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A New Myco-Heterotrophic Genus, Yunorchis, and the Molecular Phylogenetic Relationships of the Tribe Calypsoeae (Epidendroideae, Orchidaceae) Inferred from Plastid and Nuclear DNA Sequences

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We identified a new holomycotrophic orchid that is related to the myco-heterotrophic Calypsoeae. Because chloroplast genes are primarily lacking or are highly divergent, key morphological characters are either reduced or lost from many myco-heterotrophs, and the phylogenetic relationships of weakly supported paraphyletic Calypsoeae within Epidendroideae have been poorly understood in previous molecular systematic studies. Using chloroplast rbcL, psaB, and matK and nuclear Xdh and ITS sequences, we determined the circumscription and systematic positions of the new orchid and the tribe. The results indicate that the epidendroid taxa include most of the clades that are successively sister to the grade of clades representing previously recognized tribes. Calypsoeae comprising four well-supported clades with 12 genera (except for the previous temporarily placed Wullschlaegelia) is supported as a monophyletic and sister clade to Epidendreae (excluding Coeliinae). The new orchid is nested in Calypsoeae and is a sister to Dactylostalix and/or Calypso. This new holomycotrophic orchid presents a subumbel inflorescence that grows underground, and flower with a long pedicel reputing the ground to open and two fragments at the base of the hook, which are obviously morphologically different from those of Calypsoeae. To accommodate this species in the current generic circumscription, a new genus Yunorchis was created.
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RESEARCH ARTICLE
A New Myco-Heterotrophic Genus, Yunorchis,
and the Molecular Phylogenetic Relationships
of the Tribe Calypsoeae (Epidendroideae,
Orchidaceae) Inferred from Plastid and
Nuclear DNA Sequences
Guo-Qiang Zhang
1,2,4
, Ming-He Li
2,4
, Yong-Yu Su
1,3
, Li-Jun Chen
1
, Si-Ren Lan
2,4
*,
Zhong-Jian Liu
1,2,3
*
1Shenzhen Key Laboratory for Orchid Conservation and Utilization, The National Orchid Conservation
Center of China and The Orchid Conservation and Research Center of Shenzhen, Shenzhen, China,
2College of Forestry, Fujian Agriculture and Forestry University, Jinshan, Fuzhou, Fujian, China, 3College
of Forestry, South China Agricultural University, Guangzhou, China, 4Orchid Conservation and Research
Center of Fujian Agriculture and Forestry University, Jinshan, Fuzhou, Fujian, China
These authors contributed equally to this work.
*lkzx@fafu.edu.cn (SRL); liuzj@sinicaorchid.org (ZJL)
Abstract
We identified a new holomycotrophic orchid that is related to the myco-heterotrophic Calyp-
soeae. Because chloroplast genes are primarily lacking or are highly divergent, key mor-
phological characters are either reduced or lost from many myco-heterotrophs, and the
phylogenetic relationships of weakly supported paraphyletic Calypsoeae within Epidendroi-
deae have been poorly understood in previous molecular systematic studies. Using chloro-
plast rbcL,psaB, and matK and nuclear Xdh and ITS sequences, we determined the
circumscription and systematic positions of the new orchid and the tribe. The results indi-
cate that the epidendroid taxa include most of the clades that are successively sister to the
grade of clades representing previously recognized tribes. Calypsoeae comprising four
well-supported clades with 12 genera (except for the previous temporarily placed Wulls-
chlaegelia) is supported as a monophyletic and sister clade to Epidendreae (excluding Coe-
liinae). The new orchid is nested in Calypsoeae and is a sister to Dactylostalix and/or
Calypso. This new holomycotrophic orchid presents a subumbel inflorescence that grows
underground, and flower with a long pedicel reputing the ground to open and two fragments
at the base of the hook, which are obviously morphologically different from those of Calyp-
soeae. To accommodate this species in the current generic circumscription, a new genus
Yunorchis was created.
PLOS ONE | DOI:10.1371/journal.pone.0123382 April 22, 2015 1/17
OPEN ACCESS
Citation: Zhang G-Q, Li M-H, Su Y-Y, Chen L-J, Lan
S-R, Liu Z-J (2015) A New Myco-Heterotrophic
Genus, Yunorchis, and the Molecular Phylogenetic
Relationships of the Tribe Calypsoeae
(Epidendroideae, Orchidaceae) Inferred from Plastid
and Nuclear DNA Sequences. PLoS ONE 10(4):
e0123382. doi:10.1371/journal.pone.0123382
Academic Editor: Genlou Sun, Saint Mary's
University, CANADA
Received: October 24, 2014
Accepted: February 5, 2015
Published: April 22, 2015
Copyright: © 2015 Zhang et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: Funds were provided by the Science and
Technology Project of Guangdong (Grant No.
2011B060400011) and the Scientific Research
Foundation of the Graduate School of Fujian
Agriculture and Forestry University (Grant No.
1122yb006). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
Introduction
Nonphotosynthetic mycorrhizal plants have long attracted the attention of botanists and my-
cologists and have been the target of unabated controversy and speculation [1]. Worldwide,
there are more than 400 species (approximately 90 genera) of fully myco-heterotrophic/holo-
mycotrophic and almost 20 000 partially myco-heterotrophic/heteromycotrophic flowering
plants [2]. Myco-heterotrophic angiosperms comprise part of the Burmanniaceae, Corsiaceae,
Ericaceae, Gentianaceae, Iridaceae, Orchidaceae, Petrosaviaceae, Polygalaceae, Thismiaceae,
and Triuridaceae families. Among these families, Orchidaceae occupies a large number of
groups [3]. The majority of holomycotrophic flowering plants are restricted to the tropics, but
myco-heterotrophic Ericaceae and some Orchidaceae have been observed in temperate forests
[2]. Here, we conducted a field study in the tropics of Yunnan, China, and identified a new
holomycotrophic orchid that is morphologically related to the Calypsoeae tribe.
The tribe Calypsoeae (Orchidaceae, Epidendroideae) comprises approximately 12 genera
and 70 species [4]. Recently, Zhai et al. [5] added one new holomycotrophic member Dan-
xiaorchis singchiana. The species of the Calypsoeae tribe are typically terrestrial or myco-het-
erotrophs (such as Corallorhiza,Danxiaorchis, and Yoania) with nodes on a pseudobulb, a
shrimp ridge velamen, and an occasionally lateral inflorescence with several to many flowered
four pollinia in two pairs, except for Wullschlaegelia, which is characterized by two pollinia and
was temporarily placed in Calypsoeae by Pridgeon et al. [4] based on the phylogenies of the
chloroplast genes. Apart from the Corallorhiza (including approximately 11 species), Govenia
(20), Oreorchis (16), and Tipularia (7), which occupy a majority of the species of this tribe, and
other genera are monotypic or oligotypic, especially the saprophytic genera. Calypsoeae are dis-
tributed from Europe, northern Asia and the Americas and absent from Africa, Australia, and
the islands of the East Indies and Pacific Ocean [6].
Because of the reduction or loss of key morphological characters, the taxonomic affinities of
many myco-heterotrophic Calypsoeae have remained elusive for many decades [2]. The posi-
tion and circumscription of the tribe Calypsoeae has been variable, and the genera that belong
to this tribe have been previously included in different tribes or subtribes [7,8,9], e.g., Dressler
placed Wullschlaegelia and Govenia in Gastrodiinae and Cymbidieae, respectively [8]. In addi-
tion, Yoania australis, a species that is endemic to New Zealand, was renamed Danhatchia aus-
tralis and placed in Cranichideae (Orchidoideae) based on the anatomical features [4]. The
molecular tools for inferring the phylogenetic position of myco-heterotrophic plants have re-
cently become available [10]. The results of recent phylogenetic analyses of combined chloro-
plast genes [11,12,13,14] have provided new insight into the systematics of the tribe
Calypsoeae with a paraphyly in the subfamily Epidendroideae, the first group comprising Ca-
lypso,Changnienia and Tipularia, and the second group comprising Aplectrum,Corallorhiza,
Cremastra,Danxiaorchis,Govenia, and Oreorchis. However, the relationships between the two
groups in the context of the remainder of Epidendroideae remain unclear [4].
