre and H. angustilabre), Peristylus (P. nematocaulon), and the monotypic Smithorchis (S.
calceoliformis) are deeply nested within Platanthera s.l. These species are restricted in alpine region with elevation ranging from
3500 to 4300 m. The taxonomy has been complicated by these species having minute flower (the smallest in Orchidinae) and a
greatly reduced column which is occupied almost entirely by the
anther. Most genera of Asian Orchideae were distinguished on
the basis of floral characters, especially of the column (Lang,
1999; Pridgeon et al., 2001). Our observations and morphological
comparison established that these species have oblong to fusiform
rootstock (which are near horizontal in S. calceoliformis), entire and
spurred lip, naked viscidium, and a relative obvious anther connective, which supports the transfer of these species into Platanthera.
Dozens of Platanthera species from the Himalayas, such as P.
bakeriana, P. leptocaulon, P. roseotincta, and others, have two projecting stigmatic lobes (stalked stigma lobes) and an entire lip
(Fig. 3b). Duthie (1906), Hooker (1890), King and Pantling (1896,
1898), and Tuyama (1966, 1971, 1975) treated these species as
Habenaria, while Kraenzlin (1901), Lindley (1835), Lang (1998,
1999), and Soó (1929) placed them in Platanthera. Lang (1998)
even proposed a subgenus, subgen. Stigmatosae, to contain these
species. Our results indicate that stalked stigma lobes have evolved
at least twice in Platanthera, one in P. yadongensis (Jin et al., 2013),
the other in P. exelliana and its relative species. This renders the
morphological distinction between Habenariinae and Orchidinae,
which is mainly based on the morphology of the stigma,
problematic.
4.2.2. Aceratorchis, Aorchis, Galearis, and Neolindleya (Clade II)
Both Aceratorchis (Fig. 3c) and Neolindleya are little-known
monotypic genera. Bateman et al. (2009) considered Aceratorchis
to be a peloric form of Galearis. Neolindleya was previously considered as Gymnadenia (Bateman et al., 2003), then it was separated as
a distinctive genus and sister to Galearis (Bateman et al., 2009;
Efimov et al., 2009). Pridgeon et al. (2001) considered Aorchis as
congeneric with Galearis. In our phylogenetic analyses, Aceratorchis, Aorchis, Galearis and Neolindleya form a weak supported clade
(PP = 86) (Fig. 1), while Aceratorchis and Neolindleya were resolved
as successive sister to Galearis and Aorchis. These four genera typically share a stolon-like rhizome, anther loculi parallel with distinct rostellum between them, concave stigma, and viscidium
enclosed in bursicle. Considering the weak support of the clade,
we tentatively proposed to subsume all other three genera into
Galearis. However, this needs to be tested by further studies.
4.2.3. Amitostigma, Neottianthe, and Ponerorchis (Clade VII)
The generic borders between Ponerorchis and Amitostigma are
unclear due to the difficulty of distinguishing the supposed diagnostic characters, such as appendages of column and the bursicle,
and several species have been transferred back and forth between
these genera. However, it is nevertheless somewhat unexpected
that Amitostigma, Neottianthe and Ponerorchis are nested together
and form a moderately supported group (Fig. 1). This clade mainly
occurs in Eastern Asia, with one or two species extending into the
northern temperate zone. Members of this clade share an ovoid
tuber, slender plants with 1–3 slightly fleshy leaves, flowers
secund along rachis, dorsal sepal and petals forming a hood, the
base of lip more or less connecting the column, column short, lateral appendages obvious, parallel anther cells, stigma lobes situated and confluent under rostellum. The subclade formed by
three species of Neottianthe (including the type, Neottianthe cucullata) and Amitostigma gracile (type of Amitostigma) is strongly supported and well characterized by morphological characters, such as
leaves basal, elliptic to ovate, petals and three sepals forming a
hood, lip papillose. Ponerorchis is said to differ from Amitostigma
and Neottianthe by having a viscidium enveloped in a bursicle
(see Pridgeon et al., 2001). Our results indicate that a bursicle
has independently evolved four times in Orchideae (Clade II, VII,
VIII, XIV). Moreover, our fieldwork established that the presence
of a bursicle is difficult to determine even in living specimens,
and some species of Amitostigma, such as A. monanthum (Fig. 3g),
A. yuanum (Fig. 3h), do have bursicles. It is almost impossible to
assign some species, such as Amitostigma yuanum (Fig. 3h), Ponerorchis nana (Fig. 3i), and A. farreri, to Amitostigma 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
to unite Amitostigma and Neottianthe into Ponerorchis.
4.2.4. Hemipilia, Hemipiliopsis,and Tsaiorchis (Clade VIII)
Both Hemipiliopsis (Fig. 3d, e) and Tsaiorchis (Fig. 3f) are littleknown and narrowly distributed monotypic genera. Garay and
Kettredge (1985) placed Tsaiorchis neottianthoides (= Habenaria
keiskeoides) in Amitostigma, Pridgeon et al. (2001) treated Tsaiorchis
as Diphylax. Bateman et al. (2009) positioned Tsaiorchis neottianthoides in Platanthera. Bateman et al. (2003) stated that the monotypic Hemipiliopsis is closely related to Ponerorchis brevicalcarata,
and suggested these two species should be assigned to Hemipilia.
