A New Molecular Phylogeny and a New Genus, Pendulorchis, of the Aerides–Vanda Alliance (Orchidaceae: Epidendroideae)

Background The Aerides–Vanda alliance is a complex group in the subtribe Aeridinae (subfamily Epidendroideae, Orchidaceae). Some phylogenetic systems of this alliance have been previously proposed based on molecular and morphological analyses. However, several taxonomic problems within this alliance as well as between it and its allies remain unsolved. Methodology/Principal Findings We utilized ITS and five plastid DNA regions in this phylogenetic analysis. Consensus trees strongly indicate that the Aerides–Vanda alliance is monophyletic, and the 14 genera of this alliance can be grouped into the following clades with 14 subclades: 1. Aerides, comprising two subclades: Rhynchostylis and Aerides; 2. Ascocentropsis; 3. Papilionanthe; 4. Vanda, comprising five subclades: Neofinetia, Christensonia, Seidenfadenia, Ascocentrum, and Vanda–Trudelia, in which Vanda and Trudelia form a subclade; 5. Tsiorchis, comprising three subclades: Chenorchis, Tsiorchis, and two species of Ascocentrum; 6. Paraholcoglossum; and 7. Holcoglossum. Among the 14 genera, only Ascocentrum is triphyletic: two species of the Ascocentrum subclade, an independent subclade Ascocentrum subclade in the Tsiorchis clade; the Ascocentrum subclade in the Vanda clade; and one species in the Holcoglossum clade. The Vanda and Trudelia species belong to the same subclade. The molecular conclusion is consistent with their morphological characteristics. Conclusions We elucidate the relationship among the 14 genera of the Aerides–Vanda alliance. Our phylogenetic results reveal that the Aerides–Vanda alliance is monophyletic, but it can be divided into 14 genera. The data prove that Ascocentrum is triphyletic. Plants with elongate-terete leaves and small flowers should be treated as a new genus, Pendulorchis. Saccolabium himalaicum (Ascocentrum himalaicum) should be transferred to Pendulorchis. Ascocentrum pumilum, endemic to Taiwan, should be transferred to Holcoglossum. A new combination, Holcoglossum pumilum, was also established. Trudelia should not be recognized as an independent genus. Two new species, Pendulorchis gaoligongensis and Holcoglossum singchianum, were described as well.