The presence of unusual winter leaves in some of the members of the two supported groups
(paraphyletic Calypsoeae within Epidendroideae) is indicative of a close relationship [4]. The
results of Zhai et al. [5] suggest that Calypsoeae is a monophyletic tribe that is sister to Gastro-
dia, with a PP of 52 based on 26 genera samplings (except for 13 genera of Calypsoeae) in Epi-
dendroideae. Although Xiang et al. [15] sampled a total of 96 genera for the phylogenetic
analysis of Epidendroideae based on the combined chloroplast genes (rbcL,matK and psaB),
these authors sampled only one supported groups (two genera, Calypso and Tipularia)of
Calypsoeae, and the results indicated the two genera is sister to Dendrobieae/Malaxideae in a
Bayesian inference with a PP of 0.80 and unsupported in Maximum parsimony analyses. Addi-
tionally, Pridgeon et al. [4] indicated that the two Japan endemic genera Dactylostalix and
A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
PLOS ONE | DOI:10.1371/journal.pone.0123382 April 22, 2015 2/17
Competing Interests: The authors have declared
that no competing interests exist.
Ephippianthus form a well-supported clade within Calypsoeae based on the morphological
characters. The family-wide analyses of Zhai et al. [5] indicated these two genera (without mo-
lecular data) formed a clade as sister to the Calypso clade based on the combined molecular
markers (rbcL,matK and ITS) and morphological character matrix; however, the tribal-wide
analyses formed a sister clade to the Calypso and Govenia clades. ITS was popular to permit
systematic comparisons of low levels of taxa and chloroplast genes were preliminary lacking or
divergent in many myco-heterotrophic Calypsoeae, both of which caused biases in inferred
phylogenies [2,16]. These weak evidence and sparse sampling/molecular markers indicate that
the tribal phylogenetic position within Epidendroideae and the circumscription of myco-het-
erotrophic Calypsoeae remain unresolved.
In this study, we used DNA sequences for the chloroplast genes rbcL,psaB and matK and
the nuclear ITS and low-copy protein-coding gene Xdh to determine the circumscription and
systematic positions of new myco-heterotrophic orchids and the Calypsoeae tribe in Epiden-
droideae and to infer molecular phylogenetic relationships within Calypsoeae.
Results
Sequence characteristics
The general features of the DNA regions that were used are presented in Table 1. The attempt
to amplify psaB from the new myco-heterotrophic orchid (Yunorchis pingbianensis) failed. It is
possible that this region is lacking. A total of 1047 bp of rbcL and 1458 bp of matK sequences
(GenBank accession KM526763 and KM526774) were assessed, and none obvious deletion/ad-
dition loci were observed among the sequences.
In the subfamily-wide matrix, 104 DNA sequences of rbcL and matK,87ofpsaB, and 70 of
Xdh were obtained. The most variable dataset was Xdh with 47.25% potentially informative
sites (excluding uninformative characters). Parsimony analyses resulted in a parsimonious tree
of 2150 steps (TL) with a consistency index (CI) = 0.4647 and a retention index (RI) = 0.5843.
The second variable dataset was matK (31.92% potentially informative sites), which had the
lowest CI = 0.3560 and RI = 0.5182. The third most variable dataset was rbcL with 16.05% po-
tentially informative sites. The least variable dataset was psaB with 11.75% potentially informa-
tive sites, which had the highest CI = 0.5835 and RI = 0.6291. In the combined three
chloroplast markers (rbcL,matK,psaB) matrix, which comprised 4646 aligned nucleotides
Table 1. Parsimony statistics from phylogenetic analyses of the various datasets.
Data Taxa Aligned length Information site TL CI RI
Subfamily-wide matrix
rbcL 104 1346 216 (16.05%) 1182 0.5051 0.5755
matK 104 1623 518 (31.92%) 2384 0.3560 0.5182
psaB 87 1677 197 (11.75%) 862 0.5835 0.6291
rbcL+matK+psaB 104 4646 931 (20.04%) 5140 0.4811 0.5309
Xdh 70 963 455 (47.25%) 2150 0.4647 0.5843
combined 104 5609 1386 (24.71%) 7333 0.4739 0.5438
Tribal-wide matrix
rbcL 29 1296 113 (8.72%) 360 0.8139 0.6042
matK 33 1704 209 (12.27%) 497 0.8390 0.9378
rbcL+matK 36 3000 322 (10.73%) 913 0.8160 0.8918
ITS 32 673 205 (30.46%) 641 0.7285 0.7573
combined 36 3673 527 (14.35%) 1593 0.7659 0.8362
doi:10.1371/journal.pone.0123382.t001
A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
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with 931 potentially informative sites (20.04%), TL = 5140, CI = 0.4811 and RI = 0.5309. In the
combined chloroplast and nuclear Xdh markers (963 bp) matrix, which comprised 5609
aligned nucleotides with 1386 potentially informative sites (24.71%), TL = 7333, CI = 0.4739
and RI = 0.5438.
In the tribe-wide matrix, 29 DNA sequences of rbcL,33ofmatK, and 32 of ITS were ob-
tained. The most variable dataset was ITS, with 30.46% potentially informative sites. Parsimony
analyses resulted in a parsimonious tree of 641 steps with the lowest CI = 0.7285 and
RI = 0.7573. The second most variable dataset was matK with 12.27% potentially informative
sites, which had the highest CI = 0.8390 and RI = 0.9378. The least variable dataset was rbcL
with 8.72% potentially informative sites. In the combined two chloroplast markers (rbcL,
matK) matrix, which comprised 3000 aligned nucleotides with 322 potentially informative sites
(10.73%), TL = 913, CI = 0.8160 and RI = 0.8918. In the combined two chloroplast and nuclear
ITS markers (673 bp) matrix, which comprised 3673 aligned nucleotides with 527 potentially
informative sites (14.35%), TL = 1593, CI = 0.7659 and RI = 0.8362 (Table 1).
Phylogeny of the subfamily Epidendroideae
The results from the single and combined analyses were presented by the Bayesian trees. The
values of the Bayesian Posterior probabilities (PP) and Bootstrap percentage (BS
ML
and BS
MP
)
are indicated at each node. The branches that collapse in the strict consensus tree are indicated
by -. In the nuclear Xdh analysis (S1 Fig), the results with added new molecular data are not
completely in accordance with the results of Górniak et al. [11], especially the topology. The
tribe Calypsoeae, including the new molecular taxa (Dactylostalix and Danxiaorchis), is sup-
ported as monophyletic with weak support (PP = 0.36, BS
ML
= 30, and BS
MP
= 60) and sister to
Epidendreae (excluding Coelia).
No strongly supported, incongruent patterns of relationship were detected in the individual
chloroplast markers analyses, so a combined analysis of all of the chloroplast data (rbcL,matK,
and psaB) was performed. The trees of this analysis are shown in S2 Fig. The paraphyletic
Calypsoeae (two supported groups) were observed, and the results were consistent with previ-
ous molecular studies. The nuclear and chloroplast trees that were generated by the Bayesian
inference analyses (BI) were congruent with those that were retrieved by the ML and MP analy-
ses except for the poorly supported nodes (PP <0.90).
Except for Calypsoeae and/or Wullschlaegelia, there were no strongly supported incongru-
ent results in the topology of the Xdh trees and the combined chloroplast trees. Although the
Calypsoeae is paraphyletic in chloroplast trees, the weak support and the presence of winter
leaves in some members (except for the Wullschlaegelia) of the two supported groups indicate
a close relationship. Therefore, the chloroplast and nuclear DNA were combined into a single
dataset for the phylogenetic analyses (Fig 1). Our findings are consistent with the overall topol-
ogy of the trees that are produced by the BI, ML, and MP methods, except for a few collapsed
nodes (such as Dendrobieae and Malaxideae). The Posterior Probability values (PP) were often
higher than the Bootstrap values (BS). The tree that was recovered in the combined analysis is
more similar to that based on the Xdh than the combined chloroplast.