In our analyses, Hemipiliopsis is nested within Hemipilia with
robust support (PP = 100, BS = 97), while Tsaiorchis is resolved as
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 elongate and erect rostellum. On the other hand, Tsaiorchis differs
greatly from Hemipiliopsis and Hemipilia both in vegetative and floral characters. Hemipiliopsis and Hemipilia are characterized by
cylindrical to ovoid-globose tubers, leaf solitary, basal, prostrate
fleshy, more or less distinctly spotted, inflorescence with purple
spots (Fig. 3d, e), stigma lobes confluent. In contrast to these, Tsaiorchis (Fig. 3f) is characterized by hairy and horizontally extending
rhizomes, one to two chartaceous and green leaves, inflorescence
green, two lateral appendages longer than anther, rostellum elongate, stigma lobes separate, linear and extending at the base of lip.
Although Luo and Chen (2003) established Hemipiliopsis on the
basis of its less developed rostellum and the shape of spur, Hemipiliopsis is characterized by the well-developed and erect rostellum
as long as half of anther locus (Fig. 3e). The sister taxon of Hemipiliopsis, Ponerorchis brevicalcarata, fits well in Hemipilia. In addition,
Hemipiliopsis is sympatric with Hemipilia. Based on these findings,
we propose to broaden Hemipilia to include Hemipiliopsis, and
maintain Tsaiorchis as a distinctive genus.
4.2.5. Sirindhornia (Clade IX)
Sirindhornia is a recently established and morphologically distinctive genus. There are three species and they are mainly distributed in border regions in Thailand, Myanmar, 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
et al. (2002) stated that Sirindhornia is close to Hemipilia, Ponerorchis and reminiscent of Orchis. Instead, Chen et al. (2009) included
Sirindhornia monophylla (type of Sirindhornia) in Ponerorchis.
In our phylogenetic analyses, Sirindhornia is unambiguously
resolved as sister to the remaining genera, including Hemipilia, Tsaiorchis, in Superclade B, which is supported by morphological characters of Sirindhornia. Sirindhornia is characterized by the elongate
tuber, solitary and fleshy leaf, leaf convolute and not spreading,
stem and ovary papillose–pubescent, ciliate bract, lip spurred,
stigma lobes concave, and viscidium enveloped in a bursicle
(Fig. 3j). In addition, species of Sirindhornia typically grow in limestone regions, and flower from April to June, which is one to two
months earlier than other Orchidinae from Eastern Asia. Based on
these findings, we recognize Sirindhornia as a distinct genus.
4.2.6. Androcorys, Herminium, and Porolabium (Clade X, XI, XII, and
XIII)
Morphologically, it is difficult to determine the systematic positions of Androcorys, Herminium, and Porolabium in Orchideae due to
the relative small flowers, short column occupied almost by anther,
greatly reduced stigma and rostellum (Pridgeon et al., 2001).
Pridgeon et al. (2001) proposed to broaden Androcorys to include
some members of Herminium with spurred lip; Bateman et al.
(2003) suggested that some temperate members of Peristylus
should be transferred to Herminium. In our analyses, Androcorys,
Herminium, and Porolabium are deeply embedded within Habenariinae, and Herminium is not monophyletic. Some misplaced
members have to be excluded and assigned to other genera, while
some species currently residing in other genera need to be transferred to it.
Herminium lanceum is resolved as immediate sister to groups
formed by clade X plus XI with moderate support (PP = 99,
BS < 50). Our observations indicate that H. lanceum differs from
other species of Herminium by having parallel anther loculi, one
pulvinate stigma swelling at the base of the column, and transverse
rostellum, while most Herminium (including the type, H. monorchis) have more or less divergent anther loculi, two stalked stigma
lobes extending along the base of column, and a cylindrical
rostellum.
A little known species of Herminium is resolved as immediate
sister to clade X, XI, XII and XIII with weak support. Our morphological studies show that this entity is similar to Herminium and
shares many morphological characters, such as ovoid tubers, viscidium involute and hornlike, two stalked stigma lobes extending at
the base of column. This entity differs from Herminium by having
parallel anther loculi with a long (as long as half of anther locus)
and slender appendage between them. More molecular data and
a better understanding of the morphological characters are needed
to elucidate the phylogeny of this little known entity.
Tang and Wang (1940) separated Porolabium from Herminium
on the basis of having a lip with two pores. Pridgeon et al. (2001)
stated that the rostellum of Porolabium is identical to Herminium;
Pearce and Cribb (2002) suggested that Androcorys ophioglossoides
resembles Porolabium biporosum in morphological characters. Our
results indicate that Porolabium is nested within Androcorys with
strong support, and these two genera are sympatric. We found that
Androcorys and Porolabium both have a pulvinate stigma at the
base of the column. There are several divergent taxonomic options
to classify Herminium and its alliance into one or more monophyletic groups. One is to broaden Herminium to include Androcorys
and its alliance (Clade X, XI, XII, XIII). Another is to recognize each
clade in this alliance (Clade X, XI, XII, XIII) as a distinctive genus.
However, with the currently available sampling and weak supported interrelationships among these clades, it would be premature to make any firm decision.
4.2.7. Ponerorchis chrysea (Habenaria chrysea, Orchis chrysea, Clade
XIV)
Ponerorchis chrysea (Fig. 3h) may have the largest flowers in
Orchidinae, however, its systematic position is poorly understood.