Introduction
Orchidaceae is possibly the largest family of angiosperms with almost 25 000 species. The traditional typological classification divides Orchidaceae into Apostasioideae, Cypripedioideae, Spiranthoideae, Orchidoideae, and Epidendroideae [1]. Recently, Spiranthoideae has been integrated into Orchidoideae, where a new subfamily Vanilloideae was established [2] based on the affinity analysis of internal transcribed spacer (ITS), trnL-F, and matK sequences. Epidendroideae is a subfamily that accounts for more than 80% of the orchid species. The Aerides-Vanda alliance described in this study is a member of the subtribe Aeridinae and an advanced but complex group in Epidendroideae.
In the Aerides-Vanda alliance, Garay [15] placed Papilionanthe between Vanda and Aerides, but it is more closely linked to Aerides. By contrast, Jin [16] considered Vanda as a relatively primal genus of this alliance.
The members of Ascocentrum considerably differ in the shapes of their leaves, which can be divided into two types: subterete and nearly lorate leaves. Based on the morphological analysis of the mid-lobe of the lip, stipe, and spur of this genus, Jin [16] stated that Ascocentrum maybe grouped between Aerides and Seidenfadenia because the bilobed uplifted rostellum is unique to Ascocentrum and Seidenfadenia in the Aerides-Vanda alliance, and the leaves of some Ascocentrum species are similar to those of Seidenfadenia. However, Christenson [4] classified Ascocentrum under the same branch where Vanda belongs because of its notch-tipped leaves. This finding indicates that the Ascocentrum species with terete leaves do not belong to the Vanda-Ascocentrum branch.
Christenson [4] inferred that Holcoglossum and Seidenfadenia are two parallel evolutive branches, whereas Jin [16] considered Seidenfadenia to be more evolutive because of the special structure of its spur and rostellum. Most Seidenfadenia, Holcoglossum, and Ascocentrum species have closely similar vegetative organs, specifically, a very short stem and subterete leaves with a ventrally longitudinal groove. Their distribution areas usually overlap with one another, and they have very similar habits. However, the floral structures of these three genera are distinct, particularly their rostellum, pollinia, and stipe. The vegetative comparison in these three genera can be considered as their adaptation to similar habitats [16]. Given that this finding is only a speculation, molecular confirmation is still necessary.
Rhynchostylis is relatively close to Vanda in terms of its morphological structures, including its robust habit, entirely or slightly trilobed labellum, bilaterally compressed spur with its apex pointing backward, and two cleft pollinia. This species is a relatively primitive genus in the Aerides-Vanda alliance. However, its stipe is long and narrows downwards, which make it appear specialized.
Christenson [4] performed the initial branch analysis of the Aerides-Vanda alliance and stated that the generic relation of this alliance may have undergone considerable changes after thoroughly researching each genus. The character status of some genera has changed, but some rather ambiguous genera are reclassified. For example, although Holcoglossum is polymorphic, it has been treated as a single genus until recently [17][18][19]. However, Liu et al. [11] divided the Holcoglossum alliance into three genera, namely, Holcoglossum, Tsiorchis, and Paraholcoglossum, based on further molecular and morphological analyses of more taxa under this alliance and its allied groups. The two new genera were treated by Jin [18] and Fan et al. [19] as either subgeneric or sectional rank.
Although some molecular and morphological systems of this alliance have been proposed in previous studies [2,20], the relationships among the members of this alliance are unclear. Two species of Papilionanthe have been placed in the section Nujiangensia of Holcoglossum after molecular analysis [11]. Thus, to seek clarification of the Aerides-Vanda alliance phylogenetically, molecular and morphological analyses of more species are necessary.
The two recently published genera, Ascocentropsis and Chenorchis, which genetically belong to the Aerides-Vanda alliance, are both monotypic. Ascocentropsis has been established based on Ascocentrum pusillum [12], whereas Chenorchis is perceived to be genetically related to Holcoglossum and Ascocentrum [10].
In this study, we focused on improving the sampling of the Aerides-Vanda alliance to facilitate a more accurate reconstruction of the phylogenetic relationships. We collected specimens of 68 species under the 14 genera of the Aerides-Vanda alliance and its three allied genera, with emphasis on Holcoglossum, Paraholcoglossum, Tsiorchis, Chenorchis, Ascocentrum, Neofinetia, Seidenfadenia, Christensonia, Trudelia, and Ascocentropsis. Based on the molecular and morphological analyses, we provided a well-supported phylogenetic resolution for the placement of the 14 genera in the Aerides-Vanda alliance.

Results
The DNA sequences of 70 taxa, including 68 species of 17 allied genera and two species of Cymbidium as outgroup, were obtained and analyzed. The DNA sequences of all species of Holcoglossum, three species of Paraholcoglossum, two species of Tsiorchis, six species of Ascocentrum and one new species similar to Ascocentrum himalaicum, one species of Chenorchis, three species of Neofinetia, six species of Vanda, two species of Papilionanthe, and three species of Aerides were newly obtained, and other species were accessed from GenBank. Tables 1 and 2 provide the detailed sequence information, aligned length, numbers of variable sites, parsimony informative sites, tree statistics for maximum parsimony (MP) analysis, and the best-fit model selected by Modeltest.

ITS Analysis
A total of 67 taxa were analyzed. Most genera form independent clades or subclades. The generic relationships are mostly well resolved. However, the phylogenetic positions of some species are unclear, such as those of Paraholcoglossum auriculatum, Seidenfadenia mitrata, Rhynchostylis gigantean, Rhynchostylis retusa, and Aerides odorata. Figs. S1, S2, and S3 provide the detailed results.

