YMPEV 5128 No. of Pages 8, Model 5G 28 February 2015

Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx 1 Contents lists available at ScienceDirect

Molecular Phylogenetics and Evolution

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

2 Short Communication

6 4 A molecular phylogeny of (: ) 7 5 inferred from multiple nuclear and chloroplast regions

a,b,1 a,1 b a,b,c,⇑ a,⇑ 8 Long-Hai Zou , Jiu-Xiang Huang , Guo-Qiang Zhang , Zhong-Jian Liu , Xue-Ying Zhuang

9 a College of Forestry, South Agricultural University, Guangzhou, China 10 b Shenzhen Key Laboratory for Orchid Conservation and Utilization, The National Orchid Conservation Center of China and The Orchid Conservation and Research Center of 11 Shenzhen, Shenzhen, China 12 c The Center for Biotechnology and Biomedicine, Graduate School at Shenzhen, Tsinghua University, Shenzhen, China

1413 15 article info abstract 1730 18 Article history: The subtribe Aeridinae, which contains approximately 90 genera, is one of the most diverse and 31 19 Received 12 August 2014 taxonomically puzzling groups in Orchidaceae. In the present study, the phylogenetic relationships of 32 20 Revised 6 January 2015 Aeridinae were reconstructed utilizing five DNA sequences (ITS, atpI-H, matK, psbA-trnH, and trnL-F) from 33 21 Accepted 17 February 2015 211 taxa in 74 genera. The results of the phylogenetic analyses indicate that Aeridinae is monophyletic 34 22 Available online xxxx and that the subtribe can primarily be grouped into 10 clades: (1) clade, (2) 35 clade, (3) clade, (4) clade, (5) clade, (6) clade, (7) 36 23 Keywords: clade, (8) Abdominea clade, (9) clade, and (10) clade. In our examination, most 37 24 Aeridinae genera of Aeridinae were well-supported as monophyletic, and several genera, namely, , 38 25 cpDNA 26 Molecular phylogenetics Cleisostoma, , , , and , were found to be polyphyletic as currently 39 27 ITS circumscribed. In addition, several classifications of intra-genera, such as the subgenus Codonosepalum 40 28 Orchidaceae of and the section Gastrochilus of Gastrochilus, were also revealed to be paraphyletic. 41 29 Due to the many questions raised by our phylogenies, the present study may serve as a reference for 42 future taxonomic studies of Aeridinae. 43 Ó 2015 Published by Elsevier Inc. 44 45

46 47 48 1. Introduction addressed relationships in the group. With the exception of several 63 studies focusing on some individual groups, such as the Aerides– 64 49 The subtribe Aeridinae (=Sarcanthinae), a monopodial group Vanda alliance (e.g., Kocyan et al., 2008; Gardiner et al., 2013; 65 50 with approximately 1350 in approximately 90 genera that Xiang et al., 2012; Zhang et al., 2013) and Phalaenopsis (e.g., 66 51 are primarily distributed throughout the warm-temperate and tro- Padolina et al., 2006; Tsai et al., 2010; Yukawa et al., 2005), the phy- 67 52 pical areas of , , and the eastern Pacific islands, forms a logeny of Aeridinae was reconstructed by Topik et al. (2005) and 68 53 large proportion of tribe in Orchidaceae (Chase et al., Topik et al. (2012) based on the analyses of two DNA regions (ITS 69 54 2003). Historically, several orchidologists have attempted to trace and matK) in 75 taxa belonging to 62 genera and 82 taxa belonging 70 55 the spine of the relationships among the genera in Aeridinae based to 66 genera, respectively. Both studies suggested that the mono- 71 56 on the characteristics of the foot (Schlechter, 1913) and phyly of Aeridinae was tentatively supported. However, the topolo- 72 57 pollinium numbers (Dressler, 1993; Holttum, 1959; Senghas, gies between the two studies are inconsistent in 14 major clades in 73 58 1988). However, both the column foot and pollinium numbers have the former study and 11 clades in the latter. Moreover, many inner 74 59 independently evolved several times in this group (Topik et al., branch nodes with weak support were observed in the phylogenetic 75 60 2005), which indicates that these morphological characteristics trees, and many unexpected relationships were found in both stud- 76 61 are useless for determining the phyletic lines of the subtribe. Recent ies. As these findings indicated, these relationships may have 77 62 phylogenetic analyses that relied on nucleotide sequences have resulted from sparse samplings. In fact, sparse samplings may give 78 rise to negative effects on phylogenetic analyses, such as low sup- 79