Schlechter (1924) treated Habenaria chrysea as a member of Orchis
s.l., Soó (1966) transferred it to Ponerorchis, Hunt (1971) included it
in Chusua. In our analyses, Ponerorchis chrysea is resolved as sister
to the entity comprised of Clades X–XIII, with moderate support
(PP = 100, BS = 69) (Fig. 2). Most members of these clades have
basal and green leaf (or leaves), yellow, yellowish or green flowers,
which strikingly differ from Ponerorchis. Our detailed morphological examination indicate that Ponerochis chrysea is a quite out of
place in Ponerorchis on account of several morphological characters, such as leaves basal, sheaths long and clasping peduncle, floral
bracts similar to leaf and enclosing the spur, pedicel longer than
ovary, anther connective elongate and drawn out, three slightly
convex stigma lobes confluent under rostellum. Some of these
characters, such as pedicel longer than ovary, connective elongate
and drawn out, are even unique in Orchideae in Eastern Asia. On
these grounds, we propose to establish a new genus, Hsenhsua, to
accommodate this taxon.
Hsenhsua X.H. Jin, Schuit. et W.T. Jin, gen. nov. Figs. 3k and 4.
Type species: Hsenhsua chrysea (W.W. Sm.) X.H. Jin, Schuit., W.T.
Jin et L.Q. Huang.
Included taxa: Hsenhsua chrysea (W.W. Sm.) X.H. Jin, Schuit.,W.T.
Jin et L.Q. Huang, comb. nov. (Basionym Habenaria chrysea W.W.
Sm., Notes from the Royal Botanic Garden, Edinburgh 13(63–64):
204–205. 1921.)
Diagnosis: Tuber subglobose. Stem 2 leaved. Leaves green, basal.
Inflorescence 1-flowered. Floral bracts foliaceous, sheathing,
enclosing the spur. Pedicel longer than ovary; anther connective
elongate and drawn out; rostellum protruding with two arms;
three slightly convex stigma lobes confluent under rostellum.
Morphological evidence: Morphologically, Hsenhsua is similar to
Ponerorchis s.l. by sharing subglobose tuber, three-lobed lip with
a distinct spur, rostellum protruding with two arms, but differs
from the latter by having foliaceous bracts sheathing and enclosing
the spur, pedicel longer than ovary, anther connective elongate and
drawn out. Hsenhsua can be readily distinguished from Herminium
s.l. by its foliaceous bracts, elongate anther connective, three
slightly convex stigma lobes confluent under rostellum.
Molecular evidence: In the current study, the combined analyses
of the datasets of three markers (two chloroplast, one nuclear)
shows moderate support for a sister group relationships of Hsenhsua with Herminium s.l. (including Androcorys, Herminium, Porolabium and other species) (PP = 100, BS = 69).
Habit and Distribution: Growing in humid alpine grassland and
shrub between 3600 and 4200 m in the Eastern Himalayas, including China (NW Yunnan, SE to S Xizang), and Bhutan.
Etymology: Hsenhsua is named in honor of Prof. Hsen-Hsu Hu,
the renowned Chinese scientist who was a pioneer and founder
of modern botany in China.
4.2.8. Habenaria and its alliance (Clade XV, XVII, and XVIII)
Habenaria is one of the largest genera of orchid family and
widespread across the tropical and subtropical regions of the world
(Pridgeon et al., 2001). There is much debate about the generic
delimitation and infrageneric systems (see Batista et al., 2013;
Pridgeon et al., 2001). Bateman et al. (2003) stated that Habenaria
is polyphyletic and needs to be dismantled into many monophyletic genera. On the other hand, Batista et al. (2013) indicated that
Neotropical Habenaria is monophyletic and closely related to some
African congeners, and suggested that a revision of the infrageneric
system rather than an extensive generic fragmentation is most
appropriate. In our phylogenetic analyses with 36 sampled species
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, petal 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).
Clade XV includes nine Asian species mainly distributed in subtropical region, such as H. dentata, H. rhodocheila and Pecteilis
gigantea, and is sister to the predominantly alpine Herminium s.l.
clade (Clades X–XIII) and Ponerorchis chrysea. Clade XVII, mainly
including African and South American Habenaria, and Bonatea, is
resolved as sister to two monotypic genera, Nujiangia and Gennaria.
It is interesting to note that five Asian Habenaria are nested within
Clade XVII. Asian tropical Habenaria stenopetala is resolved as sister
to the African-Neotropical H. heyneanna plus H. foliosa with strong
support (PP = 100, BS = 100). Asian alpine H. intermedia is resolved
as sister to African montane H. praestans with strong support
(PP = 100, BS = 100); both belong to Habenaria sect. Mutipartitae.
Two monotypic and morphologically isolated genera in Habenariinae, Gennaria and Nujiangia, were uncovered as sister to African and American Habenaria with strong support. These two
genera share several morphological characters, such as two alternate leaves, densely flowered inflorescence, 3-lobed lip with a
short spur, auricles longer than anther, and a convex stigma (Jin
et al., 2012).
Although with limited sampling of Habenaria and its alliance
from Africa and Neotropics, our phylogenetic analyses indicate that
the taxonomy of Habenaria is even more complicated than previously thought. It is clear that as currently defined Habenaria is patently polyphyletic. There have been several highly divergent
taxonomic proposals to delimit Habenaria to be monophyletic.
One is the generic fragmentation of Habenaria into many genera,
such as Bonatea, Habenella, and many others; another is to broaden
Habenaria to include nearly all genera, such as Androcorys, Herminium, and Peristylus, in Habenariinae. The third is to redefine clade
XVII as Habenaria s.s., with typification of Habenaria macroceratitis,
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.