In the Lower or basalEpidendroideae, highly supported clades include a monophyletic
clade (PP = 1.00 and BS = 100) consisting of Neottieae sister to all of the remaining Epiden-
droideae, followed by Sobralieae (PP = 1.00 and BS >50) and the other four tribes with a rela-
tively weak bootstrap and an unstable topology. Higher Epidendroideae taxa include most of
the clades that are successively (PP >0.90 and BS >50) sister to the grade of clades represent-
ing previously recognized tribes. Arethuseae are monophyletic as sister to all of the other
Higher Epidendroideae with strong support (PP = 1.00 and BS >90), followed by Cymbidieae
A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
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(PP = 0.90 and BS = 65), and then Malaxideae plus Dendrobieae with a relatively weak boot-
strap and an unstable topology. The next most supported clade (PP = 0.99) is divided into
three tribes: Collabieae, Podochileae and Vandeae (excluding Agrostophyllinae) and strongly
support (PP >0.90 and BS >75) were observed between them. The last clade is divided into
four groups, comprising the first group Epidendreae (excluding Coelia), sister to the second
group Calypsoeae (excluding Wullschlaegelia) with a stable topology, the third group Coelia
(PP = 0.95) with an unstable topology, and the fourth group Agrostophyllinae with strong sup-
port (PP = 1.00 and BS >75). The new holomycotrophic orchid, as a sister to Calypso,isa
member of the tribe Calypsoeae.
Phylogeny of Calypsoeae
Based on the ITS sequence data, the relationships in the strict consensus tree for most of the
genera are resolved and well supported (PP >0.95 and BS >75) with only one genus
(Oreorchis) receiving a paraphyly (S3 Fig). The basal group comprises Calypso,Changnienia,
Dactylostalix, and Yunorchis. A relatively weak bootstrap in Bayesian posterior probabilities
(PP = 0.61) and stable topologies in bootstrap analyses (BS = 100) between this group and oth-
ers were observed. The new orchid Yunorchis pingbianensis is a sister to Dactylostalix ringens
with a PP of 0.96, BS
ML
of 64 and BS
MP
of 85.
The 12 genera can be distinguished easily from the phylograms that are based on the chloro-
plast sequence data analysis. The phylogenetic topologies that are generated from BI are consis-
tent with those from the ML and MP analyses (S4 Fig). The basal group comprises Calypso,
Changnienia,Dactylostalix,Tipularia, and Yunorchis.Yunorchis pingbianensis is a sister to Ca-
lypso bulbosa with a PP of 0.96, BS
ML
of 85 and BS
MP
of 83. The chloroplast sequence data anal-
ysis showed a relatively stronger bootstrap between this group and others, with PP = 1.00 and
BS >75.
In this study, ITS, matK and rbcL were combined into a single dataset. The results showed
that the currently defined Calypsoeae is subdivided into four clades (Fig 2). Clade I, comprising
the genera Calypso,Changnienia,Dactylostalix,Tipularia and Yunorchis, is strongly supported
(PP = 1.00 and BS = 100) and is followed by the others. Yunorchis pingbianensis, as a sister to
Dactylostalix ringens, has been recognized as a natural genus within this tribe. Clade II includes
only the genus Govenia, which is strongly supported (PP = 1.00, BS
ML
= 72, and BS
MP
= 99)
and is followed by a complex group (clade III), which includes Aplectrum,Cremastra,Dan-
xiaorchis,Oreorchis, and Yoania. The topologies of this group were different, with a PP of 1.00
and a relatively weak bootstrap and an unstable topology of ML and MP phylograms (no
show), and were subdivided into four subclades (Fig 2). The last clade contains a single genus,
Corallorhiza, which comprises 13 taxa, although the phylograms showed that the data are not
intragenetically well supported.
Discussion
The chloroplast genome loci rbcL and matK have been used extensively in angiosperm phylo-
genetic reconstruction because of their relatively high substitution rate in combination with a
conserved overall structure. Leafless, putatively achlorophyllous orchids such as Corallorhiza
Fig 1. Phylogenetic relationships of the subfamily Epidendroideae based on the chloroplast DNA
rbcL,matK, and psaB and the nuclear DNA Xdh. The numbers near the nodes are Bayesian posterior
probabilities and bootstrap percentages (PP left, BS
ML
middle, and BS
MP
right). *indicates that the node is
100% supported. -indicates that the node is incongruent between the topology of the MP/ML trees and the
Bayesian tree.
doi:10.1371/journal.pone.0123382.g001
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often experience deletions in their plastid genomes, and in some cases this can be severe, e.g.,
Rhizanthella (Orchidoideae), with only 59190 bp, is the least gene-rich plastid genome known
to date apart from the fragmented plastid genome of some dinoflagellate [17]. Freudenstein
and Doyle [18] found much less deletion in the plastid genome of Corallorhiza than in other
leafless angiosperms that have been examined. The rbcL and matK have not been found in Rhi-
zanthella [17] and a lot of deletion loci/ fragment of them were observed in Corallorhiza [18]
and Yoania [5], but few loci were loss in Danxiaorchis and the new orchid. This gradually lost
pattern seems that the leafless habit of orchids has developed several times and at least three
times within Calyposeae.
Subfamily-wide analysis of Epidendroideae
The use of multiple genes is helpful in the accuracy of phylogenetic reconstruction [19,20].
Xiang et al. [15] combined the rbcL,matK, and psaB sequences into a single dataset for the
analysis of Epidendroideae, but the sampling and data support for Calypsoeae was weak. In
this study, the data for the combined chloroplast genes (rbcL,matK, and psaB) indicate that the
tribe Calypsoeae is divided into two supported groups (PP <0.50, BS <50). The results of
these chloroplast analyses are consistent with those that were obtained in previous studies
[11,13,14]. Górniak et al. [11] indicated that 1) the low-copy nuclear protein-coding gene of
Xdh is appropriate for resolving the intrafamilial relationships, 2) the phylogenetic relation-
ships of Xdh within Orchidaceae are consistent with those of previous analyses using other
DNA data, 3) Xdh is more variable than the plastid genes matK and ycf1 [21] and also can be
used at the subtribal and generic levels, and 4) the difficultCalypsoeae taxa have been re-
solved better using Xdh. In this study, the results of the Xdh analyses are consistent with those
of Górniak et al. [11] and those of previous studies [4,5,21], but the topologies and higher sup-
ports were not entirely consistent with the results of previous studies. The chloroplast genes in
many myco-heterotrophs are primarily lacking or are highly divergent, which causes biases in
inferred phylogenies [2,16]. The Calypsoeae primarily comprise holomycotrophs or heteromy-
cotrophs that obtain carbon from a symbiotic fungus; this arrangement suggests that the chlo-
roplast genes are lacking, deleted or highly divergent [2,22]. These results indicate that the
phylogenetic systematic position of the results for Calypsoeae, which lack some chloroplast
genes, would be more reasonable based on the combination of rbcL,matK,psaB, and Xdh into
a single dataset for the phylogenetic analysis of Epidendroideae. The sister relationship of
Calypsoeae and Epidendreae was also recovered in the result of Nuebig et al. [21] based on the
plastid gene ycf1 using parsimony analysis.
The relationships of Coelia (Coeliinae) to other genera have been problematic for more than
a century. Based on multiple DNA (rbcL,matK,trnL-F, and ITS) analyses, van den Berg et al.