⇑ Corresponding authors at: Shenzhen Key Laboratory for Orchid Conservation port values and abnormal systematic relationships (Soltis et al., 80 and Utilization, The National Orchid Conservation Center of China and The Orchid 2011). Moreover, based on the sparse samplings, the examination 81 Conservation and Research Center of Shenzhen, Shenzhen, China (Z.-J. Liu). of the monophyly of many genera was not possible. Hence, satisfac- 82 E-mail addresses: [email protected] (Z.-J. Liu), [email protected] tory conclusions regarding the phylogenetic relationships within 83 (X.-Y. Zhuang). Aeridinae cannot yet be elucidated. 84 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ympev.2015.02.014 1055-7903/Ó 2015 Published by Elsevier Inc.

Please cite this article in press as: Zou, L.-H., et al. A molecular phylogeny of Aeridinae (Orchidaceae: Epidendroideae) inferred from multiple nuclear and chloroplast regions. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.02.014 YMPEV 5128 No. of Pages 8, Model 5G 28 February 2015

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85 In the present study, to avoid the negative effects resulting from phorchis lowii was sister to + Monanthochilus (Abdomi- 140 86 sparse taxon sampling, we focused on facilitating a more accurate nea clade) with weak support (PP 50); (b) the ITS analysis strongly 141 87 reconstruction of the phylogenetic relationships of Aeridinae by supported Abdominea + + Cleisomeria + Monanthochilus 142 88 improving the samplings in both species (and genera) and DNA (Abdominea clade) as sister to Thrixspermum (PP 100), but the cpDNA 143 89 sequences. A total of 211 species in 74 genera and five DNA mark- analysis showed that the Abdominea clade and lowii 144 90 ers (ITS, matK, trnL-F, psbA-trnH, and atpI-H) were employed. The form an independent clade; (c) Tsiorchis was found to be sister to 145 91 objective of the present study was to clarify the generic relation- Pendulorchis (PP 94) in the Vanda clade through the ITS analysis 146 92 ships within subtribe Aeridinae. but emerged as sister to the Saccolabium clade in the cpDNA analysis 147 with weak support (PP 50); and (d) in the Gastrochilus clade, the ITS 148 149 93 2. Materials and methods analysis supported the inclusion of Haraella retrocalla in Pomatocal- pa, but the cpDNA analysis indicated that Haraella is sister to 150 151 94 2.1. Taxon sampling Gastrochilus with PP 100. These conflicts between the distinct gen- omes may be a result of intergeneric hybridization in the evolution 152 153 95 A total of 219 species in 81 genera, including 211 species in 74 of Aeridinae (Zhang et al., 2013). 96 genera from subtribe Aeridinae, were analyzed. According to the 154 97 classification of Vandeae proposed by Chase et al. (2003), three 3.2. Combined analyses 98 species from subtribe , two species from subtribe Aer- 155 99 angidinae, and three species from subtribe were The BI analyses yielded better-supported trees than those 156 100 chosen as the outgroup. Fresh specimens of 92 species were inferred by maximum parsimony. Hence, the BI consensus tree 157 101 obtained from cultivation and wild collection. The GenBank acces- (Figs. 1 and 2) inferred from the combined dataset was chosen 158 102 sion numbers of the newly produced sequences and other DNA for the discussion of phylogenetic relationships. All of the topolo- 159 103 samples obtained from GenBank are listed in Table A1. gies indicate that Aeridinae is monophyletic. Our results recognize 10 well-distinguished clades: (1) Saccolabium clade, (2) Chiloschista 160 clade, (3) Phalaenopsis clade, (4) Thrixspermum clade, (5) Vanda 161 104 2.2. Collection of DNA sequences clade, (6) Aerides clade, (7) Trichoglottis clade, (8) Abdominea clade, 162 (9) Gastrochilus clade, and (10) Cleisostoma clade. 163 105 Total genomic DNA extractions, polymerase chain reaction 106 (PCR) assays and DNA sequencing were performed using the proto- (1) The Saccolabium clade (PP 100) comprises 33 species from 164 107 cols suggested by Zhang et al. (2013). The primers employed for 22 genera. It is subdivided into three subclades: Pteroceras 165 108 the amplification and sequencing of the five DNA regions investi- subclade, subclade, and Saccolabium subclade. 