Chloroplast DNA Analysis
In this analysis, chloroplast DNA (cpDNA), including trnL-F, matK, psbA-trnH, atpI-atpH, and trnS-trnfM regions were combined as a dataset for analysis. A total of 68 taxa were analyzed. Most genera form independent clades or subclades. The generic relationships are mostly well resolved. However, the position of Papilionanthe hookeriana and the phylogenetic relationships of Ascocentrum, Christensonia and Seidenfadenia mitrata are unclear. Figs. S4. S5, and S6 provide the detailed results.

Combined Dataset Analysis
We also combined all datasets into a single dataset for the phylogenetic analysis of the Aerides-Vanda alliance. The strict consensus trees strongly suggest that the Aerides-Vanda alliance is monophyletic, and the 14 genera under this alliance can be divided into the following 7 clades with 14 subclades: 1. Aerides, comprising two subclades: Rhynchostylis and Aerides; 2. Ascocentropsis; 3. Papilionanthe; 4. Vanda, comprising five subclades: Neofinetia, Christensonia, Seidenfadenia, Ascocentrum, and Vanda; 5. Tsiorchis, comprising three subclades: Chenorchis, Tsiorchis, as well as one species of Ascocentrum and one new species, Pendulorchis gaoligongensis; 6. Paraholcoglossum; and 7. Holcoglossum. Among these clades, only Ascocentrum is triphyletic and comprises two subclades. One is the Ascocentrum subclade that is related to the Seidenfadenia subclade; the other is the Ascocentrum himalaicum and Pendulorchis gaoligongensis form an independent subclade that is much more closely related to Tsiorchis than to Ascocentrum subclade. Another species, Ascocentrum pumilum, should be transferred to the Holcoglossum clade. Trudelia species do not form an independent subclade but belong to the Vanda subclade. The molecular conclusion is consistent with their morphological characteristics. Fig. 1 provide the detailed results.

Data Analysis
In this study, six DNA regions were utilized, including one nuclear (ITS) and five plastid (trnL-F, matK, psbA-trnH, atpI-atpH, and trnS-trnfM) regions. The results show that most phylogenetic relationships based on ITS agree with the plastid datasets and their combination, but some genera such as Rhynchostylis, Siedenfadenia and Paraholcoglossum appear to have some different phylogenetic relationships between ITS and plastid data. Such differences may result from intergeneric hybridization or introgression at some point during the evolution of these genera, which should need further study to testify. We performed an incongruence length difference test between ITS with cpDNA, and the result shows incongruence to a certain extent between the ITS and plastid regions (P = 0.01), but it did not affect the whole phylogenetic relationship. In fact, different genes (including ITS and cpDNA) are incongruent in many cases. Different genes are known to have different evolutionary rates and can provide different evolutionary information. Thus, we need to use more than one gene to assess their phylogenetic relationship because of their incongruence. Based on our experience and those of other researchers, the obtained phylogenetic relationship is better when more genes are used. The combination dataset still produced the best trees. Among them (Fig. 1), most species belong to their phylogenetic clades or subclades and most nodes have good support. Therefore, we believe that combining a nuclear ITS and plastid regions to solve the phylogenetic relationship is appropriate.  Overall Tree and Effect of Taxon Sampling The phylogenetic analyses identified the following seven major clades: 1. Aerides, comprising two subclades: Rhynchostylis and Aerides; 2. Ascocentropsis, monotypic; 3. Papilionanthe, comprising four species; 4. Vanda, comprising five subclades, Neofinetia, Christensonia, Seidenfadenia, four species of Ascocentrum, and Vanda; 5. Tsiorchis, comprising three subclades: Chenorchis, Tsiorchis, as well as two species of Ascocentrum and Pendulorchis gaoligongensis (PP 1.00, BS ML 100 and BS MP 97); 6. Paraholcoglossum; and 7. Holcoglossum, comprising all species of Holcoglossum and one species of Ascocentrum (PP 1.00, BS ML 95 and BS MP 63). Among the 14 genera, only Ascocentrum is triphyletic and comprises three subclades. First is the Ascocentrum subclade that is related to the Seidenfadenia subclade; second is the Ascocentrum himalaicum and Pendulorchis gaoligongensis form an independent subclade that is much more closely related to Tsiorchis than to Ascocentrum; and third is a species, Ascocentrum pumilum, that should be transferred to the Holcoglossum clade. The parsimony, maximum likelihood, and Bayesian approaches for the combined dataset result in similar tree topologies, with the identification of seven wellsupported major clades (Fig. 2). This topology partially agrees with the proposal of Liu et al. [11].
The seven clades mostly receive moderate to high support (over 50). Species of the Holcoglossum clade are related to the species of the Paraholcoglossum clade (PP 1.00, BS ML 97 and BS MP 68). Ascocentrum himalaicum, often placed in Holcoglossum, is related to the Tsiorchis subclade (PP 0.99, BS ML 88 and BS MP 78). The Ascocentrum species are divided into three groups, belonging to the Tsiorchis, Vanda, and Holcoglossum clades respectively.