[23] described a circumscription of Epidendreae that includes only five New World subtribes,
namely, Bletiinae, Chysinae, Laeliinae, Pleurothallidinae, and Ponerinae, and concluded that
Coelia is a sister to Calypsoeae. However, Pridgeon et al. [4] placed Coelia as a subtribe within
Epidendreae based on their unpublished analyses. In our analyses (Fig 1 and S1 Fig), Coelia is a
sister to Epidendreae plus Calypsoeae. Additionally, van den Berg et al. [23] indicated that the
subtribe Agrostophyllinae of Vandeae is a sister to the core Vandeae(Aeridinae, Angraeci-
nae, and Polystrachyinae) plus the tribe Cymbidieae, but the node was weakly supported. In
Bayesian analysis based on the Xdh gene, Górniak et al. [11] recovered similar relationships;
Fig 2. Phylogenetic relationships of the tribe Calypsoeae based on the combined nrDNA ITS and chloroplast DNA rbcL,matK.The numbers that are
near the nodes are Bayesian posterior probabilities and bootstrap percentages (PP left, BS
ML
middle, and BS
MP
right). *indicates that the node is 100%
supported. -indicates that the node is incongruent between the topology of the MP/ML trees and the Bayesian tree.
doi:10.1371/journal.pone.0123382.g002
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Earina (Agrostophyllinae) was far from the core Vandeae, being a sister to Epidendreae plus
Calypsoeae. In our chloroplast analysis (combining rbcL,matK, and psaB), Agrostophyllinae
was a sister to one of the groups of Calypsoeae (S2 Fig). In the nuclear Xdh (S1 Fig) and com-
bined rbcL,matK,psaB and Xdh results (Fig 1), the Agrostophyllinae was a sister (PP >0.90
and BS >75) to the group that was formed by Coeliinae being a sister to Calypsoeae/Epiden-
dreae. Therefore, the inclusion of Coeliinae (only Coelia) and Calypsoeae in Epidendreae and
the system position of Agrostophyllinae warrant further study.
The systematic position of neotropical Wullschlaegelia has long been in trouble. Szlachetko
[24] described the genus as a monotypic subtribe within Gastrodieae based on the similar seed
morphology, sectile pollinia, cellular viscidium, and the lack of chlorophyll. However, these
morphological data are not supported by chloroplast DNA data, suggesting that this genus
nests in Calypsoeae. Based on the sequences of the chloroplast genes rbcL and matK, Pridgeon
et al. [4] and Zhai et al. [5] placed this genus within Calypsoeae. Based on the sequence of nu-
clear Xdh, Górniak et al. [11] indicated that this genus is a sister to Xerorchis within the
lowerEpidendroideae. Szlachetko [24] also indicated differences in the floral morphology
and the position of the anther with Gastrodieae. Because Wullschlaegelia is a holomycotrophic
plant [25], the nuclear evidence appears to be more credible [2]. Moreover, Wullschlaegelia has
walls in a striate-rugulate pattern with free-standing baculae between them or coarsely verru-
cate-striate or gemmate [4]. The pollinia of Orchidaceae have been considered informative
sources of both taxonomic and phylogenetic information and are thought to be less susceptible
to parallelism than other floral features [26]. Indeed, the number of pollinia in Wullschlaegelia
is two, and the members of Calypsoeae are four. These data suggest that this genus and Calyp-
soeae are not closely related. Therefore, we conducted analyses with Wullschlaegelia, which is a
sister to Xerorchis, based on our results (Fig 1 and S1 and S2 Figs) and the conclusion of Gór-
niak et al. [11].
Phylogenetic relationships within Calypsoeae
The present phylogenetic study was primarily conducted based on the molecular dataset that
was described by Zhai et al. [5]. In this study, we performed an additional DNA sequence anal-
ysis using the excluded highly homoplasious morphological characters. Although the phyloge-
netic relationships were fully assessed and discussed in the previous study using the combined
analysis of molecular and morphological characters, most of the support was weak. Based on
the new molecular systematic evidence with greater support and in the interest of nomenclatur-
al stability, we divided this tribe into four clades, and we redefined the four subtribes, namely,
Aplectrinae, Cremastrinae, Wullschalaegeliinae (the genus Oreorchis in this study), and Yoanii-
nae, which were circumscribed by Zhai et al. [5], as four subclades of clade II.
In the phylogenetic analyses (Fig 2 and S3 and S4 Figs), the new holomycotrophic orchid
were not nested into the same group with the other three (Corallorhiza,Danxiaorchis and Yoa-
nia) holomycotrophic genera of Calyposeae. The Corallorhiza comprising 11 species that are
confined to the northern hemisphere of the New World except for one circumboreal species
(C.trifida), is one of the largest leafless genera in Orchidaceae [9], and the species are charac-
terized by the coralloid rhizome and unlobed labellum narrowing to a claw at base with two
free lamellae [4]. Danxiaorchis is founded in Guangdong, China, and the species possesses a la-
bellum with a large Y-shaped callus and fleshy seeds [5]. Yoania is sister to Danxiaorchis in the
phylogeny of combined chloroplast rbcL and matK (Fig 2 and S4 Fig). The same relationships
were observed in Zhai et al. studies [5] based on the combined molecular markers and/or mor-
phological character matrix. These indicated that Yoania is most closely related to Dan-
xiaorchis. Additionally, the inflorescences of Yoania stand straight up, the labellum bears a
A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
PLOS ONE | DOI:10.1371/journal.pone.0123382 April 22, 2015 9/17
prominent spur and the pollinarium have four superposed pollinia [4]. However, the subumbel
inflorescences of the new orchid grow underground, the sac-shaped labellum has hook and
two fragments at the base of the hook, the pollinarium are in two pairs and the two pollinia of
each pair are unequal in size. These features make it easy to distinguish the new orchid from
the other holomycotrophic genera of Calyposeae.
The new orchid, Yunorchis, which is a sister to Dactylostalix (Fig 2 and S1 Fig) and/or Ca-
lypso (S2 Fig), Changnienia, and Tipularia, form an independent root clade (clade I) of Calyp-
soeae. The genera Calypso,Dactylostalix, and Yunorchis have a trilobed labellum. The lip lobe
of Calypso occupies the first half of the sac border, the lobe base has tufted setae, and the label-
lum tip has two calcariform objects. This green leaf plant has an erect raceme and is primarily
distributed in temperate regions. While the lip lobe of Yunorchis is small and is located in the
middle of the labellum sac, the sac border before the lobe closes upward and forms the unci-
form tip. The entire plant, including the inflorescence, grows underneath the ground, and the
flower stretches toward the earths surface when blooming; therefore, only a few flowers are ob-
served scattering on the ground, and this plant is completely saprophytic and is distributed in
tropical regions. Both genera have obvious plant morphology and habitat differences and are
different from Changnienia and Tipularia, which have a distinct calcar in the labellum, green
leaves, a scape growing along the ground, and inflorescences with one or many tiny flowers.
Pridgeon et al. [4] indicated that the Dactylostalix and Ephippianthus have viscidia with only
the rudiments of a stipe and are unusual in having only slightly thickened rhizomes and no
corms. Moreover, both genera show leaves in some seasons. Unfortunately, the molecular mate-
rials of the other genus Ephippianthus could not be obtained. Zhai et al. [5] indicated that the
genus formed a sister with Dactylostalix based on morphological characteristics. The inflores-
cence of Dactylostalix, which has one or two sheathing bracts, solitary flowers, pollinarium with
four superposed pollinia and a viscidium with a rudimentary stipe [4], and the few-flowered
plants of Ephippianthus, which have membranous bracts, an entire labellum, and pollinarium
with four superposed pollinia [4], are significantly different from the new holomycotrophic or-
chid and the other orchids of the Calypso Clade.
Conclusions
The new orchid entity is restricted to Pingbian County in southern Yunnan, China, and is char-
acterized by plants that have subumbel inflorescences growing underground. The flowers of
these plants have a long pedicel rupturing the ground, a sac-shaped labellum, and a sac mouth
that is connate on the front half of the edges, forming a hook that has two fragments at the
base. These features distinguish the new orchid from all of the other known orchids.