166 109 gated in this study are listed in Table A2. Editing, identification, The Pteroceras subclade, which is sister to the other two sub- 167 110 and alignment of the DNA sequences were performed according clades, includes five genera: Pteroceras, , Grosour- 168 111 to Zhang et al. (2013). dya, Ascochilopsis, and . Pteroceras emerges as 169 paraphyletic because P. decipiens is allied with Ascochilus 170 112 2.3. Phylogenetic analyses emarginatus but not with Pteroceras semiteretifolium. With 171 weak support (PP 61), the Sarcochilus subclade contains Sar- 172 113 Phylogenetic analyses based on maximum parsimony (MP) and cochilus, , , , , and 173 114 ⁄ Bayesian inference (BI) were performed with PAUP version 4.0b10 Taeniophyllum. The Saccolabium subclade comprises 11 gen- 174 115 (Swofford, 2002) and MrBayes 3.2.2 (Ronquist et al., 2012), respec- era: , , , Saccolabium, 175 116 tively. Missing data were coded with ‘‘?’’ in the MP analyses and , , , , Dyakia, 176 117 excluded from the BI calculation. In the MP analyses, all of the , and Cryptopylos. 177 118 characters were equally weighed and unordered, and a heuristic (2) The Chiloschista clade is composed of C. yunnanensis, C. luni- 178 119 search with 1000 random addition sequence replicates and tree-bi- fera, C. pusilla C. parishii and one unidentified Chiloschista 179 120 section-reconnection branch swapping were performed. In the BI species with strong support (PP 100). 180 121 analyses, the best-fit models (Table A3) for each dataset were (3) The Phalaenopsis clade (PP 100) comprises Phalaenopsis, 181 122 selected by Modeltest3.7 (Posada and Crandall, 1998) under the and Ornithochilus. Hygrochilus is clustered with 182 123 Akaike Information Criterion, and the following settings were Ornithochilus, and these are sister to Phalaenopsis. 183 124 used: sampling frequency = 1000, temp = 0.1, burn-in = 3750, and (4) Dimorphorchis is sister to Thrixspermum in the Thrixspermum 184 125 number of Markov Chain Monte Carlo generations = 15,000,000. clade with weak support (PP 73). The monophyly of 185 Thrixspermum was tentatively confirmed in this analysis. 186 126 3. Results (5) The Vanda clade (PP 100) is composed of four distinct sub- 187 clades: s.l. (PP 100), Vanda s.l. (PP 100), 188 127 3.1. ITS and cpDNA analyses subclade (PP 100) and Ascocentropsis. Holcoglossum s.l. 189 includes Holcoglossum, Paraholcoglossum, Tsiorchis, Pendu- 190 128 For the ITS matrix, cpDNA matrix, and each individual dataset, lorchis, and Penkimia. Vanda s.l. contains Ascocentrum, Chris- 191 129 the parameters, numbers of variables, parsimony informative sites, tensonia, , Vanda, and Neofinetia, and these 192 130 and tree statistics for MP analysis are listed in Table 1. genera are monophyletic. The Luisia subclade comprises Lui- 193 131 The results of the incongruence length difference (ILD) test sia, , and . As the eldest sister 194 132 (p = 0.01) for ITS + cpDNA suggested the existence of incongruence within the Vanda clade, Ascocentropsis pusilla forms an inde- 195 133 in the datasets. The main incongruences detected in the ITS pendent subclade by itself. 196 134 (Fig. A1) and cpDNA (Fig. A2) analyses based on BI analyses are (6) The Aerides clade (PP 82) is composed of two species from 197 135 the following: (a) the placement of Dimorphorchis as sister to Ptero- , two species from , four species from 198 136 ceras + Brachypeza + + Ascochilopsis + Ascochilus (Sacco- , six species from Aerides, and bella. 199 137 labium clade) was observed in the ITS analysis (PP 75), but the Rhynchostylis is sister to the remaining members of this group. 200 138 cpDNA analysis indicated that Dimorphorchis rossii was sister to However, the relationships among Aerides, Esmeralda, Arach- 201 139 Thrixspermum in the Thrixspermum clade (PP < 50) and that Dimor- nis, and Renanthera are poorly resolved with low support. 202

Please cite this article in press as: Zou, L.-H., et al. A molecular phylogeny of Aeridinae (Orchidaceae: Epidendroideae) inferred from multiple nuclear and chloroplast regions. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.02.014 YMPEV 5128 No. of Pages 8, Model 5G 28 February 2015

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Table 1 Statistics of the chloroplast and nuclear datasets from the parsimony analysis.