Future work with more comprehensive sampling and more molecular data will undoubtedly provide new insights into the taxonomy
of Habenaria.
4.2.9. Peristylus (Clade XVI)
Peristylus is a widespread genus in Asia with about 70 species
(Pridgeon et al., 2001), however, it is almost neglected in previous
studies of Orchideae. There is some debate on its distinction from
Habenaria. Seidenfaden (1977) proposed to define Peristylus on the
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
Peristylus typically have pulvinate stigmatic swellings at the base
of the column and adnate to the base of the lip. Comber (2001) also
suggested a set of morphological characters to define Peristylus. In
our phylogenetic analyses, Peristylus is resolved as an unambiguously supported clade within Habenariinae, and immediate sister
to the group formed by Clades X to XV (Fig. 2). Some misplaced
species of Peristylus, such as P. nematocaulon and P. coeloceras,
are nested within Platanthera or Herminium, whereas at least one
misplaced Platanthera with trilobed lip and ovoid tubers, namely
P. biermanniana, is nested within Peristylus. Geographically, Peristylus is mainly distributed in Asia in tropical to subtropical regions,
while Platanthera and Herminium mainly occur in alpine regions.
In our phylogenetic analyses, the currently delimited Peristylus
s.s. is strongly supported by molecular evidence and is more uniform in morphological characters, characterized by having ovoid
to globose tubers, flowers small, dorsal sepal and petals forming
a hood, lip trilobed and spurred, spur shorter than ovary, and pulvinate stigmas adnate to the base of lip. Therefore, we suggest to
make some intergeneric transfers to retain Peristylus as a monophyletic genus which mainly includes members from tropical
and subtropical regions.
4.3. Biogeography
Orchidinae are mostly distributed in northern temperate or
alpine regions, however, recent studies indicate that the widespread tropical genus Brachycorythis is sister to the remaining
Orchidinae (Bateman et al., 2003; Inda et al., 2010). There are
two centers of biodiversity of Orchidinae. One is the Mediterranean
Region, the other is the Pan-Himalayas (including Himalayas and
the Hengduan Mountains) (Pridgeon et al., 2001; Lang, 1998).
Inda et al. (2012) stated that Orchidinae have diversified in the
Mediterranean Region in the last 15 Million years. Many taxa of
Superclade A, such as Himantoglossum, Ophrys, Pseudorchis and Serapias, are predominantly Mediterranean (Pridgeon et al., 2001). On
the other hand, many genera in Superclade A, such as Platanthera
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
et al. (2012) showed that Platanthera and its alliance are deeply
nested within the Mediterranean Region group. Our results indicate that Clade III, IV, V, and VI (the Mediterranean Region group)
are resolved as successive sister to Platanthera and its alliance.
Members of Superclade B, including Hemipilia, Ponerochis, Sirindhornia and Tsaiorchis, are mostly restricted to mountain regions
in Eastern Asia, especially the Pan-Himalayas. Tropical/subtropical
genera, such as Sirindhornia and Tsaiorchis, are resolved as successive sister to alpine taxa (Fig. 1).
In contrast to Orchidinae, Habenariinae are widespread in tropical/subtropical regions around the world and only a few species
occur in alpine regions. Recent studies indicate that tropical African genera, such as Cynorkis and, Stenoglottis, are basal groups in
the subtribe (Bateman et al., 2003; Batista et al., 2013; Inda
et al., 2010, 2012). Our results indicate that alpine groups, such
as Herminium and its alliance (including Clade X, XI, XII, XIII, and
XIV, most endemic in Pan-Himalayas), are deeply nested within
Habenariinae (Fig. 2).
Taken together, it seems that the tropical ancestral group of
Orchideae radiated in the Mediterranean region in the last 15 million years and in the Pan-Himalayas in the last 8 million years
(Geographical history of Pan-Himalayas see Shi et al., 1998; Biogeography of Pan-Himalayas see Sun, 2002a, 2002b).
5. Main conclusions
Based on three DNA markers (plastid matK, rbcL, and nuclear
ITS), morphological characters and comprehensive sampling, our
current study greatly advances our understanding of the phylogeny of Orchidinae and Asian Habenariinae. Orchidinae are subdivided into two sister groups: one is Superclade A + Clade VI
(including Gymnadenia, Ophrys, Orchis and its alliance, and Platanthera and its alliance) with diversity centers in the Mediterranean
region and Pan-Himalayas, the other is Superclade B (Hemipilia,
Ponerorchis, Sirindhornia, and Tsaiorchis) and is mainly distributed
in Eastern Asia. Habenaria is subdivided into two distant related
groups: one group mainly distributed in tropical and subtropical
Asia, the other group (including type of Habenaria) is widespread
in both Old and New Tropics. Elements of alpine regions are deeply
nested within each tropical/subtropical group. Many genera, such
as Herminium, Platanthera, Peristylus and Ponerorchis, are not
monophyletic. Many previously undetected phylogenetic relationships, such as clades formed by Ponerorchis chrysea, the Platanthera
latilabris group, Sirindhornia, and Tsaiorchis, are well supported by
both molecular and morphological evidence. We propose to combine Hemipiliopsis with Hemipilia, to broaden Ponerorchis to include
Amitostigma and Neottianthe, to combine Aorchis, Aceratorchis, and
Neolindleya with Galearis, and to establish a new genus to accommodate Ponerorchis chrysea. Tsaiorchis and Sirindhornia are recognized as two distinctive genera. A nomenclatural revision is
provided in the Appendix A. At the same time, the relationships
of many groups, such as Herminium and its alliance (including Androcorys, Herminium, Platanthera latilabris group, and Porolabium)
and Galearis and its alliance (including Aceratorchis, Aorchis, Galearis, and Neolindleya) remain unresolved. Further work based on
broader sampling and more markers is needed.