Morphological Characteristics and Distribution Analyses
The morphological characteristics of the Aerides-Vanda alliance support its division into the following 14 genera. (1) Rhynchostylis, which includes two species that are both large plants with flat leaves, laterally compressed and backward-pointing spur, cleft pollinia with narrow stipe much longer than the pollinia, and a small viscidium. This genus is widespread in tropical Asia north to south China. (2) Aerides includes 17 species with flat leaves, elongated column, appendiculate spur, bifid rostellum, cleft pollinia, and semicircular viscidium. This genus is widespread in tropical Asia from north China to the Himalayas. (3) Ascocentropsis is a monotypic genus similar to Ascocentrum, from which it differs by having more or less cross-shaped pollinarium with sulcate or split pollinia, visible caudicle, narrowly linear stipe much longer than either the pollinia or viscidium, and strongly incurved side-lobes of the lip. This genus is found in Thailand. (4) Papilionanthe includes four species with terete leaves, large flowers, trilobed lip attached to the column foot, elongated rostellum, and cleft pollinia attached by a broadly triangular or subquadrate stipe to a large viscidium. This genus is distributed in China, India, and Southeast Asia. (5) Neofinetia has flat narrow leaves, cleft pollinia, footless column, and narrow stipe much longer than pollinia. This genus is found in China, Japan, and Korea. (6) Christensonia is a monotypic genus that exhibits a mosaic of characters found in its closely related genera Aerides and Rhynchostylis. This genus, distributed in Vietnam, differs from the former by having a footless column and from the latter by a clearly three-lobed lip [9]. (7) Seidenfadenia, a monotypic genus similar to Holcoglossum, from which it differs by having a pouched and backward-pointing spur, is found in Thailand and Myanmar. (8) Ascocentrum miniatum, Ascocentrum ampullaceum, Ascocentrum curvifolium, and Ascocentrum aurantiacum have flat leaves, similar sepals and petals, lip firmly adnate to the column base with suberect lateral lobes adnate to column, and oblong mid-lobe thickening at the base. These species are found in Southeast Asia and the Himalayas. (9) Vanda includes five species of Vanda and two species transferred back from Trudelia (T. alpina and T. pumila), with cleft pollinia, footless column, and a broad stipe shorter than pollinia. This genus is widespread in tropical Asia extending to New Guinea and Australia. (10) Chenorchis is a monotypic genus with clavate rachis, three-lobed lip with its side-lobes from both lower sides of the mid-lobe, and large rostellum conspicuously broader than the column. This species is distributed in China (West Yunnan).

Holcoglossum Clade
This clade comprises all species of Holcoglossum, including one species transferred from Ascocentrum. Our data strongly suggest (PP1.00) that the Holcoglossum clade is related to the Paraholcoglossum clade. However, Tsiorchis should be placed distantly outside the Holcoglossum clade. Papilionanthe is treated as an independent clade outside the Holcoglossum clade. The aforementioned four genera have marked differences in morphology (Fig. 1). The phylogenetic relationships between Holcoglossum and its allied genera are well resolved, and all data sets supported the recognition of Papilionanthe. The Holcoglossum alliance is divided into the Tsiorchis, Paraholcoglossum, and Holcoglossum clades (PP1.00).
Holcoglossum is treated as a genus of 13 species, including a new species, H. singchianum, and a new combination. They all combine to form a homologous subclade. The new species is related to Holcoglossum quasipinifolium, but distinguishable from it by some marked characteristics and should be treated as a new species.