Information nrITS atpI-atpH matK psbA-trnH trnL-F cpDNA Combined No. taxa 218 121 187 124 149 188 219 Aligned length 824 1298 1643 1451 2370 6771 7595 No. Variable Characters 713 528 665 414 1084 2696 3175 No. parsimony-informative characters 368 281 433 156 562 1434 1802 Tree length 2899 984 1810 672 2250 5908 8968 Consistency index(CI) 0.3029 0.688 0.4917 0.7396 0.6302 0.5907 0.4871 Retention index(RI) 0.7063 0.7766 0.7115 0.6615 0.732 0.7047 0.6948

203 (7) The Trichoglottis clade includes eight genera. , Ade- Pteroceras semiteretifolium. Smith (1920) claimed that Sarcochilus 254 204 noncos, , and form a distinct subclade with emarginatus (=Ascochilus emarginatus) was the nearest ally of Ptero- 255 205 strong support (PP 97), and the remaining genera, namely ceras decipiens. In fact, the two species share common features an 256 206 Trichoglottis, , Ceratochilus, and , unmovable lip and no authentic spur. 257 207 form the other subclade (PP 100). Both parviflora Although the sister relationship between Taeniophyllum and Pte- 258 208 and Micropera pallida are nested within Acampe. roceras was strongly supported by the molecular data reported by 259 209 (8) The Abdominea clade (PP 51) is composed of Abdominea Topik et al. (2005) and Topik et al. (2012), a different relationship 260 210 minimiflora, Microsaccus griffithii, Cleisomeria pilosulum, in which Taeniophyllum is sister to Bogoria and Rhinerrhiza in the 261 211 Monanthochilus chrysanthus, and congestum. Sarcochilus subclade was resolved in our analyses. The members 262 212 (9) The Gastrochilus clade contains nine species of Gastrochilus, of the Sarcochilus subclade are characterized by four pollinia, no col- 263 213 one species of Haraella, and four species of . umn foot, and a less elongated gynostemium. Schlechter (1913) 264 214 Haraella is sister to Gastrochilus with weak support (PP 65). subdivided Taeniophyllum into two subgenera, Codonosepalum and 265 215 (10) The Cleisostoma clade comprises 50 species belonging to 14 Eutaeniophyllum, based on the basal edges of perianths coherent 266 216 genera. Three subclades, namely the Cleisostoma subclade, or free, respectively. However, the relationships within Taeniophyl- 267 217 subclade, and subclade, are recognized lum revealed in the present study do not appear to be congruent 268 218 for discussion in detail. The Cleisostoma subclade (PP 100) with Schlechter’s view. In our examination, subgenus Codonosepa- 269 219 contains 10 species of Cleisostoma, one species of Cleisocen- lum represented by T. complanatum, T. glandulosum, and T. aphyllum 270 220 tron, two species of , and two species of Stere- is monophyletic, but the perianth-segmented species belonging to 271 221 ochilus. The Uncifera subclade (PP 86) includes U. the subgenera Eutaeniophyllum, T. fasciola, T. biocellatum and T. 272 222 acuminata, Robiquetia succisa, helferi, S. micran- smithii are not monophyletic. Because Taeniophyllum comprises 273 223 tha, and Cleisostoma nangongense. The Schoenorchis subclade approximately 180 species (Chase et al., 2003), the best approach 274 224 (PP 76) contains five species of , eight species of to clarify the inner relationships is the sampling of a large propor- 275 225 Cleisostoma, six species of Schoenorchis, and two species of tion of its taxa. 276 226 Echioglossum. Cleisostoma is triphyletic because its species In the Saccolabium subclade, Parapterocearas elobe, the type spe- 277 227 are located in the three aforementioned subclades. Other cies of Parapteroceras, is not clustered with Tuberolabium. 278 228 taxa that were not organized into groups are Diploprora trun- This result is not congruent with that reported by Christenson 279 229 cata, D. championii, Vandopsis gigantea, V. lissochiloides, V. (1992), who considered Parapteroceras a marginal genus of Tubero- 280 230 undulate, Malleola baliensis, M. ligulata, Robiquetia spatulata, labium. In fact, Parapteroceras is morphologically different from 281 231 and philippinensis. The genera Robiquetia, Malleola, Tuberolabium because it has an articulation between the lip and 282 232 and Diploprora are polyphyletic. the column (Tsi et al., 1999). Wood (1990) placed T. escritorii in 283 233 Parapteroceras. However, the present analyses suggest that Tubero- 284 234 4. Discussion labium escritorii, a species with no joint at the base of the lip, must 285 be retained in Tuberolabium. 286 235 In the present study, an updated phylogeny of Aeridinae was 236 proposed based on more comprehensive samplings of both species 4.2. Chiloschista clade 287 237 and DNA sequences. The monophyly of Aeridinae has been verified 238 by prior investigations (Carlsward et al., 2006; Topik et al., 2005) In the Chiloschista clade, Chiloschista is defined by leaflessness 288 239 and the present study, but heated debates of the infra-subtribe (at least in the flowering phase), four pollinia, and a spurred or 289 240 composition continue. Our analyses provide new insights into the pouched disk with a fingerlike and hirsute-pubescent callus. Topik 290 241 of Aeridinae, and based on our findings, some reassess- et al. (2005) found consanguinity between Chiloschista and Ornitho- 291 242 ments and modifications are necessary. chilus, but our analyses do not support this relationship. Chiloschista 292 shares no similarity in lip structure or vegetative morphology with 293 243 4.1. Saccolabium clade the latter. In GenBank, we found evidence that the predecessors 294 may have reconstructed the relationship based on pseudo-matK 295 244 The Saccolabium clade, which is sister to all other members of sequences. In fact, employing pseudogenes for phylogenetic ana- 296 245 Aeridinae, is characterized by an undeveloped and abbreviated lyses may lead to incorrect topologies (Meimberg et al., 2006). 297 246 middle lobe that is smaller than the lateral lobes of the lip. Three 247 subclades are well-distinguished in this group: the Pteroceras sub- 4.3. Phalaenopsis clade 298 248 clade with two cleft pollinia (except Grosourdya with entire polli- 249 nia), the Saccolabium subclade with two entire pollinia (except The monophyly of Phalaenopsis, Hygrochilus and Ornithochilus is 299 250 Cryptopylos and Macropodanthus), and the Sarcochilus subclade strongly supported (PP 100). These taxa are characterized by an 300 251 with four pollinia. abbreviated stem (except Hygrochilus parishii), fleshly and flat 301 252 In the Pteroceras subclade, Pteroceras is resolved as non-mono- leaves similar to those in genus Phalaenopsis, a well-developed 302 253 phyletic because P. decipiens is allied with Ascochilus but not with middle lobe of the lip with a longitudinal keel, four pollinia (except 303