Acknowledgements
Funds were provided by grants from the National Natural Science Foundation of China (Grant Nos. 31107176, 31311120061)
and the Chinese Special Fund for Medicine Research in the Public
Interest (201407003).
Appendix A
A.1. Nomenclatural revision
1. Galearis Raf.
(1) Galearis camtschatica (Cham.) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Orchis camtschatica Cham., Linnaea 3: 27 (1828).
2. Hemipilia Lindl.
Synonym.
Hemipiliopsis Y.B. Luo et S.C. Chen, syn. nov.
(2) Hemipilia purpureopunctata (K.Y. Lang) X.H. Jin, Schuit. et
W.T. Jin, comb. nov.
Basionym Habenaria purpureopunctata K.Y. Lang, Acta Phytotax.
Sin. 16(4): 127 (1978).
3. Peristylus Blume.
W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53
(3) Peristylus biermannianus (King et Pantl.) X.H. Jin, Schuit. &
W.T. Jin, comb. nov.
Basionym Habenaria biermanniana King et Pantl., J. Asiat. Soc.
Bengal. 64: 343 (1895).
4. Platanthera Rich.
Synonym.
Smithorchis Tang et F.T. Wang, syn. nov.
(4) Platanthera angustilabris (King et Pantl.) X.H. Jin, Schuit. et
W.T. Jin, comb. nov.
Basionym Herminium angustilabre King et Pantl. J. Asiat. Soc.
Bengal, 65(2): 131. 1895 (1896).
(5) Platanthera calceoliformis (W.W. Sm.) X.H. Jin, Schuit. et
W.T. Jin, comb.nov.
Basionym Herminium calceoliforme W.W. Sm., Notes Roy. Bot.
Gard. Edinburgh. 13: 211 (1921).
(6) Platanthera carnosilabris (Tang et F.T. Wang) X.H. Jin,
Schuit. et W.T. Jin, comb. nov.
Basionym Herminium carnosilabre Tang et F.T. Wang, Bull. Fan
Mem. Inst. Biol. Bot. 10: 32 (1940).
5. Ponerorchis Rchb. f.
Synonyms.
Amitostigma Schltr., syn. nov.
Neottianthe (Rchb.) Schltr., syn. nov.
(7) Ponerorchis alpestris (Fukuy.) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Amitostigma alpestre Fukuy., Bot. Mag. Tokyo 49: 664
(1935).
(8) Ponerorchis amplexifolia (Tang et F.T. Wang) X.H. Jin,
Schuit. et W.T. Jin, comb. nov.Basionym Amitostigma amplexifolium Tang et F.T. Wang, Bull. Fan Mem. Inst. Biol. Bot. 7: 3
(1936).
(9) Ponerorchis basifoliata (Finet) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Peristylus tetralobus f. basifoliatus Finet, Rev. Gén. Bot.
13: 525, pl. 13(C). (1901).
(10) Ponerorchis bidupensis (Aver.) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Hemipilia bidupensis Aver., Lindleyana 14: 222 (1999).
(11) Ponerorchis bifoliata (Tang et F.T. Wang) X.H. Jin, Schuit.
et W.T. Jin, comb. nov.
Basionym Amitostigma bifoliatum Tang et F.T. Wang, Bull. Fan
Mem. Inst. Biol. Bot. 7: 127 (1936).
(12) Ponerorchis capitata (Tang et F.T. Wang) X.H. Jin, Schuit. et
W.T. Jin, comb. nov.
Basionym Amitostigma capitatum Tang et F.T. Wang, Bull. Fan
Mem. Inst. Biol. Bot. 7: 4–5 (1936).
(13) Ponerorchis camptoceras (Rolfe) X.H. Jin, Schuit. et W.T.
Jin, comb. nov.
Basionym Habenaria camptoceras Rolfe, J. Linn. Soc., Bot., 29:
319 (1892).
(14) Ponerochis compacta (Schltr.) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Neottianthe compacta Schltr., Acta Horti Gothob. 1:
136 (1924).
(15) Ponerorchis cucullata (L.) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Orchis cucullata L., Sp. Pl.: 939 (1753).
(16) Ponerorchis cucullata var. calcicola (W.W. Sm.) X.H. Jin,
Schuit. et W.T. Jin, comb. nov.
Basionym Gymnadenia calcicola W.W. Sm., Notes Roy. Bot. Gard.
Edinburgh. 8: 188 (1914).
(17) Ponerorchis dolichocentra (Tang, F.T. Wang et K.Y. Lang)
X.H. Jin, Schuit. et W.T. Jin, comb. nov.
Basionym Amitostigma dolichocentrum Tang, F.T. Wang et K.Y.
Lang, Acta Phytotax. Sin. 20(1): 84 (1982).
51
(18) Ponerorchis faberi (Rolfe) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Habenaria faberi Rolfe, Kew Bull. 1896: 201 (1896).
(19) Ponerorchis farreri (Schltr.) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Amitostigma farreri Schltr., Repert. Spec. Nov. Regni
Veg. 20: 378 (1924).
(20) Ponerorchis gonggashanica (K.Y. Lang) X.H. Jin, Schuit. et
W.T. Jin, comb. nov.