Based on the combined sequences of the chloroplast genes rbcL,psaB, and matK and the nu-
clear low-copy protein-coding gene Xdh, the subfamily-wide molecular analysis revealed better
topology and higher support compared to previous studies, with strong evidence that Calyp-
soeae is a monophyletic sister tribe to Epidendreae (except for Coelia) and that Wullschlaegelia
is not a member of Calypsoeae. Based on the combined sequences of the chloroplast genes rbcL
and matK and the nuclear ITS gene, the subfamily-wide molecular analysis revealed that
Calypsoeae comprises four well-supported clades with 12 genera. The newly identified orchid
has several distinct features, and molecular analyses indicate that this plant represents an inde-
pendent lineage under the tribe Calypsoeae. This lineage should be treated as a new genus in
the Calypso Clade with the following classification:
Subfamily: Epidendroideae
Tribe: Calypsoeae
A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
PLOS ONE | DOI:10.1371/journal.pone.0123382 April 22, 2015 10 / 17
Yunorchis pingbianensis Z. J. Liu, G. Q. Zhang et M. H. Li, gen. et sp. nov. (Figs 3and 4)
[urn:lsid:ipni.org:names: 771453311] Type: China, Yunnan, Pingbian, in a forest, alt. 2100 m,
2013. 5.31. Z. J. Liu 7103 (holotype, NOCC).
Fig 3. Yunorchis pingbianensis Z. J. Liu, G. Q. Zhang et M. H. Li: A. Flowering plant; B. Flower, front view; C. Lateral sepal, petal, dorsal sepal and
lip; D. Lip and column, side view; E. Lip, longitudinal section; F. Pollinarium.
doi:10.1371/journal.pone.0123382.g003
A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
PLOS ONE | DOI:10.1371/journal.pone.0123382 April 22, 2015 11 / 17
Etymology. The generic name alludes to the type locality, Yunnan, and incorporates the
Greek name for orchid; Yunorchis refers to an orchid genus in Yunnan province, China. The
specific epithet pingbianensis refers to the type of species of Yunorchis that grows in Pingbian
county of Yunnan.
Fig 4. Yunorchis pingbianensis Z. J. Liu, G. Q. Zhang et M. H. Li: A. Flowering in nature habitat; B. Flowering plant; C. Inflorescence; D. Pedicel and
ovary; E. Lip, bottom view; F. Flower, front view; G. Floral part; H. Pollinarium.
doi:10.1371/journal.pone.0123382.g004
A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
PLOS ONE | DOI:10.1371/journal.pone.0123382 April 22, 2015 12 / 17
Diagnosis. This new remarkable genus is distinct from all known orchid genera; it com-
prises an entire plant with a subumbel inflorescence growing underground, flowers that each
have a long pedicel that ruptures the ground to open and that has a sac-shaped labellum, a sac
mouth that is connate on the front half edges, forming a hook and two fragments at the base of
the hook, and four waxy pollinia in two pairs, each containing two pollinia that are unequal in
size, and there are no conspicuous caudicles that are attached directly to a common viscidium.
Description. Holomycotrophic plants 69 cm tall, rhizome tuberous, cylindrical, 46cm
long, 57 mm thick, with many short branches. Entire plant growing underground, scape aris-
ing from the terminal of rhizome, 1 or 2 inflorescences, terete, pale red-brow, 46-sheathed;
sheaths, cylindrical, clasping scape stem, membranous, 1.01.5 cm long; subumbel inflores-
cence, 0.50.8 cm long, 26-flowered; flowers rupturing the ground when open, floral bracts
broadly ovate or ovate-elliptic, 11.6 cm long, obtuse at apex; pedicel and ovary 3.85.0 cm
long, glabrous, twisted; sepals pale purple-red; petals white; lip white, spotted with purple-red
at apex and inside; dorsal sepals oblong, 22.3 cm long, 6.07.5 mm wide, obtuse at apex; later-
al sepals ovate-elliptic, concave, with incurved margins, 2.02.3 cm long, 0.81.0 cm wide, ob-
tuse at apex; petals narrowly oblong, 1.92.1 cm long, 1.61.8 cm wide, obtuse at apex; lip
semiglobose-saccate, 1.71.9 cm long; sac mouth connate on front half edges forming a hook,
with the lateral lobes reduced at the base of hook; disk with a purple prominent central papil-
late-pubescent callus extending from base to apex, column 78 mm long, 5.05.5 mm wide,
with a 1.71.9 mm long fingerlike process projecting on either side of the anther at the apex,
with a 23 mm long column foot at the base; anther terminal, anther cap ellipsoid, with a horn
at the apex; four pollinia, in two pairs, narrowly elliptic, waxy, each pair containing two pollinia
that are unequal in size, lacking conspicuous caudicles, attached directly to a common visci-
dium; and stigma concave, broad and large; rostellum conspicuous; capsule fusiform. Fl. May
June.
Materials and Methods
Ethics statement
The locations of the field studies are neither private lands nor protected areas but are controlled
by the State Forestry Administration of China, with which our institution is affiliated. The
State Forestry Administration authorized us to conduct scientific observations or tests in the
regions that it controls. A valid permit was obtained for testing the genes of Yunorchis.
Taxon and gene sampling
To reconstruct a robust phylogeny for determining the circumscription and systematic posi-
tions of Calypsoeae, we sampled as many taxa as possible from all of the tribes recognized with-
in Epidendroideae by Chase et al. [27] and Pridgeon et al. [4,28,29]. A total of 104 genera were
sampled. The outgroup included two genera from the subfamily Cypripedioideae and one
genus from the subfamily Apostasioideae. The sampled strategy is not only important for ex-
amining relationships, but the inclusion of DNA sequence data from four nuclear and chloro-
plast regions was expected to permit possible reinterpretation of previous hypotheses that
included only few of these genomes at a time [11,12,13]. A composite of slowly evolving genes
was used to infer the phylogenetic relationships at the tribal/subtribal levels. All of the terminal
taxa represent a single genus and include at least three of the four DNA markers (rbcL,matK,
psaB and Xdh), except for the new orchid and Agrostophyllum, which include two markers
(rbcL and matK). The Dactylostalix is sampled for the first time in a broad molecular phyloge-
netic study. The voucher information and GenBank accession numbers are listed in S1 Table.
A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
PLOS ONE | DOI:10.1371/journal.pone.0123382 April 22, 2015 13 / 17
A second series of analyses focused on the tribe Calypsoeae. A composite of slowly (rbcL
and matK) and quickly (ITS) evolving genes and DNA regions was used to infer the phyloge-
netic relationships. The analysis is primarily based on the molecular dataset that was published
by Zhai et al. [5] based on the combined analysis of molecular and morphological characters.
In this study, we performed additional DNA extraction (six DNA sequences of rbcL, four of
matK, and five of the ITS were newly obtained) excluding the highly homoplasious morpholog-
ical characters for molecular phylogenetic analyses. This tribal-wide matrix included 34 in-
group species and two outgroups (Agrostophyllum majus and Earina autumnalis). The voucher
information and GenBank accession numbers are listed in S2 Table. DNA extraction, PCR am-
plification, sequencing, sequence editing and assembly were performed according to Zhang
et al. [30]. The primers are showed in S3 Table.
Phylogenetic analyses
Phylogenetic analyses were performed under Bayesian inference (BI) and the MP and ML
methods. We performed the BI analysis using MrBayes 3.1.2 [31]. For the ML analysis, we used
RAxML version 7.2.8 with 100 bootstrap replicates and settings as described in Stamatakis
et al. [32]. The homogeneities of the subfamily Epidendroideae between nuclear Xdh and com-
bined chloroplast DNA and the tribe Calypsoeae between nuclear ITS data and the combined
chloroplast DNA dataset were tested using the incongruence length difference (ILD) test [33],
as implemented in PAUPversion 4.0b10 [34]. The ILD test was conducted with 1000 repli-
cates, each with 10 random addition sequence replicates and TBR branch swapping, maintain-
ing no more than 100 trees per random addition replicate. Following Cunningham [35], a
significance level of P = 0.01 was used for this test. The MP analyses were performed using
PAUPversion 4.0b10 [34]. The test settings included 1000 replications of randomly added se-
quences and a heuristic search with tree bisection-reconnection branch swapping. Table 1 lists
the tree length (TL), consistency index (CI), and retention index (RI).