Please cite this article in press as: Zou, L.-H., et al. A molecular phylogeny of Aeridinae (Orchidaceae: Epidendroideae) inferred from multiple nuclear and chloroplast regions. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.02.014 YMPEV 5128 No. of Pages 8, Model 5G 28 February 2015

4 L.-H. Zou et al. / Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx

Fig. 1. Part A of the Bayesian tree obtained from analysis of the combined dataset showing the detailed relationships of the outgroup and the Saccolabium, Chiloschista,

Phalaenopsis, Thrixspermum, and Vanda clades. The numbers near the nodes are the Bayesian posterior probabilities (100Â) and the bootstrap percentages (PP left, BPMP right).

Please cite this article in press as: Zou, L.-H., et al. A molecular phylogeny of Aeridinae (Orchidaceae: Epidendroideae) inferred from multiple nuclear and chloroplast regions. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.02.014 YMPEV 5128 No. of Pages 8, Model 5G 28 February 2015

L.-H. Zou et al. / Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx 5

Fig. 2. Part B of the Bayesian tree obtained from analysis of the combined dataset showing the detailed relationships of the Aerides, Trichoglottis, Abdominea, Gastrochilus, and

Cleisostoma clades. The numbers near the nodes are the Bayesian posterior probabilities (100Â) and the bootstrap percentages (PP left, BPMP right).