Basionym Amitostigma gonggashanicum K.Y. Lang, Acta Phytotax. Sin. 22(4): 315 (1984).
(21) Ponerorchis gracilis (Blume) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Mitostigma gracile Blume, Mus. Bot. Lugd.-Bat. 2: 190
(1856).
(22) Ponerorchis keiskei (Finet) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Gymnadenia gracilis var. keiskei Finet, Bull. Soc. Bot.
France 47: 280 (1900).
(23) Ponerorchis kinoshitae (Makino) X.H. Jin, Schuit. et W.T.
Jin, comb. nov.
Basionym Gymnadenia kinoshitae Makino, Bot. Mag. (Tokyo) 23:
137 (1909).
(24) Ponerorchis lepida (Rchb. f.) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Gymnadenia lepida Rchb. f., Otia Bot. Hamburg.: 51
(1878).
(25) Ponerorchis luteola (K.Y. Lang et S.C. Chen) X.H. Jin, Schuit.
et W.T. Jin, comb. nov.
Basionym Neottianthe luteola K.Y. Lang et S.C. Chen, Acta Phytotax. Sin. 35(6): 545 (1996).
(26) Ponerorchis monantha (Finet) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Peristylus monanthus Finet, Rev. Gén. Bot. 13: 323
(1901).
(27) Ponerorchis oblonga (K.Y. Lang) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Neottianthe oblonga K.Y. Lang, Acta Phytotax. Sin.
35(6): 544 (1997).
(28) Ponerorchis ovata (K.Y. Lang) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Neottianthe ovata K.Y. Lang, Acta Phytotax. Sin. 35(6):
542 (1997).
(29) Ponerochis papilionacea (Tang, F.T. Wang et K.Y. Lang)
X.H. Jin, Schuit. et W.T. Jin, comb. nov.
Basionym Amitostigma papilionaceum Tang, F.T. Wang et K.Y.
Lang, Acta Phytotax. Sin. 20(1): 83 (1982).
(30) Ponerorchis parciflora (Finet) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Peristylus tetralobus f. parciflorus Finet, Rev. Gén. Bot.
13: 525 (1902) (as ‘parceflorus’).
(31) Ponerorchis physoceras (Schltr.) X.H. Jin, Schuit. et W.T.
Jin, comb. nov.
Basionym Amitostigma physoceras Schltr., Acta Horti Gothob. 1:
133 (1924).
(32) Ponerorchis pinguicula (Rchb. f. et S. Moore) X.H. Jin,
Schuit. et W.T. Jin, comb. nov.
Basionym Gymnadenia pinguicula Rchb. f. et S. Moore., J. Bot. 16:
135 (1878).
(33) Ponerochis simplex (Tang et F.T. Wang) X.H. Jin, Schuit. et
W.T. Jin, comb. nov.
Basionym Amitostigma simplex Tang et F.T. Wang, Bull. Fan
Mem. Inst. Biol. Bot. Ser. 10: 25 (1940).
(34) Ponerorchis secundiflora (Hook. f.) X.H. Jin, Schuit. et W.T.
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:
518 (1898).
(35) Ponerorchis tetraloba (Finet) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Peristylus tetralobus Finet, Rev. Gén. Bot. 13: 524
(1901).
(36) Ponerorchis thailandica (Seidenf. et Thaithong) X.H. Jin,
Schuit. et W.T. Jin, comb. nov.
Basionym Amitostigma thailandicum Seidenf. et Thaithong in G.
Seidenfaden, Contr. Orchid Fl. Thailand 13: 8 (1997).
(37) Ponerorchis tibetica (Schltr.) X.H. Jin, Schuit. et W.T. Jin,
comb. nov.
Basionym Amitostigma tibeticum Schltr., Repert. Spec. Nov. Regni Veg. 20: 379 (1924).
(38) Ponerorchis trifurcata (Tang, F.T. Wang et K.Y. Lang) X.H.
Jin, Schuit. et W.T. Jin, comb. nov.
Basionym Amitostigma trifurcatum Tang, F.T. Wang et K.Y. Lang,
Acta Phytotax. Sin. 20(1): 80, pl. 1(5–8) (1982).
(39) Ponerorchis wenshanensis (W. H. Chen, Y. M. Shui et K.Y.
Lang) X.H. Jin, Schuit. et W.T. Jin, comb. nov.
Basionym Amitostigma wenshanense W. H. Chen, Y. M. Shui et
K.Y. Lang, Acta Bot. Yunnan. 25(5): 521 (2003).
(40) Ponerorchis yuana (Tang et F.T. Wang) X.H. Jin, Schuit. et
W.T. Jin, comb. nov.
Basionym Amitostigma yuanum Tang et F.T. Wang, Bull. Fan
Mem. Inst. Biol. Bot. 10: 26 (1940).
6. Tsaiorchis Tang et F.T. Wang.
(41) Tsaiorchis keiskeoides (Gagnep.) X.H. Jin, Schuit. et W.T.
Jin, comb. nov.
Basionym Habenaria keiskeoides Gagnep., Bull. Soc. Bot. Fr.
78:71. (1931).
Synonym Tsaiorchis neottianthoides Tang et F.T. Wang, Bull. Fan
Mem. Inst. Biol. Bot. 7: 133 (1936).
Appendix B. Supplementary material
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.ympev.2014.
04.004.