Nomenclature acts
The electronic version of this article in Portable Document Format (PDF) in a work with an
ISSN or ISBN will represent a published work according to the International Code of Nomen-
clature for algae, fungi, and plants; therefore, the new names that are contained in the electron-
ic publication of a PLoS ONE article are effectively published under that Code from the
electronic edition alone. There is no longer any need to provide printed copies.
In addition, the new names that are contained in this work have been submitted to IPNI,
from where they will be made and are available to the Global Names Index. The IPNI LSID
771453311 can be resolved and the associated information viewed through any standard web
browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The
online version of this work is archived and available from the following digital repositories:
PubMed Central, LOCKSS.
Supporting Information
S1 Fig. Phylogenetic relationships of the subfamily Epidendroideae based on the nuclear
DNA Xdh. The numbers near the nodes are Bayesian posterior probabilities and bootstrap
percentages (PP left, BS
ML
middle, and BS
MP
right). indicates that the node is 100% sup-
ported. -indicates that the node is incongruent between the topology of the MP/ML trees
and the Bayesian tree.
(TIF)
A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
PLOS ONE | DOI:10.1371/journal.pone.0123382 April 22, 2015 14 / 17
S2 Fig. Phylogenetic relationships of the subfamily Epidendroideae based on the chloro-
plast DNA rbcL,matK, and psaB.The numbers near the nodes are Bayesian posterior proba-
bilities and bootstrap percentages (PP left, BS
ML
middle, and BS
MP
right). indicates that the
node is 100% supported. -indicates that the node is incongruent between the topology of the
MP/ML trees and the Bayesian tree.
(TIF)
S3 Fig. Phylogenetic relationships of the tribe Calypsoeae based on the nrDNA ITS. The
numbers near the nodes are Bayesian posterior probabilities and bootstrap percentages (PP
left, BS
ML
middle, and BS
MP
right). indicates that the node is 100% supported. -indicates
that the node is incongruent between the topology of the MP/ML trees and the Bayesian tree.
(TIF)
S4 Fig. Phylogenetic relationships of the tribe Calypsoeae based on the combined chloro-
plast rbcL and matK.The numbers near the nodes are Bayesian posterior probabilities and
bootstrap percentages (PP left, BS
ML
middle, and BS
MP
right). indicates that the node is
100% supported. -indicates that the node is incongruent between the topology of the MP/
ML trees and the Bayesian tree.
(TIF)
S1 Table. Taxa, voucher, and GenBank accession numbers of the Epidendroideae that were
used in this study. A dash (-) indicates missing data, an asterisk () denotes the sequences that
were obtained in this study (the voucher and location are listed in S2 Table), and the remaining
sequences are from GenBank.
(DOC)
S2 Table. Taxa, voucher, and GenBank accession numbers of the Calypsoeae that were used
in this study. A dash (-) indicates missing data, an asterisk () denotes sequences that were ob-
tained in this study, and the remaining sequences are from GenBank.
(DOC)
S3 Table. Primers that were used for amplification and sequencing in this study.
(DOC)
Acknowledgments
The authors are grateful to Wei-Rong Liu and Wen-Hui Rao for assistance with the fieldwork
and to Gui-Zhen Chen for the analysis of the molecular sequence data.
Author Contributions
Conceived and designed the experiments: ZJL SRL MHL GQZ. Performed the experiments:
GQZ MHL. Analyzed the data: MHL GQZ. Contributed reagents/materials/analysis tools: ZJL
GQZ YYS LJC. Wrote the paper: MHL ZJL GQZ. Revised the manuscript draft: ZJL SRL MHL
GQZ.
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A New Genus and Molecular Phylogenetic Relationships of Calypsoeae
PLOS ONE | DOI:10.1371/journal.pone.0123382 April 22, 2015 17 / 17
... Most recently, two new leafless genera were proposed and one genus has been subsumed under another. The two new genera, Danxiaorchis and Yunorchis, were published in association with phylogenetic analyses of the subtribe by Zhai et al. (2013) and Zhang et al. (2015), respectively. Zhai et al. (2013) analyzed the subtribe based on ITS, matK and rbcL sequences, as well as morphology. ...
... Their tree had poor support for much of its backbone, leaving fundamental relationships in the group unclear. Zhang et al. (2015) named Yunorchis based in part on the position of this new taxon in their phylogenetic analysis. The new genus is morphologically highly similar to Yoania, but their analysis suggested that it is only relatively distantly related to Yoania. ...
... Cremastra aphylla was not included in their analysis, but the placement of Cremastra in the tree would necessitate a second origination of holomycotrophy in this genus. Zhang et al.'s (2015) analysis placed Danxiaorchis + Yoania japonica as sister to Oreorchis with poor support and placed their newly described genus, Yunorchis, as sister to Dactylostalix, also with poor support. Yunorchis in that case would represent a separate loss of leaves and Cremastra aphylla would represent a third, with Corallorhiza then a fourth instance of leaf loss in the group. ...
Article
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Calypsoinae are a small, largely temperate subtribe that are diverse with respect to trophic strategy (many species have an unusual winter leaf and several are leafless) and pollinium stalk morphology. Here we sought to re-evaluate the placement of Yoania and recently proposed genera and to examine trophic strategy, pollinium stalk type, and geographic distribution in a phylogenetic context. We analyzed ITS and matK sequences from all accepted genera using maximum likelihood and parsimony on individual and combined data sets. The only supported disagreement among trees was between the combined ML and parsimony analyses for the placement of Dactylostalix and Ephippianthus; the trees from one analysis reflected the nuclear pattern, while the other resembled the plastid pattern. A group of genera related to Calypso and a group related to Corallorhiza were resolved. Yoania is closely related to Calypso; the recently proposed Yunorchis is a species of Yoania, while Didiciea is part of Tipularia. Examining leaf morphology on the tree revealed two originations of the winter leaf morphology and four losses of leaves (and shifts to mycoheterotrophy). Pollinium stalks evolved in three directions, two of which resulted in epidermally-derived stalks (tegulae) and one that comprises the entire rostellum (a hamulus). Biogeographic analysis suggests a New World origin for the subtribe, with two major shifts to the Old World and one shift back to the New World.
... The newly described orchids included 23 more genera (29 new genera and six combined genera) and 141 more species than FRPS. Subsequently, more than 10 combined genera and 20 new or newly recorded genera (e.g., Chen & Liu, 2010;Li et al., 2011Li et al., , 2014aLi et al., , 2015Liu et al., 2011;Huang et al., 2012;Xiang et al., 2012;Yang et al., 2013;Zhai et al., 2013Zhai et al., , 2014Zhang et al., 2013Zhang et al., , 2015aJin et al., 2014Jin et al., , 2015Tang et al., 2015a), and 60 species (e.g., Hu et al., 2013Hu et al., , 2014Jin et al., 2013;Li & Yan, 2013;Peng et al., 2013;Guan et al., 2014;Li et al., 2014b;Meng et al., 2014;Tian et al., 2014;Xu et al., 2014;Zou et al., 2014;Su et al., 2015) have been reported in recent years. The completion of the FRPS, Flora of China, and the updated taxa of orchids have contributed greatly to the understanding of Chinese orchid classification and diversification. ...
... Within the higher epidendroids, support for the relationships of these clades is not high, and the sequences of certain genera are lacking; thus, the relationships among the tribes and monophylies of Epidendreae are not clearly refuted on the basis of the TL results. The topology of the RT is nearly consistent with previous molecular analyses (G orniak et al., 2010;Luo et al., 2014;Givnish et al., 2015;Zhang et al., 2015a) and the classification of Chase et al. (2015) and the tribe and subtribe levels are successively sister to the next; thus, we chose the results of the RT to elucidate the arrangement of the suprageneric levels. ...
... The bestsampled analysis in terms of data was carried out by , who provided a mixed result depending on the type of analysis (ML vs. MP). Based on the combined chloroplasts of rbcL, matK, and psaB, and the nuclear Xdh, the results of Zhang et al. (2015a) showed that Arundina is sister to Coelogyninae. ...