Please cite this article in press as: Zou, L.-H., et al. A molecular phylogeny of Aeridinae (Orchidaceae: Epidendroideae) inferred from multiple nuclear and chloroplast regions. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.02.014 YMPEV 5128 No. of Pages 8, Model 5G 28 February 2015

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304 some species of Phalaenopsis), obovate or cuneiform stipes and a the other genera because it has no spur or sac at the base of the 366 305 larger-sized viscidium than the base of the stipe. Our results indi- lip and a footless column. However, it shares slender and cylindrical 367 306 cate that Doritis, Nothoritis, Lesliea, and Kingidium must be treated leaves with Papilionanthe and Paraphalaenopsis. In some cases, the 368 307 as synonyms of Phalaenopsis (e.g., Padolina et al., 2006; Topik floral traits do not mirror phylogeny, which is attributed to pollina- 369 308 et al., 2005; Tsai et al., 2010; Yukawa et al., 2005). The systematic tor-mediated evolution (e.g., Kocyan et al., 2008; Xiang et al., 2012). 370 309 position of is a problem that has long puzzled taxonomists 310 (Topik et al., 2005; Kocyan et al., 2008), and Li et al. (2014) recently 4.6. Aerides clade 371 311 identified Sedirea as congeneric with Hygrochilus based on DNA and 312 morphological data. Our results also resolve these relationships. Flat leaves, larger viscid discs than stipes and moderately large 372 flowers are the traits shared by the members of the Aerides clade. 373 313 4.4. Thrixspermum clade Through additional sampling, our analyses propose close relation- 374 ships among Aerides, Arachnis, Esmeralda and Renanthera, which is 375 314 The characteristic shared by the members of the Thrixspermum not in agreement with the close relationships between Aerides and 376 315 clade is a longitudinal prominence on the disk. The sister relation- Christensonia + Turdelia obtained in the study conducted by Topik 377 316 ship between Thrixspermum and Dimorphorchis obtained in our et al. (2005). Our results suggest that Aerides, Arachnis, and Renan- 378 317 analysis is consistent with the results reported by Topik et al. thera are monophyletic. With the exception of the resolved sister- 379 318 (2005). Dimorphorchis is a special group characterized by long relationship between Esmeralda and Arachnis, the other internal 380 319 hairy inflorescences with two distinctly different flowers and relationships remain unclear. 381 320 huge-size vegetation. This genus had been previously taxonomical- 321 ly placed in Vanda, Renanthera, Arachnanthe, Arachnis, and Vandop- 4.7. Trichoglottis clade 382 322 sis. Due to the inconsistency of the systematic relationships of 323 Dimorphorchis between nuclear and DNA, we speculate that Four pollinia, an immovable lip, and a longitudinal flange across 383 324 the genus may be the result of natural hybridization. the lip characterize the Trichoglottis clade. With the exceptions of 384 Micropera, the other taxa share the consistent trait of a hairy or 385 325 4.5. Vanda clade papillose lip. Topik et al. (2005) recognized the close relationship 386 among Trichoglottis, Staurochilus, Ceratochilus, Ventricularia, and 387 326 Of all of the taxa in Aeridinae, this group has recently received . However, our results indicate that V. lis- 388 327 the most attention (Christenson, 1987; Gardiner, 2012; Gardiner sochiloides is eliminated from the affinity of this group. Sharing 389 328 et al., 2013; Liu et al., 2011; Xiang et al., 2012; Zhang et al., its main characteristic of flowers with Trichoglottis, Staurochilus 390 329 2013). The Vanda clade is characterized by two pollinia, a fixed has been considered congeneric with the former (summary in 391 330 lip and larger middle lobes than laterals. Seidenfaden, 1992). In fact, the greatest difference between the 392 331 In Holcoglossum s.l., Tsiorchis is imbedded in a subclade with two genera is that Trichoglottis has short inflorescences and the lat- 393 332 Pendulorchis and Penkimia, and another subclade contains Para- ter has long inflorescences. However, T. latisepala, which possesses 394 333 holcoglossum and Holcoglossum. Based on DNA and vegetative data, short inflorescences, is embedded in Staurochilus. The unexpected 395 334 Xiang et al. (2012) suggested that Holcoglossum (including Para- position of T. latisepala suggests that more sampling is necessary 396 335 holcoglossum and Tsiorchis) was paraphyletic, containing Penkimia when inspecting these two genera. Our topologies indicate that 397 336 and Pendulorchis (Ascolabium). Hence, they proposed a Holcoglos- Adenoncos parviflora and Micropera pallida are nested in Acampe. 