See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/230997222 Phylogenetic analysis of the grape family (Vitaceae) based on the noncoding plastid trnC-petN, trnH-psbA, and trnL-F sequences Article in Taxon · June 2011 CITATIONS READS 30 279 7 authors, including: Limin Lu Quentin Luke 11 PUBLICATIONS 88 CITATIONS 46 PUBLICATIONS 338 CITATIONS Institute of Botany CAS SEE PROFILE National Museums of Kenya SEE PROFILE Dianxiang Zhang Zhiduan Chen 129 PUBLICATIONS 611 CITATIONS 133 PUBLICATIONS 3,488 CITATIONS Chinese Academy of Sciences SEE PROFILE Chinese Academy of Sciences SEE PROFILE Some of the authors of this publication are also working on these related projects: Phylogeny and diversification of Tetrastigma (Vitaceae) View project island biodiversity View project All content following this page was uploaded by Limin Lu on 19 December 2016. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. TAXON 60 (3) • June 2011: 629–637 Ren & al. • Plastid phylogeny of Vitaceae M O L E C U L A R PH Y LO G E N E T I C S A N D B I O G E O G R A PH Y Phylogenetic analysis of the grape family (Vitaceae) based on the noncoding plastid trnC-petN, trnH-psbA, and trnL-F sequences Hui Ren,1,3 Li-Min Lu,2,3 Akiko Soejima,4 Quentin Luke, 5 Dian-Xiang Zhang,1 Zhi-Duan Chen2 & Jun Wen2,6 1 South China Botanical Garden, the Chinese Academy of Sciences, Guangzhou 510650, China 2 State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China 3 Graduate School of the Chinese Academy of Sciences, Beijing 100039, China 4 Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kurokami, Kumamoto 860-8555, Japan 5 East African Herbarium, National Museums of Kenya, Nairobi 00502, Kenya 6 Department of Botany, National Museum of Natural History, MRC166, Smithsonian Institution, Washington, D.C. 20013-7012, U.S.A. Hui Ren and Li-Min Lu contributed equally to this paper. Author for correspondence: Jun Wen, wenj@si.edu Abstract The phylogeny of Vitaceae was reconstructed sampling 114 accessions of Vitaceae and the outgroup Leea of Leeaceae, using three noncoding plastid markers: trnC-petN, trnH-psbA, and trnL-F. Six 5-merous genera including Parthenocissus, Yua, Ampelocissus, Vitis, Nothocissus, and Pterisanthes form a well-supported clade. Ampelopsis, Rhoicissus, and the Cissus striata complex form a clade sister to the clade containing all the other taxa of Vitaceae. The core Cissus clade is resolved to be sister to the Cayratia-Tetrastigma-Cyphostemma clade, forming a clade of taxa with 4-merous flowers. The ParthenocissusYua clade is sister to the Ampelocissus-Vitis-Nothocissus-Pterisanthes clade. The Old World Cissus is paraphyletic, with the New World core Cissus nested within it. The intercontinental disjunction between Africa and Asia may have evolved at least twice in Cissus. Cayratia is paraphyletic with four Asian species sampled grouping with Tetrastigma and the African species forming another clade. Keywords phylogeny; trnC-petN ; trnH-psbA; trnL-trnF ; Vitaceae INTRODUCTION Vitaceae (the grape family) consist of 14 genera and about 900 species primarily distributed in tropical regions in Asia, Africa, Australia, the Neotropics, and the Pacific islands, with two genera (Parthenocissus Planch. and Ampelopsis Michx.) disjunctively distributed in the north temperate regions (Soejima & Wen, 2006; Wen, 2007a; Wen & al., 2007). Vitaceae is economically highly important containing Vitis vinifera L. as source of grapes and raisins, as well as some taxa of Parthenocissus as ornamentals (e.g., Parthenocissus quinquefolia (L.) Planch. and P. tricuspidata (Sieb. & Zucc.) Planch.). Taxa of Vitaceae are usually woody climbers or herbaceous vines, and occasionally become succulent trees (Chen & al., 2007; Wen, 2007a). This family can be easily recognized by its leaf-opposed tendrils, unique seed morphology usually with a pair of ventral infolds and a dorsal chalaza, presence of “pearl” glands (small multicellular spherical caducous epidermal structures with a short stalk), and calcium oxalate crystals contained in parenchyma (Metcalfe & Chalk, 1950; Arnott & Webb, 2000; Chen & Manchester, 2007; Wen, 2007a; Chen, 2009). Recent molecular analyses generally supported the Vitaceae