References
Aceto, S., Caputo, P., Cozzolino, S., Gaudio, L., Moretti, A., 1999. Phylogeny and
evolution of Orchis and allied genera based on ITS DNA variation: morphological
gaps and molecular continuity. Mol. Phylogenet. Evol. 13, 67–76.
Bateman, R.M., Hollingsworth, P.M., Preston, J., Luo, Y.B., Pridgeon, A.M., Chase,
M.W., 2003. Molecular phylogenetics and evolution of Orchidinae and selected
Habenariinae (Orchidaceae). Bot. J. Linn. Soc. 142, 1–40.
Bateman, R.M., James, K.E., Luo, Y.B., Lauri, R.K., Fulcher, T., Cribb, P.J., Chase, M.W.,
2009. Molecular phylogenetics and morphological reappraisal of the Platanthera
clade (Orchidaceae: Orchidinae) prompts expansion of the generic limits of
Galearis and Platanthera. Ann. Bot.-London 104, 431–445.
Batista, J.A.N., Borges, K.S., de Faria, M.W.F., Proite, K., Ramalho, A.J., Salazar, G.A.,
van den Berg, C., 2013. Molecular phylogenetics of the species-rich genus
Habenaria (Orchidaceae) in the new world based on nuclear and plastid DNA
sequences. Mol. Phylogenet. Evol. 67, 95–109.
Bellusci, F., Pellegrino, G., Palermo, A.M., Musacchio, A., 2008. Phylogenetic
relationships in the orchid genus Serapias L. based on noncoding regions of
the chloroplast genome. Mol. Phylogenet. Evol. 47, 986–991.
Box, M.S., Bateman, R.M., Glover, B.J., Rudall, P.J., 2008. Floral ontogenetic evidence
of repeated speciation via paedomorphosis in subtribe Orchidinae
(Orchidaceae). Bot. J. Linn. Soc. 157, 429–454.
Bytebier, B., Bellstedt, D.U., Linder, H.P., 2007. A molecular phylogeny for the large
African orchid genus Disa. Mol. Phylogenet. Evol. 43, 75–90.
Chase, M.W., Cameron, K.M., Barret, R.L., Freudenstein, J.V., 2003. DNA data and
Orchidaceae systematics: a new phylogenetic classification. In: Dixon, K.W.,
Kell, S.P., Barret, R.L., Cribb, P.J. (Eds.), Orchid Conservation. Natural History
Publications, Kota Kinabalu, pp. 69–89.
Chen, S.C., Lang, K.Y., Stephan, W.G., Phillip, J.C., Paul, O., 2009. Subfam.
Orchidoideae. In: Wu, Z.Y., Raven, P.H., Hong D.Y. (Eds.), Flora of China. vol.
25. Science Press, Beijing, Missouri Botanical Garden, St. Louis, pp. 45–166.
Comber, J.B., 2001. Orchids of Sumatra. The Royal Botanic Gardens Kew, London.
Cunningham, C.W., 1997. Can three incongruence tests predict when data should be
combined? Mol. Biol. Evol. 14, 733–740.
Devos, N., Raspé, O., Jacquemart, A., Tyteca, D., 2006. On the monophyly of
Dactylorhiza Necker ex Nevski (Orchidaceae): is Coeloglossum viride (L.)
Hartman a Dactylorhiza? Bot. J. Linn. Soc. 152, 261–269.
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
nuclear ribosomal ITS sequences. Am. J. Bot. 86, 887–899.
Dressler, R.L., 1981. The Orchids: Natural History and Classification. Harvard
University Press, Cambridge.
Dressler, R.L., 1993. Phylogeny and classification of the orchid family. Dioscorides
Press, Portland.
Duthie, J.F., 1906. The orchids of the North-Western Himalaya. Ann. Roy. Bot. Gard.
(Calcutta) 9, 85–211.
Efimov, P.G., Lauri, R.K., Bateman, R.M., 2009. Neolindleya Kraenzl. (Orchidaceae), an
enigmatic and largely overlooked autogamous genus from temperate East Asia.
Kew. Bull. 64, 661–671.
Farris, J.S., Kälersjö, M., Kluge, A.G., Bult, C., 1995. Constructing a significance test for
incongruence. Syst. Biol. 44, 570–572.
Felsenstein, J., 1988. Phylogenies from molecular sequences: inference and
reliability. Annu. Rev. Genet. 22, 521–565.
Gamarra, R., Galán, P., Herrera, I., Ortúňez, E., 2008. Seed micromophology supports
the splitting of Limnorchis from Platanthera (Orchidaceae). Nordic J. Bot. 26, 61–
65.
Garay, A., Kettredge, W., 1985. Notes from the Ames Orchid Herbarium. Bot. Mus.
Leaf. Harv. Univ. 30, 47–59.
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,
95–98.
Hapeman, J.R., Inoue, K., 1997. Plant-pollinator interactions and floral radiation in
Platanthera (Orchidaceae). In: Givnish, T.J., Sytsma, K.J. (Eds.), Molecular
Evolution and Adaptive Radiation. Cambridge University Press, Cambridge, pp.
433–454.
Hooker, J.D., 1890. The flora of British India 6: Orchideae. Reeve, London.
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
intron sequences to the phylogenetics of tribe Orchideae (Orchidaceae): do the
distribution and sequence of this intron tell us something about its evolution?
Taxon 59, 1053–1064.
Inda, L.A., Pimentel, M., Chase, M.W., 2012. Phylogenetics of tribe Orchideae
(Orchidaceae: Orchidoideae) based on combined DNA matrices: inferences
regarding timing of diversification and evolution of pollination syndromes. Ann.