Paraholcoglossum Clade
From the tree of the combined dataset, the Paraholcoglossum clade is related to the Holcoglossum clade. Paraholcoglossum comprises three species: P. subulifolium, P. auriculatum, and P. amesianum. Morphologically, this genus differs from Holcoglossum by a lip that is saccate (not spurred) at the base, a ridged (not crested or fleshy) callus at the sac entrance (not mid-lobe base), and an oblong (not tapering) stipe [11].

Rhynchostylis-Aerides Clade
This clade consists of the two subclades Rhynchostylis and Aerides. Rhynchostylis has three or four species, and its two Chinese species, which combine to form a subclade, were analyzed in this study. This study further suggests that Rhynchostylis is an independent genus. Aerides has approximately 20 species, among which 16 species were analyzed in this study. They combine to form a basal subclade of the Aerides-Vanda alliance and another basal subclade related to the Rhynchostylis subclade. The topology of Aerides partially agrees with that described by Kocyan et al. [2].

Papilionanthe Clade
This clade is composed of all 12 Papilionanthe species. The four species selected in this study combine to form a group distantly related to Holcoglossum, which contradicts previous findings [11]. Papilionanthe has an elongated stem, terete leaves, two-or threelobed mid-lobe of the lip, cylindrical or horn-shaped spur, and short cleft pollinia. These characteristics relatively leave an evolutionary trace in Tsiorchis, Paraholcoglossum, and Holcoglossum.

Ascocentropsis Clade
The Ascocentropsis clade includes a monotypic genus formerly established based on Ascocentropsis pumila [12]. The molecular data from this study strongly support the independent position of this clade at the generic level, and prove that the Ascocentropsis clade is a basal clade compared with the Holcoglossum clade.

Vanda (Trudelia)-Ascocentrum-Neofinetia Clade
This group is a multifarious clade composed of the following subclades: (1) Neofinetia comprising the monophyletic genus Neofinetia with three species; (2) Christensonia, a monotypic genus that is considered independent at the generic level and related to the Seidenfadenia and Ascocentrum subclades; (3) Seidenfadenia, also a monotypic genus and a subclade related to the Ascocentrum subclade; (4) Ascocentrum, comprising four species with big flowers and flat leaves, i.e., a subclade related to the Seidenfadenia subclade. One other species and one new species (Pendulorchis gaoligongensis) with small flowers and terete leaves are considered independent genera placed in the Pendulorchis-Tsiorchis-Chenorchis clade. Pendulorchis gaoligongensis has unique characteristics and should be treated as a new species. Ascocentrum pumilum should be transferred to Holcoglossum; and (5) Vanda, comprising approximately 40 species, seven of which were selected in this study and combined to form a subclade. Trudelia alpina and Trudelia pumila should be transferred back to Vanda.