clade (including Leeaceae) as sister to all other rosids (Soltis & al., 2000, 2005, 2007; Jansen & al., 2006; Wang & al., 2009). APG III (2009) placed Vitaceae as sister to the fabids + malvids clade (eurosids I + II) and recognized it in its own order Vitales following Takhtajan (1997). The genus Leea L. has been excluded from Vitaceae and treated as the monogeneric family Leeaceae by Vitaceae/Leeaceae specialists (e.g., Planchon, 1887; Suessenguth, 1953; Ridsdale, 1974; Shetty & Singh, 2000; Latiff, 2001; Ren & al., 2003; Chen & al., 2007; Wen, 2007a,b). Leeaceae has been supported as the closest relative of Vitaceae based on DNA molecular phylogenetic and morphological data (Soejima & Wen, 2006; Wen, 2007a,b; Wang & al., 2009). Generic delimitation in Vitaceae has been problematic (Soejima & Wen, 2006; Wen, 2007a; Wen & al., 2007). Linnaeus (1753) established the first two genera—Cissus L. and Vitis L.—in Vitaceae and different classification systems have been proposed ever since (Hooker, 1862; Baker, 1871; Lawson, 1875; Planchon, 1887). The number of genera has also increased from two (Linnaeus, 1753) through ten (Planchon, 1887; Suessenguth, 1953) to presently 14 (Wen, 2007a). Planchon (1887) delimited ten genera largely based on characteristics of the inflorescences and nectariferous discs. The 14 genera are Acareosperma Gagnep., Ampelocissus Planch., Ampelopsis, Cayratia Juss., Cissus, Clematicissus Planch., Cyphostemma (Planch.) Alston, Nothocissus (Miq.) Latiff, Parthenocissus, Pterisanthes Blume, Rhoicissus Planch., Tetrastigma (Miq.) Planch., Vitis, and Yua C.L. Li. (Soejima & Wen, 2006; Wen, 2007a; Wen & al., 2007). Clematicissus has been recently expanded from a monotypic genus from Western Australia to also include the former Cissus opaca F. Muell. (Jackes & Rossetto, 2006). 629 Ren & al. • Plastid phylogeny of Vitaceae The phylogeny of Vitaceae has been reconstructed with several markers (Ingrouille & al., 2002; Rossetto & al., 2001, 2002; Soejima & Wen, 2006; Wen & al., 2007). With 37 taxa sampled in the combined analyses, Soejima & Wen (2006) reconstructed the phylogeny of Vitaceae based on three chloroplast markers (trnL-F region, atpB-rbcL spacer, the rps16 intron), which supported three major clades: (1) the Ampelopsis-RhoicissusParthenocissus-Vitis-Nothocissus-Pterisanthes-Ampelocissus clade; (2) the core Cissus clade (except the South American Cissus striata complex); and (3) the Cayratia-TetrastigmaCyphostemma clade. Wen & al. (2007) sampled eleven genera and 95 species and infraspecific taxa of Vitaceae to reconstruct the relationships within Vitaceae with the nuclear GAI1 sequences. The three major clades formerly recognized by Soejima & Wen (2006) were strongly supported by the GAI1 data. Particularly, the first clade was 100% supported by the GAI1 data compared to a less than 50% bootstrap (BS) value in the three plastid markers, and a close relationship between the core Cissus clade and the 5-merous clade was well supported. Different from the plastid phylogeny, the GAI1 data recognized Ampelopsis as the closest relative of Parthenocissus instead of Vitis, although the support values were low. Rossetto & al. (2007) constructed the phylogeny of Australian Vitaceae using plastid trnL-F and nuclear internal transcribed spacer sequences. Their data supported a robust sister relationship between Clematicissus and a clade of two South American Cissus (Cissus tweediana (Baker) Planch. and Cissus striata Ruiz & Pav.) and further supported the paraphyly of Cayratia. With the limited taxon sampling in the previous plastid dataset and the low support values of several major clades, it is necessary to construct the phylogeny of Vitaceae using plastid markers with more extensive taxon sampling. We herein expanded the taxon sampling to represent the morphological diversity and geographic range of Vitaceae (especially with enhanced sampling in Africa, southeastern Asia and South America). Our main objectives of this study are to construct the plastid phylogeny of Vitaceae using three noncoding intergenic spacers, trnC-petN, trnH-psbA, and trnL-F, and to compare the phylogeny with the previous analyses of the family. MATERIALS AND METHODS