398 337 sum sensu lato. However, our analyses revealed that each of the Including the above-mentioned traits, Adenoncos and Acampe share 399 338 five genera presented in this manuscript are monophyletic, which other morphological features, such as short inflorescences and a 400 339 is congruent with the findings reported by Zhang et al. (2013). The rather fleshy labellum. For these reasons, Adenoncos parviflora 401 340 separation of Tsiorchis from Holcoglossum was elaborated by Liu should be treated as a member of Acampe. However, Micropera pal- 402 341 et al. (2011). Due to the markedly divergent floral characteristics lida is markedly different from Acampe because it has a long spur 403 342 among these genera (Xiang et al., 2012; Zhang et al., 2013), the and a non-fleshy lip without hair. Carlsward et al. (2006) suggested 404 343 retention of a narrowly defined Holcoglossum may be a more accu- that there is no kinship between this species and Acampe. Hence, 405 344 rate classification. without stronger evidence, the inclusion of Micropera pallida in 406 345 In Vanda s.l., Ascocentrum is successively sister to Christensonia, Acampe necessitates additional reconsideration. 407 346 Seidenfadenia, Vanda, and Neofinetia. The monophyly of Ascocen- 347 trum was supported by Zhang et al. (2013), who corrected the 4.8. Abdominea clade 408 348 placements of the spurious species Holcoglossum pumilum (=Asco- 349 centrum pumilum) and Pendulorchis himalaicum (=Ascocentrum The Abdominea clade, which is characterized by four pollinia, 409 350 himalaicum). Recently, a combination of Vanda s.s. with related comprises Abdominea, Microsaccus, Cleisomeria, Monantochilus, 410 351 genera, namely Ascocentrum, Ascocentropsis, Christensonia, Epar- and Diplocentrum. The affinity of the former four genera was recog- 411 352 matostigma and Neofinetia, was proposed by Gardiner and col- nized by Topik et al. (2005) and in our study. However, the analysis 412 353 leagues (Gardiner, 2012; Gardiner et al., 2013). These arguments weakly supports (PP 51) the finding that Diplocentrum is sister to 413 354 have focused on the phylogenetic positions of the taxa referred the other four taxa. Hence, the systematic placement of Diplocen- 414 355 from only cpDNA data but not the specific morphological charac- trum still needs to be examined in a future study. 415 356 ters used to delimit Vanda. The taxa of Vanda s.s. have large-sized 357 perianths with twisted or wavy edges, a distorted base, a shortly 4.9. Gastrochilus clade 416 358 spurred lip and an inappendiculate mid-lobe. However, Ascocen- 359 trum and Neofinetia possess flat perianths, a long spur and a mid- Composed of Gastrochilus, Haraella, and Pomatocalpa, the Gas- 417 360 lobe with appendages. Furthermore, Ascocentropsis was resolved trochilus clade is characterized by an immovable lip and a sad- 418 361 as the earliest diverging lineage of all of the other members of dle-shaped or concave viscidium. We found that Haraella,a 419 362 the Vanda clade but was not found in Vanda s.l. Hence, a morpho- monotypic genus, is allied with Gastrochilus. This result shows no 420 363 logically diversiform Vanda still needs to be reconsidered. differences from that reported by Yukawa (cited in Watthana 421 364 A close relationship among Luisia, Papilionanthe and Parapha- et al., 2006). Nine species of Gastrochilus were examined in this 422 365 laenopsis was resolved in the Luisia subclade. Luisia differs from study, and the genus was resolved as monophyletic. Although the 423