Pendulorchis-Tsiorchis-Chenorchis Clade
This clade is composed of the following subclades. (1) Chenorchis comprising a monotypic genus described in 2008 in China [10]. This species was previously believed [13] to be identical with the Indian plant Penkimia nagalandensis [24]. However, further molecular comparative analysis between these two species is needed. The sample in our study is from the type specimen of Chenorchis, which provides evidence that it is genetically related to Tsiorchis (original Holcoglossum) and Pendulorchis (original Ascocentrum). Thus, these subclades are all related. (2) Tsiorchis comprises a genus of two species and originally belongs to Holcoglossum. Tsiorchis is related to the Pendulorchis subclade, but it is quite distinct from Holcoglossum and Paraholcoglossum due to its cleft, not its porate. The pollinia each have a distinct caudicle attached to a common stipe and lip marked with purple or dark purple on the mid-lobe and side-lobes. (3) Pendulorchis subclade includes a genus of one species that originally belongs to Ascocentrum and one new species. These two species are pendulous and have tiny, not fully open flowers. Pendulorchis is closely related to Tsiorchis and to a lesser extent to Chenorchis. These species form the Pendulorchis-Tsiorchis-Chenorchis clade.
Our molecular analysis and morphological observation strongly support the recognition of the following as independent genera: Holcoglossum, Paraholcoglossum, Tsiorchis, Chenorchis, Christensonia, Seidenfadenia, Ascocentropsis, Neofinetia, Ascocentrum, Pendulorchis, Papi-lionanthe, Aerides, Rhynchostylis, and Vanda. However, the branch analysis by Christenson [4] is only partially supported. We do not support the placement of Papilionanthe between Vanda and Aerides, as previously treated by Garay [15]. Instead, we propose that Papilionanthe should be placed between the Aerides-Rhynchostylis and Vanda-Ascocentrum-Neofinetia alliances. Although this study covered all types of species, not all species of the genera in the Aerides-Vanda alliance were considered. Thus, further research is needed to confirm whether all the species in these genera are homologous.

Conclusion
The Aerides-Vanda alliance is confirmed to be monophyletic, but it can be divided into 14 genera, including the recently established Chenorchis, Ascocentropsis, Christensonia, Seidenfadenia, Paraholcoglossum, and Tsiorchis. We elucidate the relationship among the 14 genera of the Aerides-Vanda alliance, which comprises 7 main clades with 14 subclades. Its basal groups are Rhynchostylis and Aerides. Our molecular data prove that Ascocentrum is triphyletic. Ascocentrum pumilum is closely related genetically to Holcoglossum and should be transferred to this genus. One species of Ascocentrum and one new species with terete leaves and tiny flowers are treated in this study as a new genus, Pendulorchis, which is more closely related to Tsiorchis than to Ascocentrum. This study suggests that Vanda alpina and Vanda pumila should not be separated from Vanda to form an independent genus, Trudelia. A new genus, Pendulorchis, and two new species, Holcoglossum singchianum, are also described. Three new combinations, Pendulorchis gaoligongensis, Pendulorchis himalaica, and Holcoglossum pumilum, are established as well. Description. Epiphytic pendulous plants, with many long and flattened roots. Stem often 13 cm to 24 cm long, enclosed by leaf sheaths, often branched. Leaves 6 to 14, fleshy, deep green, subterete, 30 cm to 60 cm long, 3 mm to 5 mm in diameter, channeled adaxially, jointed and sheathed at the base. Inflorescences often 6 to 15, paniculate or racemose, arising from the axils of the lower leaves, with 17 to 39 flowers; flowers 4 mm to 5 mm in diameter, not fully open; sepals, petals, and lip reddish; dorsal sepal oblong, abaxially carinate; lateral sepals elliptic; petals obovate-elliptic; lip 3-lobed; side-lobe erect, oblong, toward abaxial base strongly concave forming a callus-like structure; mid-lobe spreading forward, obovate, adaxially with three longitudinal midveins; spur cylindric; column stout and short; pollinia two globose, cleft, attached by a common stipe to a large suborbicular viscidium.
The new species is similar to H. linearifolium, differing by its nearly ascending stem, leaves 3-4 mm thick, inflorescence 12-to 16-flowered, lip with subobovate-rhombic mid-lobe, and yellow side-lobes spotted with brown.

