A New Orchid Genus, Danxiaorchis, and Phylogenetic Analysis of the Tribe Calypsoeae

Background Orchids have numerous species, and their speciation rates are presumed to be exceptionally high, suggesting that orchids are continuously and actively evolving. The wide diversity of orchids has attracted the interest of evolutionary biologists. In this study, a new orchid was discovered on Danxia Mountain in Guangdong, China. However, the phylogenetic clarification of this new orchid requires further molecular, morphological, and phytogeographic analyses. Methodology/Principal Findings A new orchid possesses a labellum with a large Y-shaped callus and two sacs at the base, and cylindrical, fleshy seeds, which make it distinct from all known orchid genera. Phylogenetic methods were applied to a matrix of morphological and molecular characters based on the fragments of the nuclear internal transcribed spacer, chloroplast matK, and rbcL genes of Orchidaceae (74 genera) and Calypsoeae (13 genera). The strict consensus Bayesian inference phylogram strongly supports the division of the Calypsoeae alliance (not including Dactylostalix and Ephippianthus) into seven clades with 11 genera. The sequence data of each species and the morphological characters of each genus were combined into a single dataset. The inferred Bayesian phylogram supports the division of the 13 genera of Calypsoeae into four clades with 13 subclades (genera). Based on the results of our phylogenetic analyses, Calypsoeae, under which the new orchid is classified, represents an independent lineage in the Epidendroideae subfamily. Conclusions Analyses of the combined datasets using Bayesian methods revealed strong evidence that Calypsoeae is a monophyletic tribe consisting of eight well-supported clades with 13 subclades (genera), which are all in agreement with the phytogeography of Calypsoeae. The Danxia orchid represents an independent lineage under the tribe Calypsoeae of the subfamily Epidendroideae. This lineage should be treated as a new genus, which we have named Danxiaorchis, that is parallel to Yoania. Both genera are placed under the subtribe Yoaniinae.


Introduction
Orchidaceae is one of the largest families of angiosperms [1]. It has been said ''The speciation rate in orchids is frequent because of their diverse flower and vegetable morphologies'' [2]. Considerable attention has been given to their extraordinary pollination, multiple adaptive strategies to various habitats, and numerous dust-like seeds that lack endosperms [3,4]. Previously, Orchidaceae has been divided into five subfamilies based on their morphological characters as follows: Apostasioideae, Cypripedioideae, Spiranthoideae, Orchidoideae, and Epidendroideae [5]. However, a phylogenetic analysis based on the internal transcribed spacer (ITS), trnL-F, and matK sequences revealed that Spir-anthoideae is a member of Orchidoideae and that Vanilla and its allies should be separated from Epidendroideae to form a new subfamily, Vanilloideae [6][7][8]. Epidendroideae is a highly evolved and diverse subfamily, in which a few species are mycotrophic and lack green leaves. Many holomycotrophic orchids are found in China, comprising approximately 20 genera. None of these orchids has a bisaccate labellum.
Vanilla, Apostasia, Cyrtosia, Palmorchis, Selenipedium, and several Neuwiedia species have wingless seeds with hard seed coats. Several members of Vanilleae, such as Epistephium and Galeola, have a hard seed coat over the embryo and a developed wing around the seed. Several Neuwiedia species have small seeds with sac-like appendages at either end. Most other orchids have a loose, rather papery seed coat around the embryo, which has a length that ranges from 0.15 mm to 6 mm. However, all of these orchid seeds are dry and lack an endosperm.
In this report, we documented a new orchid found on Danxia Mountain in Guangdong, China. The flower and seed structures of this new orchid are different from those of other known taxa in Orchidaceae. However, the phylogenetic clarification of this new orchid requires further molecular, morphological, and phytogeographic analyses.

Morphological Analysis
The new orchid entity is restricted to the Danxia Mountain in northern Guangdong, China (Fig. S1). The Danxia region, known as the Danxia Landform, is famous for its topographic features. A detailed comparison between the newly discovered orchid and other members of Orchidaceae was conducted. The new plant is characterized by a labellum with two sacs at the base, an elongated column that has a terminal concave stigma and lacks conspicuous staminodes and rostellum, four sectile pollinia attached by two caudicles to a common large viscidium, and cylindrical, fleshy seeds. These features distinguish the new orchid from all other known orchids (Figs. 1, 2, and S2).