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We estimated the molecular phylogenetic relationships of the Chinese members of the family Orchidaceae. Within the Tree of Life for the Genera of Chinese Vascular Plants using atpB, rbcL, matK, ndhF, and matR, the currently delimited subfamilies, tribes, and subtribes are highly supported as monophyletic except for the perplexing Epidendroideae. Five genes (rbcL, matK, psaB, ycf1, and Xdh), which are more universally used in Orchidaceae, were further analyzed to reconstruct the phylogeny of Epidendroideae. The reconstructed trees were in strong agreement and showed significant support for the tribal and subtribal clades. Based on the highly supported circumscription and arrangement of the suprageneric levels in the Tree of Life and reconstructed trees, we have proposed a new phylogenetic classification of Chinese Orchidaceae that includes five subfamilies, 17 tribes, and 21 subtribes.
... Phylogenetic analyses based on combined DNA sequences (ITS, matK and rbcL) and morphological characters matrix indicated that Danxiaorchis is a member of the tribe Calypsoeae in the subfamily Epidendroideae (Zhai et al. 2013). Under Calypsoeae, Danxiaorchis was inferred to be most closely related to Yoania Maximowicz (1873: 68), and this relationship was subsequently confirmed by Zhang et al. (2015). However, the only sequence for Yoania included in both Zhai et al. (2013) and Zhang et al. (2015) data sets was a partial rbcL sequence of 222 bp out of the full length of 1428 bp, and Freudenstein et al. (2017) noted that such a high proportion of missing data of Yoania would output a spurious relationship for Danxiaorchis and Yoania. ...
... Under Calypsoeae, Danxiaorchis was inferred to be most closely related to Yoania Maximowicz (1873: 68), and this relationship was subsequently confirmed by Zhang et al. (2015). However, the only sequence for Yoania included in both Zhai et al. (2013) and Zhang et al. (2015) data sets was a partial rbcL sequence of 222 bp out of the full length of 1428 bp, and Freudenstein et al. (2017) noted that such a high proportion of missing data of Yoania would output a spurious relationship for Danxiaorchis and Yoania. After including more representatives and sequences of Yoania and reanalyzing generic relationships among subtribe Calypsoinae, Freudenstein et al. (2017) found that Danxiaorchis emerges in Corallorhiza (Gagnebin 1755: 61) clade and is sister to Oreorchis (Lindley 1859: 26) + Corallorhiza, while Yoania emerges in Calypso (Salisbury 1807: 89) clade. ...
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Danxiaorchis yangii (Calypsoinae, Epidendreae, Epidendroideae), a holomycotrophic new species from Jinggangshan National Nature Reserve, western Jiangxi, eastern of China, is here illustrated and described based on both morphological and phylogenetic evidences. The new species can be easily distinguished from D. singchiana by its much smaller flowers and larger Y-shaped callus adaxially bearing a additional obovoid appendage, and by its four pollinia narrowly elliptic in shape and equal in size.
... including new genera Danxiaorchis [14], Hsenhsua [15], Shizhenia [16] and Yunorchis [17], the newly recorded genus Thaia [18] [33]; plastid phylogenomic resolution of subtribes Aeridinae [19] and Goodyerinae [34]; genera phylogenetics such as Dendrobium [35], Cymbidium [36], Holcoglossum [37] and Paphiopedilum [38] In parallel with the examination of plant's organelle genomes, opportunities are rising for using transcriptomic data for orchid phylogenetics. Several orchid phylotranscriptomics have been performed for addressing either broad-scale or shallow-scale orchid relationships including species diversification and genome evolution, as well as key traits like sexual deception [39][40][41][42][43]. ...
Article
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Orchidaceae is one of the largest, most diverse families in angiosperms with significant ecological and economical values. Orchids have long fascinated scientists by their complex life histories, exquisite floral morphology and pollination syndromes that exhibit exclusive specializations than any other plants on earth. These intrinsic factors together with human influences also make it a keystone group in biodiversity conservation. The advent of sequencing technologies and transgenic techniques represents a quantum leap in orchid research, enabling molecular approaches to be employed to resolve the historically interesting puzzles in orchid basic and applied biology. To date, 15 different orchid genomes covering four subfamilies (Apostasioideae, Vanilloideae, Epidendroideae and Orchidoideae) have been released. These genome projects have given rise to massive data that greatly empowers the studies pertaining to key innovations and evolutionary mechanisms for the breadth of orchid species. The extensive exploration of transcriptomics, comparative genomics, and recent advances in gene engineering have linked important traits of orchids with a multiplicity of gene families and their regulating networks, providing great potential for genetic enhancement and improvement. In this review, we summarize the progress and achievement in fundamental research and industrialized application of orchids with a particular focus on molecular tools, and make future prospects of orchid molecular breeding and post-genomic research, providing a comprehensive assemblage of start-of-art knowledge in orchid research and industrialization.
... including new genera Danxiaorchis [14], Hsenhsua [15], Shizhenia [16] and Yunorchis [17], the newly recorded genus Thaia [18] [33]; plastid phylogenomic resolution of subtribes Aeridinae [19] and Goodyerinae [34]; genera phylogenetics such as Dendrobium [35], Cymbidium [36], Holcoglossum [37] and Paphiopedilum [38] In parallel with the examination of plant's organelle genomes, opportunities are rising for using transcriptomic data for orchid phylogenetics. Several orchid phylotranscriptomics have been performed for addressing either broad-scale or shallow-scale orchid relationships including species diversification and genome evolution, as well as key traits like sexual deception [39][40][41][42][43]. ...
Article
Full-text available
Orchidaceae is one of the largest, most diverse families in angiosperms with significant ecological and economical values. Orchids have long fascinated scientists by their complex life histories, exquisite floral morphology and pollination syndromes that exhibit exclusive specializations than any other plants on earth. These intrinsic factors together with human influences also make it a keystone group in biodiversity conservation. The advent of sequencing technologies and transgenic techniques represents a quantum leap in orchid research, enabling molecular approaches to be employed to resolve the historically interesting puzzles in orchid basic and applied biology. To date, 15 different orchid genomes covering four subfamilies (Apostasioideae, Vanilloideae, Epidendroideae and Orchidoideae) have been released. These genome projects have given rise to massive data that greatly empowers the studies pertaining to key innovations and evolutionary mechanisms for the breadth of orchid species. The extensive exploration of transcriptomics, comparative genomics, and recent advances in gene engineering have linked important traits of orchids with a multiplicity of gene families and their regulating networks, providing great potential for genetic enhancement and improvement. In this review, we summarize the progress and achievement in fundamental research and industrialized application of orchids with a particular focus on molecular tools, and make future prospects of orchid molecular breeding and post-genomic research, providing a comprehensive assemblage of start-of-art knowledge in orchid research and industrialization.
... Thus, setae are found on the labellum of Calypso Salisb. (Zhang et al. 2015), Catasetum (Franken et al. 2016) and Pterostylis R.Br. (Janes et al. 2010), which use food deception (Tuomi et al. 2015), perfume rewarding (Milet-Pinheiro and Gerlach 2017) and sexual deception (Phillips et al. 2013) as pollination strategies, respectively. ...
Article
Floral innovations are key for pollinator specialization and play a significant role in plant diversification. Orchidaceae present many examples of floral innovations that allow its high degrees of pollinator specialization and promoted speciation. The rich neotropical genus Telipogon evolved an uncinate (=hook-like) viscidium on the pollinarium and setae-bearing flowers, which are uncommon in Orchidaceae; however, the importance of them on pollination success and whether they are floral innovations or exaptations in Telipogon have not been investigated. Here we investigate the morphology of the viscidium and floral setae within the Telipogon alliance (including the genera Hofmeisterella, Trichoceros and Telipogon), test their significance in pollination, and their occurrence and evolution across the Oncidiinae. We used Telipogon peruvianus as a model species to test whether uncinate viscidium and floral setae increased pollination success compared with a cochleariform (=spoon-like) viscidium and lack of floral setae condition. We show that the uncinate viscidium is a synapomorphy for Telipogon; setae-bearing flowers are not universally found among all species of Telipogon and evolved once in the Telipogon alliance. Furthermore, Telipogon peruvianus flowers with an uncinate viscidium have achieved higher pollinia export than those with cochleariform viscidium (ancestral condition), whereas flowers with setae have both higher success in pollinia removal, although not significant, and pollinia deposition than those lacking of setae (ancestral condition). We demonstrate that uncinate viscidia and floral setae in Telipogon are a key innovation and exaptation, respectively, that enhance pollination success and they might act as drivers of diversification and pollinator specialization in this genus.