Amplification and Sequencing
Total DNA was extracted from fresh material or silica gel-dried plant tissue using a Multisource Genomic DNA Miniprep Kit (Axygen Biosciences) following the manufacturer's instructions. The amplification reaction included total DNA, primers, Ex-Taq buffer, and Ex-Taq DNA polymerase (Takara Bio). The polymerase chain reaction (PCR) profile consisted of an initial 5 min pre-melting stage at 95uC, followed by 30 cycles of 30 s at 95uC (denaturation), 30 s at 50uC to 55uC (annealing temperature was determined based on the primer requirement), and 1 min to 3 min at 72uC (extension time was determined based on the length of the target DNA region), and a final 10 min extension at 72uC.
The amplification of the ITS region was performed using the primer pairs ITS A and ITS B [26]. The trnL-F region was amplified with primers c and f [27] or the two sets of primers developed by Liu et al. [11]. For matK sequences, amplification was performed using the primer pair matK-19F and trnK-2R [26], and several fragments were amplified using the three sets of primers developed by Liu et al. [11]. The psbA-trnH region was amplified and sequenced by the primer pairs psbAF and trnHR [28]. The atpI-atpH region was amplified and sequenced using the primer pairs atpI and atpH [29]. The trnS-trnfM region was amplified and sequenced using the primer pair trnS-trnfM [30]. Table 3 contains the detail information.
The PCR products were run on 1.5% agarose gels to assess the quality of the amplified DNA. The gels with the target products were excised, purified using DNA Gel Extraction Kits (Axygen Biosciences), and then sequenced by Life Technologies Corporation.

Sequence Editing and Assembling
Both forward and reverse sequences, as well as electropherograms were edited and assembled using DNASTAR (http://www. dnastar.com/). DNA sequences were aligned to the muscle model and manually adjusted using MEGA5.05 [31]. Aligned sequences are available from the corresponding authors upon request.

Data Analyses
The datasets included a nuclear ITS, plastid DNA (cpDNA; including the trnL-F, matK, psbA-trnH, atpI-atpH, trnS-trnfM), and their combination. Insertions, deletions, and some unavailable sequences were treated as missing. Phylogenetic analyses were performed under ML, MP, and Bayesian inference (BI) methods. The best-fit model for each dataset was selected by Modeltest 3.7 [32] under the Akaike Information Criterion ( Table 2). The homogeneities between nrDNA ITS data and the combined cpDNA dataset were tested using the incongruence length difference (ILD) test [33], as implemented in PAUP* version 4.0b10 [34]. The ILD test was conducted with 1000 replicates, each with 10 random addition sequence replicates, TBR branch swapping, and keeping no more than 100 trees per random addition replicate. Following Cunningham [35], a significance level of P = 0.01 was adopted for this test.
MP analyses were performed using the PAUP* version 4.0b10 [34]. All characters were equally weighed and unordered. Test settings included 1000 replications of random addition sequence and heuristic search with tree bisection-reconnection branch swapping. Table 1 lists the tree length, consistency index (CI), and retention index (RI) ML analysis was performed using RAxML version 7.2.8 with 100 bootstrap replicates and settings as described in Stamatakis et al. [36]. BI analysis was performed using MrBayes 3.1.2 [37]. The best-fit model for each dataset was selected using Modeltest 3.7. In the combined dataset of all datasets, the model was also based on the best fit model for each individual dataset. The following settings were applied: sampling frequency = 1000, temp = 0.1, burn-in = 10 000, and number of Markov chain Monte Carlo generations = 40 000 000. The first 10 000 trees were discarded as burn-in to ensure that the chains reached stationarity. A majority-rule consensus tree was constructed on these trees sampled after generation 10 000 000.
Phylogenetic analyses were performed under the ML, MP, and BI for each dataset. The BI, MP, and ML trees of each dataset had similar topological structures, indicating the very good repeatability of the analysis and reliability of the experimental data. The differences among the BI, MP, and ML trees were the values of bootstrap percentages or posterior probabilities in each node. Generally, the values in the BI tree were higher than those in the MP and ML trees (Figs.1, S1, S2, S3, S4, S5, S6). We submitted all data and trees to TreeBase, http://purl.org/phylo/treebase/ phylows/study/TB2:S13699.

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 Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.
In addition, new names contained in this work have been submitted to IPNI, from where they will be made available to the Global Names Index. The IPNI LSIDs 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. Figure S1 Bayesian consensus trees based on ITS data. The Bayesian posterior probability (6100) is given above the branches. (TIF)