Taxon sampling. — This study sampled 114 accessions representing 12 genera of Vitaceae with six taxa of Leeaceae as outgroups. Voucher specimens were deposited at the US National Herbarium, Washington, D.C. (see Appendix). DNA extraction, amplification, and sequencing. — Genomic DNAs were extracted from silica-gel–dried material or herbarium material using the DNeasy Plant Mini Kit protocol (Qiagen, Crawley, U.K.). Amplification protocol and primers for amplifying trnC-petN, trnH-psbA, and trnL-F were from Shaw & al. (2005), Lee & Wen (2004), and Soejima & Wen (2006), respectively. PCR products were purified by the polyethylene glycol (PEG) precipitation method (Wen & al., 2007). Purified PCR products were sequenced in both directions by standard methods using BigDye 3.1 reagents with an ABI 630 TAXON 60 (3) • June 2011: 629–637 3730 automated sequencer (Applied Biosystems, Foster City, California, U.S.A.) with the primers from the original amplification. The direct and reverse sequences were assembled and corrected using Sequencher v.4.1.4 (Gene Codes Co., Ann Arbor, Michigan, U.S.A.). Sequence alignment was initially performed using ClustalX v.1.83 (Thompson & al., 1997) and then manually adjusted in the program Se-Al v.2.0a11 (Rambaut, 2002). Phylogenetic analyses. — Phylogenetic analyses of each partition and the combined plastid DNA dataset (trnC-petN, trnH-psbA, trnL-F) were conducted using maximum parsimony (MP) (Fitch, 1971) and Bayesian inference (BI) (Rannala & Yang, 1996; Mau & al., 1999). For maximum parsimony, PAUP* v.4.0b10 (Swofford, 2003) was used with heuristic search, 10 random stepwise additions, TBR branch swapping, collapse of zero-length branches, multrees option in effect, holding one tree at each step. Gaps were either treated as missing data or coded as simple indels (Simmons & Ochoterena, 2000) using the program SeqState (Müller, 2005). Parsimony bootstrap analyses (Felsenstein, 1985) were subsequently performed employing 1000 replicates, with the random taxon addition sequence limited to 10 and branch swapping limited to 10,000,000 rearrangements per replicate. Prior to the model-based analytical approaches, Modeltest v.3.7 (Posada & Crandall, 1998) was implemented to identify the best available model for nucleotide substitutions. The generalized time reversible model (GTR + I + G model) was suggested as the best-fit model of sequence evolution for the combined plastid dataset. Bayesian inference was carried out in MrBayes v.3.1.2 (Ronquist & Huelsenbeck, 2003) with a GTR + I + G model as determined above. We performed two independent runs of 2,000,000 generations from a random starting tree with four Markov chains, sampling one tree every 100 generations. To check whether the burn-in stage had reached stationarity, the likelihood scores and number of generations were plotted. After discarding the first 2500 trees as burn-in, a 50% majority-rule consensus tree was calculated in PAUP* for the remaining trees to estimate the posterior probabilities (PP). Bayesian analyses were repeated twice to confirm results. RESULTS The three plastid markers (trnC-petN, trnH-psbA, trnL-F) had 3096 aligned positions, of which 2176 were constant, 301 were variable but parsimony-uninformative, and 619 were parsimony-informative. The aligned length of each marker was 798 from trnH-psbA, 1167 from trnC-petN and 1131 from trnL-F. Treating gaps as missing data, the parsimony search of the combined dataset yielded more than 100,000 most parsimonious trees (1549 steps, consistency index CI = 0.74, retention index RI = 0.93). The strict consensus tree of the combined dataset corresponded to the majority-rule consensus of 17,501 trees (20,001 trees minus 2500 as burn-in) derived from the BI analysis (Fig. 1). Three strongly supported clades were recognized within Vitaceae: the Ampelocissus-Vitis-NothocissusPterisanthes-Parthenocissus-Yua clade (Fig. 1B; BS = 88%, TAXON 60 (3) • June 2011: 629–637 PP = 1.00), the core Cissus clade (Fig. 1A; BS = 100%, PP = 1.00), and the Cayratia-Tetrastigma-Cyphostemma clade (Fig. 1A; BS = 100%, PP = 1.00). The core Cissus formed a clade sister to the Cayratia-Tetrastigma-Cyphostemma clade with moderate support (BS = 77%, PP = 0.96). Within the Cayratia-Tetrastigma-Cyphostemma clade, both Tetrastigma and Cyphostemma were strongly supported as monophyletic (BS = 100%, PP = 1.00 for each of the two clades). Cayratia was paraphyletic with four Asian species sampled forming a clade sister to Tetrastigma (BS = 100%, PP = 1.00) and the African species forming another well-supported clade (BS = 100%, PP = 1.00) (Fig. 1A). The African species of Cayratia were sister to the clade comprising the Asian Cayratia taxa and the monophyletic Tetrastigma. The Ampelopsis-Rhoicissus-Cissus striata clade was recognized in both MP and BI analyses, but with low support (Fig. 1B; BS < 50%, PP = 0.81). All the three plastid DNA regions contained gaps. After the ambiguous blocks in the alignment were deleted, there were 237 indel characters in the plastid DNA dataset (65 from trnHpsbA, 100 from trnC-petN and 72 from trnL-F), of which 157 were parsimony-informative. The analysis treating indels as new characters generated more than 100,000 most parsimonious trees (1868 steps, CI = 0.73, RI = 0.93). The topology of the strict consensus tree from the analyses with gaps as new characters was identical to that with gaps as missing data. The bootstrap values of most clades were similar in both analyses, except that the indel characters increased the bootstrap values of the Cissus-Cayratia-Tetrastigma-Cyphostemma clade to 82% (vs. 77% with gaps as missing data), and decreased that of the Ampelocissus-Vitis-Nothocissus-Pterisanthes-Parthenocissus-Yua clade to 79% (vs. 88% with gaps as missing data). DISCUSSION Our present analyses sampling 12 genera and 98 species of Vitaceae and three plastid markers provided a well-supported phylogeny for the family. Six 5-merous genera including Ampelocissus, Vitis, Nothocissus, Pterisanthes, Parthenocissus and Yua, form a well-supported clade. Ampelopsis, Rhoicissus and the Cissus striata complex form a clade sister to all the other taxa of Vitaceae. The core Cissus clade is resolved to be sister to the Cayratia-Tetrastigma-Cyphostemma clade. All taxa with 4-merous flowers form a clade. Five-merous taxa. — The relationships among the 5-merous taxa have never been well resolved in previous studies. The nuclear GAI1 data (Wen & al., 2007) weakly supported (BS = 63%) the Ampelopsis-Rhoicissus-Cissus striata clade as the closest relative of the Parthenocissus-Yua clade, while the plastid data by Soejima & Wen (2006) supported the close relationship between the Ampelocissus-Vitis-NothocissusPterisanthes clade and the Parthenocissus-Yua clade but with low support (BS = 63%). In the present study, Ampelocissus, Vitis, Nothocissus, Pterisanthes, Parthenocissus, and Yua form a well supported clade (BS = 88%, PP = 1.00). Within this clade, two subclades, the Ampelocissus-Vitis-Nothocissus-Pterisanthes clade and the Parthenocissus-Yua clade, are recognized Ren & al. • Plastid phylogeny of Vitaceae albeit with low support (Fig. 1B). The Ampelopsis-RhoicissusCissus striata clade was well supported by previous analyses (Soejima & Wen, 2006; Wen & al., 2007), but it is only weakly supported here. The Ampelocissus-Vitis-Nothocissus-Pterisanthes clade is composed of Nothocissus spicifera (Griff.) Latiff, Pterisanthes stonei Latiff, two Ampelocissus from Central America and 11 species of Vitis. Vitis is characterized by its dioecious reproductive biology and calyptrate petals, and two very distinct subgenera are commonly recognized: V. subg. Vitis L. and V. subg. Muscadinia (Planch.) Small (Brizicky, 1965; Wen, 2007a). Subgenus Vitis contains about 60 species distributed primarily in eastern Asia and North America to Central America, while subg. Muscadinia consists of only 2–3 species from North America, the West Indies to Mexico. The monophyly of Vitis was not resolved in the current study, but has been supported by the recent phylogenetic work on Vitis (Tröndle & al., 2010). Here species of subg. Vitis form a weakly supported clade and two species from subg. Muscadinia (Vitis popenoei J.L. Fennell, V. rotundifolia Michx.) constitute a strongly supported clade. Within subg. Vitis, species from Asia and North America form an unresolved polytomy (Fig. 1B). With 30 Vitis