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,
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.
Jin, W.T., Zhou, H.L., Jin, X.H., 2013. Platanthera yadongensis (Orchidaceae,
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.
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.
Kraenzlin, F., 1901. Orchidacearum Genera et Species, vol. 1. Mayer and Müller,
Berlin.
Kurzweil, H., Weber, A., 1992. Floral morphology of southern African Orchideae. II.
Habenariinae. Nordic J. Bot. 12, 39–61.
Lang, K.Y., 1998. A new subgenus of Platanthera (Orchidaceae). Acta Phytotaxon. Sin.
36, 449–458.
Lang, K.Y., 1999. Flora Republicae Popularis Sinicae, vol. 17. Science Press, Beijing.
Lindley, J., 1835. The Genera and Species of Orchidaceous Plants. Ridgways
Piccadilly, London.
Luer, C.A., 1975. The Native Orchids of the United States and Canada Excluding
Florida. New York Botanical Garden, New York.
Luo, Y.B., Chen, S.C., 2003. Hemipiliopsis, a new genus of Orchidaceae. Novon 13,
450–453.
Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway
for inference of large phylogenetic trees. In: Proceedings of the Gateway
Computing Environments Workshop (GCE), 14 November 2010, New Orleans,
LA, pp. 1–8.
Pearce, N.R., Cribb, P.J., 2002. Orchideae. In: Pearce, N.R., Cribb, P.J. (Eds.),The
Orchids of Bhutan. Royal Botanic Garden Edinburgh, Edinburgh, Royal
Government of Bhutan, Bhutan, pp. 122–192.
Pedersen, H.A., Suksathan, P., Indhamusika, S., 2002. Sirindhornia, a new orchid
genus from Southeast Asia. Nordic J. Bot. 22, 391–404.
Posada, D., Crandall, K.A., 1998. Modeltest: testing the model of DNA substitution.
Bioinformatics 14, 817–818.
Pridgeon, A.M., Cribb, P.J., Chase, M.W., Rasmussen, F.N., 2001. Genera
Orchidacearum, vol. 2. Oxford University Press, Oxford.
Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics 19, 1572–1574.
Schlechter, R., 1924. Additamenta ad Orchideologiam chinensem. (Orchidaceae
novae et criticae) Repert. Spec. Nov. Regni Veg. 19, 372–383.
Seidenfaden, G., 1977. Orchid genera in Thailand V: Orchidoideae. Dansk Bot. Ark.
31, 1–149.
W.-T. Jin et al. / Molecular Phylogenetics and Evolution 77 (2014) 41–53
Shi, Y.F., Li, J.J., Li, B.Y., 1998. Uplift and Environmental Changes of Qinghai-Tibetan
Plateau in the Late Cenozoic. Guangdong Science and Technology Press,
Guangzhou.
Soliva, M., Kocyan, A., Widmer, A., 2001. Molecular phylogenetics of the sexually
deceptive orchid genus Ophrys (Orchidaceae) based on nuclear and chloroplast
DNA sequences. Mol. Phylogenet. Evol. 20, 78–88.
Soó, R., 1929. Revision der Orchideae-Ophrydineae von Ostasien und dem
Himalaya. Annls Hist.-Nat. Mus. Nat. Hung. 26, 339–384.
Soó, R., 1966. Die sog. Orchis Arten der Ostasiatisch-Nordamerikanischen Flora. Acta
Bot. Acad. Sci. Hung. 12, 351–354.
Sun, H., 2002a. Tethys retreat and Himalayas-Hengduanshan Mountains uplift and
their significance on the origin and development of the Sino-Himalayan
elements and alpine flora. Acta Bot. Yunnan. 24, 273–288.
Sun, H., 2002b. Evolution of Arctic-Tertiary Flora in Himalayan-Hengduan
Mountains. Acta Bot. Yunnan. 24, 671–688.
Swofford, D.L., 2002. PAUP⁄: phylogenetic analysis using parsimony (⁄ and Other
Methods), version 4.0b10. Sinauer, Sunderland.
53
Szlachetko, D.L., Kras, M., 2006. Notes sur le genre Habenella. Richardiana 6, 33–39.
Tang, T., Wang, F.T., 1940. Contributions to the knowledge of eastern Asiatic
Orchidaceae I. Bull. Fan Mem. Inst. Biol. Bot. Ser. 10, 23–46.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The
CLUSTAL_X windows interface: flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882.
Tuyama, T., 1966. Orchidaceae. In: Hara, H. (Ed.), The Flora of Eastern Himalaya.
University of Tokyo, Tokyo, pp. 424–452.
Tuyama, T., 1971. Orchidaceae. In: H. Hara (Ed.), The Flora of Eastern Himalaya,
Second Report. University of Tokyo, Tokyo, pp. 176–196.
Tuyama, T., 1975. Orchidaceae. In: H. Ohashi (Ed.), The Flora of Eastern Himalaya,
Third Report. University of Tokyo Press, Tokyo, pp. 137–164.
Tyteca, D., Klein, E., 2008. Genes, morphology and biology – the systematics of
Orchidinae revisited. J. Eur. Orch. 40, 501–544.
Vaidya, G., Lohman, D.J., Meier, R., 2011. SequenceMatrix: concatenation software
for the fast assembly of multi-gene datasets with character set and codon
information. Cladistics 27, 171–180.