Analyses of Phylogenetic Placement
Danxiaorchis singchiana is morphologically related to the tribe Calypsoeae and, to a lesser degree, to the tribe Gastrodieae. Both of these tribes belong to the subfamily Epidendroideae. A detailed morphological character matrix (59 characters of 74 taxa) was integrated with a molecular matrix (3586 nucleotide sequences of the ITS, matK, and rbcL genes of 74 genera) to classify the plant into an appropriate phylogenetic position (Figs. 3, S3, S4, and S5).
Bayesian inference (BI) phylogram showed the monophyly of the new orchid plant. Five clades were distinguished in Orchidaceae, with a posterior probability (PP) of over 99% (Fig. 3). Based on evolutionary sequences, the five clades correspond to the subfamilies Apostasioideae, Cypripedioideae, Vanilloideae, Orchidoideae, and Epidendroideae. Calypsoeae formed an independent lineage (PP = 52%) in the subfamily Epidendroideae, under which Danxiaorchis is included together with other genera of Calypsoeae. In the family-level BI phylogram of the combined ITS, matK, and rbcL gene sequences, the Calypsoeae clade is divided into two subclades (PP = 95%, Fig.  S4). The first subclade includes Calypso, Tipularia, and Changnienia, whereas the second subclade comprises eight genera, including Danxiaorchis, which is most closely related to Yoania, as confirmed by maximum parsimony (MP) analysis (Fig. S5).

Phylogeny of Calypsoeae
Nuclear ITS sequence data analysis. The phylogenetic trees generated based on the ITS sequence data analysis clearly revealed the independence of the eight genera of this tribe. The BI phylogram with most of the clades received a strong support (PP.90%). Danxiaorchis, which forms a single clade with a PP of 99%, has been recognized as a natural genus within this tribe (Fig.  S6). However, a relatively weak bootstrap and unstable topology is found in MP phylogram (Fig. S7).
Chloroplast sequence data analysis. Similarly, the 11 genera can be easily distinguished from the phylograms based on chloroplast sequence data analysis. The phylogenetic topologies generated by BI are approximately congruent with the ones by MP analysis (Figs. S8 and S9). The basal clade is independently composed of Calypso, Tipularia, and Changnienia. The next clade is Govenia, followed by a complex clade, which includes Aplectrum, Cremastra, Danxiaorchis, Yoania, Wullschlaegelia, and Oreorchis. Corallorhiza occupies the terminal positions in both MP and BI phylograms, although it is not well-supported intragenetically in the MP phylogram.
Combined analysis. In this study, ITS, matK, and rbcL were combined into a single dataset. The strict consensus BI phylogram (Fig. S10) strongly supports the division of the Calypsoeae alliance (except Dactylostalix and Ephippianthus) into seven clades with eight subclades (PP = 100%, except for one with 77%). The first clade, which consists of the Changnienia, Tipularia, and Calypso subclades, is strongly supported as a sister to the outgroup clade, which consists of Sobralia and Nervilia (PP = 100%). The second clade, which has a single genus, Govenia, is strongly supported as a sister to the first clade (PP = 100%). The third clade is the Aplectrum genus, and the fourth is Cremastra (PP = 100%). The fifth clade contains the new genus Danxiaorchis, and its ally, Yoania (PP = 100%). The last two clades have weak support (PP = 77%). The sixth clade is comprised of Wullschlaegelia and Oreorchis. The seventh clade contains a single genus, Corallorhiza, which consists of 13 species that are further divided into two subclades. The results are in agreement with the results of the complex clades in the MP phylogram (Fig. S11).
The sequence data of each species and the morphological characters of each genus were combined into a single dataset (Dactylostalix and Ephippianthus having morphological characters only). The strict consensus BI phylogram supports the division of the 13 genera of Calypsoeae into four clades with 13 subclades, which is in agreement with the results of the combined sequence data analysis. These results show that the genera Dactylostalix and Ephippianthus belong to a single clade near the Govenia and Calypso clades (Figs. 4 and 5).