Please cite this article in press as: Zou, L.-H., et al. A molecular phylogeny of Aeridinae (Orchidaceae: Epidendroideae) inferred from multiple nuclear and chloroplast regions. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.02.014 YMPEV 5128 No. of Pages 8, Model 5G 28 February 2015

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424 study only covered the species of section Gastrochilus and section Based on a combined dataset of both nuclear and plastid markers, 485 425 Microphylla in this genus, the results indicate that section Gas- the main lineages of Aeridinae were resolved. Based on our results, 486 426 trochilus (Tsi, 1996) is polyphyletic. This is a first step to exploring the subtribe was found to be composed of 10 major clades. 487 427 the intrageneric relationship of Gastrochilus through molecular Through our morphological analyses, we investigated the 488 428 measures, and careful analyses of the whole genus should be per- relationships among members of the subtribe, namely Pteroceras, 489 429 formed in the future. Cleisostoma, Vandopsis, Diploprora, Malleola, Robiquetia, subgenus 490 Codonosepalum of Taeniophyllum and section Gastrochilus of Gas- 491 trochilus, which were found to be polyphyletic. The circumscription 492 430 4.10. Cleisostoma clade of Cleisostoma and its related taxa was incorrect in previous mor- 493 phological classifications. In addition, we found some other prob- 494 431 The polyphyly of Cleisostoma has been reported in several studies lems, such as the taxonomic status of Micropera pallida and the 495 432 (Carlsward et al., 2006; Topik et al., 2005, 2012). With greater sam- relationship between and Cleisostoma. These issues 496 433 pling, our analysis of Cleisostoma and its related genera found that should be resolved by increasing taxon sampling and including 497 434 21 species of the genus comprised the following three subclades: more DNA regions in future studies. 498 435 Cleisostoma subclade, Schoenorchis subclade, and Uncifera subclade. 436 In the Cleisostoma subclade, Sarcoglyphis, , and 437 Cleisocentron are nested among the species from sections Cleisos- 6. Uncited reference 499 438 toma and Subulatum of Cleisostoma. In fact, these genera (except 439 Cleisocentron) share the main synapomorphy of a fleshy callus on Garay (1972). 500 440 the back wall of the spur close to the entrance either alone or 441 together with a cushion of the front wall near the base of the lip. Acknowledgments 501 442 Due to the presence of the type section (sect. Cleisostoma) in this 443 subclade, these species of Sarcoglyphis and Stereochilus should be We gratefully acknowledge Xu-Hui Chen, Wen-Chieh Tsai, and 502 444 transferred to Cleisostoma. Due to the unexpected placement of Liu Qiang for providing materials. We thank Ming-He Li and two 503 445 Cleisocentron merrillianum in the subclade and because it shares anonymous reviewers for providing helpful comments regarding 504 446 no generic characteristics with Cleisostoma, we have no opinions our manuscript. This study was supported by the Development 505 447 regarding this entity. More samplings of Cleisocentron species Special Fund of Biological Industry of Shenzhen Municipality 506 448 would aid our conclusions of its systematic position. (Grant Nos. JC201005310692A and JC201005310690A). 507 449 Three branches are well recognized in the Schoenorchis sub- 450 clade: (1) Pelatantheria s.l., (2) Schoenorchis, and (3) sections Pan- 451 iculatum and Echioglossum of Cleisostoma.InPelatantheria s.l., the Appendix A. Supplementary material 508 452 species from sections Complicata, Mitriformis, and Repentia of 453 Cleisostoma are sister to Pelatantheria. The species in this group pos- Supplementary data associated with this article can be found, in 509 454 sess synapomorphies in having a spur with a septum, a fleshy callus the online version, at http://dx.doi.org/10.1016/j.ympev.2015.02. 510 455 on the inner backwall, a round of hair on the middle of the inner 014. 511 456 wall, a saddle-shaped or crescent-shaped viscidium, and an elon- 457 gated stem. The group of sections Paniculatum and Echioglossum References 512 458 shares a spur with a fleshy callus on the inner backwall and a 459 winged column. Section Echioglossum was erected by Seidenfaden Carlsward, B.S., Whitten, W.M., Williams, N.H., Bytebier, B., 2006. Molecular 513 460 (1975) based on Echioglossum Blume. Due to the absence of the type Phylogenetics of Vandeae (Orchidaceae) and the evolution of leaflessness. Am. 514 515 461 species of Echioglossum, namely E. javanica (=Cleisostoma javanica), J. Bot. 93, 770–786. Chase, M.W., Cameron, K.M., Barrett, R.L., Freudenstein, J.V., 2003. DNA data and 516 462 the recovery of Echioglossum cannot be determined. Orchidaceae systematics: a new phylogenetic classification. In: Orchid 517 463 Although having the aforementioned features found in Pelatan- Conservation. Natural History Publications, , Kota Kinabalu, pp. 69–89. 518 519 464 theria s.l., Cleisostoma nangongense in the Uncifera subclade was Christenson, E.A., 1987. 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Please cite this article in press as: Zou, L.-H., et al. A molecular phylogeny of Aeridinae (Orchidaceae: Epidendroideae) inferred from multiple nuclear and chloroplast regions. Mol. Phylogenet. Evol. (2015), http://dx.doi.org/10.1016/j.ympev.2015.02.014