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 Aeridinae (Orchidaceae: Epidendroideae) 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 China 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 1314 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) Saccolabium clade, (2) Chiloschista 35 clade, (3) Phalaenopsis clade, (4) Thrixspermum clade, (5) Vanda clade, (6) Aerides clade, (7) Trichoglottis 36 23 Keywords: clade, (8) Abdominea clade, (9) Gastrochilus clade, and (10) Cleisostoma clade. In our examination, most 37 24 Aeridinae genera of Aeridinae were well-supported as monophyletic, and several genera, namely, Pteroceras, 38 25 cpDNA 26 Molecular phylogenetics Cleisostoma, Vandopsis, Diploprora, Malleola, and Robiquetia, 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 Taeniophyllum 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 species 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 Asia, Australia, and the eastern Pacific islands, forms a logeny of Aeridinae was reconstructed by Topik et al. (2005) and 68 53 large proportion of tribe Vandeae 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 column 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 2 L.-H. Zou et al. / Molecular Phylogenetics and Evolution xxx (2015) xxx–xxx 85 In the present study, to avoid the negative effects resulting from phorchis lowii was sister to Cleisomeria + 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 + Microsaccus + 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 Dimorphorchis 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 Angraecinae, two species from subtribe Aer- 155 99 angidinae, and three species from subtribe Polystachyinae 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, Sarcochilus 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, Brachypeza, Grosour- 168 111 to Zhang et al. (2013). dya, Ascochilopsis, and Ascochilus. 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, Gunnarella, Drymoanthus, Rhinerrhiza, Bogoria, 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: Parapteroceras, Biermannia, Pennilabium, Saccolabium, 175 116 tively. Missing data were coded with ‘‘?’’ in the MP analyses and Hymenorchis, Amesiella, Tuberolabium, Ceratocentron, Dyakia, 176 117 excluded from the BI calculation.