Morphological Analysis
This study is the first to report an orchid with a bisaccate labellum and cylindrical, fleshy seeds. This orchid is difficult to classify in any known subtribe or tribe within Orchidaceae. Although the new orchid has a particular similarity to Gastrodia and its allies in terms of pollinium structure, the pollinarium of Gastrodia lacks distinct caudicles and viscidium like the new orchid does. This new orchid sharply differs from Satyrium and Corybas except for the two elongate or saccate spurs at the base of their labellum [9]. Danxiaorchis has cylindrical (1.5 mm60.5 mm) and fleshy seeds, which is similar to the seeds of underground orchid species of Rhizanthella [10]. However they're distinct from Danxiaorchis by its underground habitat, absence of roots, fleshy overlapping bracts, small flowers and minute seeds. Thus, this genus is distinguishable from all other genera of orchids.

Family-level Analysis
The results of our analyses are in agreement with those obtained by previous researchers [5,9,11,12] and support the relationships among the subfamilies Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae, and Epidendroideae. In addition, a more precise phylogenetic tree was obtained in the present study at higher categories. This finding may be attributed in part to the utilization of a more diversified nuclear genetic marker, ITS, the application of multiple genetic markers, and the integration of morphological and molecular characters.   [13] added Wullschlaegelia and Govenia to this tribe, although Dressler had placed the former in Gastrodiinae and the latter in Cymbidieae [5], both at subtribal rank. Meanwhile, Chen et al. [14] treated these genera as members of the tribe Epidendreae, with the following subtribes in China: subtribe Yoaniinae with the genus Yoania; subtribe Calypsoinae with the genera Oreorchis, Cremastra, Tipularia, Calypso, and Changnienia; and subtribe Corallorhizinae with the genus Corallorhiza. However, all of these classification systems were based only on morphological evidence.
Govenia and Corallorhiza are both monophyletic genera (PP = 100%), with the former genus having a relatively anomalous distribution in the tribe Calypsoeae [13]. Thus, their subtribal rank, the subtribes Goveniinae [5] and Corallorhizinae [14], is maintained. The phylogenetic placement and the infrageneric relationships of Corallorhizinae are very similar to those studied by Freudenstein et al. [15] Corallorhiza should be divided into two subclades. The first subclade contains C. striata Lindl., C. bentleyi Freudenst., C. involuta Greenm., and C. vreelandii Rydb. The species of this subclade possess three-veined perianth segments and a thickened labellum that are fused at the base. The second subclade contains the rest of the species in this genus. They possess a thin-textured labellum, and some species, such as C. trifida Châtel. and C. odontorhiza (Willd.) Nutt. are autogamous [15][16][17][18].
Aplectrum and Wullschlaegelia are composed of a few species that are distributed from North America to tropical South America. For Wullschlaegelia, a monotypic subtribe Wullschlaegeliinae was established in 1990 [5]. Cremastra and Oreorchis are only found in Asia [19,20]. Yoania is most closely related to Danxiaorchis and they share the same habitat. However, Danxiaorchis can be distinguished from Yoania by its rooted rhizome, bisaccate labellum, Y-shaped appendages, and caudicles [21].
Two distantly related genera, Dactylostalix and Ephippianthus [13], are distributed along the Sakhalin Peninsula in northern Japan and the Kuriles. Unfortunately, materials from these two genera could not be obtained. However, in our study, these two genera formed a sister clade with the Calypso and Govenia clades based on their morphological characters.

Conclusion
The Danxia orchid has several distinct features. Based on results obtained by applying phylogenetic methods to a matrix of morphological and molecular characters, the Danxia orchid can be treated as a new genus of Calypsoeae (subfamily Epidendroideae). Analysis of the combined datasets using maximum likelihood methods revealed strong evidence that Calypsoeae is a monophyletic tribe consisting of eight well-supported clades with 13 subclades, which are all in agreement with the phytogeography of Calypsoeae.