... The analysis of a partial sequence of rRNA gene is considered an efficient molecular tool for species identification which can indorse the phenotype-based methods which sometimes produce refuted results (Jagielski et al., 2013). The small subunit, SSU rRNA is a common molecular marker frequently used in diversity research (Lindeque et al., 2013;Fonseca et al., 2014;Wu et al., 2015) and phylogenetic studies (Petrov et al., 2014;Zhang et al., 2015). The SSU rRNA are widely used for identification of species from different taxonomic groups from bacteria to higher animals (Jagielski et al., 2013;Wu et al., 2015). ...
Article
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Ostracods are microscopic crustaceans living in a wide variety of environments. They are very important as the key food item for fish and benthic macro-invertebrates and are used as bio-indicators for environmental changes and pollution. The identification and taxonomy of the Heterocypris species are so challenging issues because of their morphological plasticity that is often related to environmental factors. The studied species is recorded for the first time in Egypt. Therefore, Morphological evidences for the identification of this species were thoroughly recorded using the scanning electron and stereo microscopes. Furthermore, DNA barcoding was used to confirm the morphological identification. The morphological examination identified the species as Heterocypris salina, where it revealed an accurate description for its carapace, valves and appendages. However, few dissimilarities with the description of that species by other authors were noted. The sequencing of partial sequences of 28S, 18S rRNA genes confirmed the results of the morphological identification. New barcodes for 28S, 18S rRNA genes and cytochrome oxidase subunit 1, COI gene, of H. salina were added to GenBank databases. Based on morphological and genetic evidences, this study adds new species to the biodiversity inventory of the aquatic environment of Egypt. Furthermore, COI sequence for Heterocypris salina has been submitted on the GenBank databases for the first time to be used as a marker for identification of such species in the future.
... The outgroup included one genus from the tribe Vandeae Lindley (1826: 14) and two genera from Epidendreae Lindley (1826: 16). The selection of outgroups was based on the phylogenetic analysis of Zhang et al. (2015), which placed the clade Malaxideae-Vandeae-Epidendreae as sister to Cymbidieae based on Xdh, psaB, rbcL and matK. ...
Article
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The subtribal delimitation and relationship of Old World Cymbidiinae and Eulophiinae within Cymbidieae remain unresolved. Relationships among all subtribes of Cymbidieae (Orchidaceae: Epidendroideae) were estimated using the coding genes psaB, rbcL, matK, ycf1 and Xdh for 103 taxa. The results indicate that most of the clades are successively sister to the grade of clades representing previously recognized subtribes, and Dipodium does not belong in its previous classification of Cymbidiinae and Eulophiinae. Instead, this genus represents an additional isolated lineage.
Article
Recently, two new genera of mycoheterotrophic orchids were described from China: Danxiaorchis Zhai, Xing & Liu (2013: e60371) and Yunorchis Liu, Zhang & Li (2015: e123382). Both were considered to be monospecific, and in each case the descriptions were accompanied by a phylogenetic analysis. It was inferred that Danxiaorchis is the sister genus of another mycoheterotrophic genus, Yoania Maximowicz (1873: 68), whereas Yunorchis was found to be closely related to the autotrophic genera Calypso Salisbury (1807: Tab. 89) and Dactylostalix Reichenbach (1878: 74). Support for the sister-group relationship of Danxiaorchis and Yoania appeared to be strong, and the clade consisting of Calypso, Dactylostalix and Yunorchis had a high Bayesian posterior probability. These analyses suggested that Yunorchis and Yoania were only distantly related. However, based on additional molecular analyses, Freudenstein et al. (2017) found that Yunorchis and Yoania formed a clade. They also argued, and we would agree, that Yunorchis and Yoania do not significantly differ morphologically.
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In this study, an updated checklist of Orchidaceae for China is presented based on all recently available literature and online resources relevant to new taxa, national or regional new records, and new taxonomic papers. This checklist includes 208 genera and 1695 taxa. Additionally, two new national records for Orchidaceae from Yunnan, viz. Rhomboda lanceolata and Porpax elwesii, are reported with detailed morphological descriptions and illustrations.
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The order Orchidales is divided into three families: Apostasiaceae, Cypripediaceae and Orchidaceae, with the last containing eight subfamilies. Appropriate new combinations at species level toalling 315 are proposed. The system proposed here is not based solely on similarities and differences in the generative structures. To avoid a too unilateral approach to the subject the author also used classical morphological data when analysing evolutionary connections between orchid groups (leaf venation, position of the inflorescence, etc.), collected during studies of herbarium materials and available in floristic works already published. -from Author
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ROGERS, DS, IF GREENBAUM, SJ GUNN, AND MD ENGSTROM. 1984. Cytosystematic value of chromosomal inversion data in the genus Peromyscus (Rodentia: Cricetidae). J. Mammal. 65: 457-465. RUVOLO, M. 1992. Molecular evolutionary processes can produce ...
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Collabieae (Orchidaceae) is a long neglected tribe with confusing tribal and generic delimitation and little-understood phylogenetic relationships. Using plastid matK, psaB, rbcL, and trnH-psbA DNA sequences and morphological evidence, the phylogenetic relationships within the tribe Collabieae were assessed as a basis for revising their tribal and generic delimitation. Collabieae (including the previously misplaced mycoheterotrophic Risleya) is supported as monophyletic and nested within a superclade that also includes Epidendreae, Podochileae, Cymbidieae and Vandeae. Risleya is nested in Collabiinae and sister to Chrysoglossum, a relationship which, despite their great vegetative differences, is supported by floral characters. Ania is a distinct genus supported by both morphological and molecular evidence, while redefined Tainia includes Nephelaphyllum and Mischobulbum. Calanthe is paraphyletic and consists four clades; the genera Gastrorchis, Phaius and Cephalantheropsis should be subsumed within Calanthe. Calanthe sect. Ghiesbreghtia is nested within sect. Calanthe, to which the disputed Calanthe delavayi belongs as well. Our results indicate that, in Collabieae, habit evolved from being epiphytic to terrestrial.
Book
In Volume Two of Genera Orchidacearum, three of the seven tribes found in the subfamily Orchidoideae-Orchideae, Diurideae, and Diseae are thoroughly described. Each of the 101 genera in this volume receive separate treatment with sections on nomenclature and synonymy, distribution, anatomy, palynomy, embryology, cytogenetics, ecology, phytochemistry, phylogenetics, pollination, taxonomic notes, and economic uses. For those genera found in hobbyist's collections, cultivation notes emphasizing aritificial propagation are provided.This book features distribution maps of each genus with details on the dispersion of the flowers; superb illustrations with over 120 color photographs and line drawings; cultivational details for orchid enthusiasts and growers; contributions from over 50 international orchid experts; and diagnostic illustrations that detail each genus.
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Resumen Calypsoeae represent a small tribe of anatomically little-known orchids with a wide distribution in the Western Hemisphere. Leaves are present in all genera, except Corallorhiza and Wullschlaegelia both of which are subterranean taxa. Stomata are abaxial (ad- and abaxial in Aplectrum) and tetracytic (anomocytic in Calypso). Fiber bundles are absent in leaves of all taxa examined except Govenia tingens. Stegmata are present in leaves of only Cremastra and Govenia. Roots are velamentous, except in filiform roots of Wullschlaegelia. Vegetative anatomy supports a relationship between Wullschlaegelia and Corallorhiza but does not support the grouping of winter-leaved Aplectrum and Tipularia nor proposed groupings of genera based on pollinarium features.
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— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.