Materials
The locations of the field studies are neither private lands nor protected areas, but are controlled by the State Forestry Administration of China, to which our institution is affiliated. The State Forestry Administration authorized us to conduct scientific observations or tests in the regions it controls.A valid permit was also obtained for testing the genes of Danxiaorchis.
A total of 74 genera were analyzed in the family-level study. Two genera, Hypoxis and Curculigo, were selected as outgroups. Three genetic markers (ITS, matK, and rbcL) of Danxiaorchis, Corallorhiza, Cremastra, Oreorchis, and Yoania were analyzed. The gene sequences of the other 61 genera were accessed from GenBank (Table S1). Danxiaorchis singchiana was collected from the Danxia Mountain in northern Guangdong, China (25uN, 113uE).
A total of 34 species (or subspecies or varieties) and 35 individuals of 13 genera were included in the tribe-level analysis, wherein Sobralia and Nervilia were selected as outgroups. The ITS, matK, and rbcL gene sequences of Danxiaorchis singchiana, Corallorhiza trifida, Changnienia malipoensis, Cremastra appendiculata, Yoania japonica, Oreorchis indica, and O. nana were applied in the same way as that in the family-level study. The other sequences were accessed from GenBank (Table S2).
Corallorhiza trifida and Oreorchis nana were collected from Huanglong in Sichuan Province. Cremastra appendiculata was cultivated in a nursery in Shenzhen, whereas Yoania japonica was obtained from the herbarium of The Orchid Conservation and Research Center of Shenzhen (NOCC, Z. J. Liu 6241).
Danxiaorchis singchiana was collected between April 2012 and May 2012 from its habitat in northern Guangdong, China. Several individual plants with young fruits were cultivated in our nursery in Shenzhen for mature fruits and seeds. Fresh flowers, especially the pollinaria, were examined using a stereoscope (Guiguang XTL-500, China). Colour photographs, black-white drawings, and descriptions were catelogued at the time. Molecular experiments were performed at the Shenzhen Key Laboratory for Orchid Conservation and Utilization of The Orchid Conservation and Research Center of Shenzhen.
All material for morphological and molecular examinations was kept in FAA (55% alcohol: glacial acetic acid: formalin at a ratio of 95:5:5) and allochroic silica gel.

Methods
Amplification and sequencing. Total DNA was extracted from fresh material, silica gel-dried plant tissue, or herbarium specimens using a modified hexadecyl trimethyl ammonium bromide method [25].
The amplification reaction included total DNA, primers, Mighty Amp buffer version 2.0, and Mighty Amp DNA polymerase (Takara Bio). The polymerase chain reaction (PCR) profile consisted of an initial 2 min pre-melt stage at 98uC; 35 cycles of 20 s at 98uC (denaturation), 20 s at 45uC to 55uC (annealing temperature was determined by the requirements of the primer), and 50 s to 90 s at 68uC (extension time was determined by the length of the target DNA region); and a final extension of 6 min to 8 min at 68uC.
Amplification of the ITS, matK, and rbcL regions was separately performed using the primer pairs ITS A and ITS B, matK-19F and trnK-2R, and rbcL [26][27][28]. Other matK and rbcL primer sets were also amplified (Table S3).
The PCR products were run on 1.5% agarose gels to check the amplified DNA quality. Gels with target products were excised, purified using DNA gel extraction kits (OMEGA BIO-TEK, USA), and sequenced by Invitrogen (Shanghai).
Sequence editing and assembling. The forward and reverse sequences as well as electropherograms were edited and assembled using DNASTAR (http://www.dnastar.com/). The DNA sequences were aligned using MEGA5.05 using Muscle method [29] and then manually adjustments were made for inserting gaps to improve the alignments [30]. The aligned sequences are available from the corresponding authors upon request.
Morphological analyses. A matrix, which consists of 59 morphological characters of 74 taxa in the family-level analysis (Morphological Character Codes S1 and Table S4) and 69 morphological characters of 35 taxa in the tribe-level analysis (Table S5), was constructed to explore the phylogenetic positions of the Danxiaorchis alliance by morphological classification.
Data analyses. Maximum Parsimony (MP) analyses were performed usingPAUP* version 4.0b10 [31]. All characters were equally weighed and unordered. The test settings included 1,000 replications of random addition sequence and heuristic search with tree bisection and reconnection branch swapping. Tree length, consistency indices (CI), and retention indices (RI) are shown in Table S6. BI analysis was performed using MrBayes3.1.2 [32]. The best-fit model for each dataset was selected using Modeltest 3.7. The model for the combined ITS, matK, and rbcL datasets was also based on the best-fit model for each individual dataset (Tables  S7 and S8). The following settings were applied: sampling frequency = 100; temp = 0.1; burn-in = 10,000; and the number of Markov Chain Monte Carlo generations = 4,000,000. The first 10,000 trees were discarded as burn-in. A majority-rule consensus phylogram was constructed based on the phylograms sampled after the 1,000,000 th generation.

Nomenclature Acts
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Supporting Information
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