species and several cultivars of V. vinifera sampled, the genus-level phylogenetic study discriminated three clades within subg. Vitis corresponding to their distribution in Europe, Asia, and North America, although the European and American clades were not well supported (Tröndle & al., 2010). Based on cpDNA polymorphisms, Péros & al. (2011) recognized a wellsupported American clade and obtained evidence supporting an Asian origin for subg. Vitis. They also strongly supported the sister relationship between subg. Vitis and subg. Muscadinia. Taxa of subg. Muscadinia differ morphologically from subg. Vitis in their simple tendrils, prominent lenticels on stems, pith continuous through the nodes, and shorter infructescences with fewer fruits (Brizicky, 1965; Wen, 2007a). Furthermore, the diploid chromosome number in subg. Vitis is 38, while it is 40 in V. popenoei and V. rotundifolia. Some workers (e.g., Bouquet, 1983) thus suggested splitting Vitis popenoei, V. rotundifolia and V. munsoniana Simpson ex Munson from Vitis and establishing a distinct genus. The close relationship between Ampelocissus and Vitis has been reported by previous studies (Soejima & Wen, 2006; Wen & al., 2007). Ampelocissus is a genus with ca. 90 species distributed in Asia, Africa, and Central America. It was segregated from Vitis by Planchon (1884) based on its 4–5-merous flowers in thyrses, inflorescences subtended by a tendril near the base, and the T-shaped endosperm in transverse section (M-shaped in Vitis) (Jackes, 1984). The Asian Ampelocissus was suggested to be more closely related to Nothocissus and Pterisanthes than to its congeneric species in Central America (Soejima & Wen, 2006). We need to expand the sampling of Ampelocissus further evaluate the relationships within the Ampelocissus-Vitis-Nothocissus-Pterisanthes clade. Within the Parthenocissus-Yua clade, three subclades are recognized. The two species of Yua form a strongly supported clade. Taxa of Yua were segregated from Parthenocissus by Li (1990) based on their 2-branched tendrils, leaf-opposed dichasia and the extent of the seed ventral infolds upward for 2/3 of the 631 Ren & al. • Plastid phylogeny of Vitaceae TAXON 60 (3) • June 2011: 629–637 seed length from the base (Li, 1998; Chen & al., 2007). Species of Parthenocissus form two separate clades corresponding to their distribution in the New and the Old World (Fig. 1B). The New World Parthenocissus includes three species with palmate 5–7-foliolate leaves, while the Old World Parthenocissus contains nine species with simple or 3–5-foliolate leaves. Three subclades are recognized within the Old World Parthenocissus, which are consistent with their leaf, tendril and inflorescence characters (Fig. 1B). The subclade of Parthenocissus dalzielii Gagnep., P. tricuspidata, and P. suberosa Hand.-Mazz. possesses synapomorphies of having simple and 3-foliolate leaves, the young apex of tendrils expanding to form ball-like structures, and a loose corymbose polychasium on extremely short branches. The 3-foliolate species are from 100 1.00 100 1.00 A 92 1.00 53 1.00 Africa Asia New World 4-merous taxa 100 1.00 100 1.00 100 1.00 94 1.00 <50 0.64 100 1.00 92 1.00 81 1.00 97 1.00 100 1.00 100 1.00 100 1.00 90 1.00 100 1.00 100 1.00 100 0.99 100 1.00 100 1.00 100 0.98 100 1.00 51 1.00 100 1.00 100 1.00 77 0.96 100 1.00 96 1.00 74 0.72 100 1.00 100 1.00 100 1.00 66 0.93 51 0.97 100 1.00 87 1.00 100 1.00 100 0.99 64 0.80 83 1.00 100 1.00 87 0.99 100 1.00 62 0.92 98 1.00 Tetrastigma sp. W5983 Tetrastigma serrulatum NM445 Tetrastigma obtectum W9121 Tetrastigma obtectum NM454 Tetrastigma triphyllum NM342 Tetrastigma triphyllum W9051 Tetrastigma yunnanense W9143 Tetrastigma xishuangbannaense R55108 Tetrastigma sp. W8370 Tetrastigma bioritsense W9451 Tetrastigma hemsleyanum NM451 Tetrastigma erubescens R55116 Tetrastigma garrettii NP s.n. Tetrastigma jinghongense W8471 Tetrastigma lanyuense W9404 Tetrastigma pachyphyllum W8319 Tetrastigma planicaule R55071 Tetrastigma siamense 03439 Cayratia corniculata W9461 Cayratia japonica W8330 Cayratia japonica W6140 Cayratia japonica W9262 Cayratia japonica W9263 Cayratia maritima W9403 Cayratia trifolia R55095 Cayratia trifolia R55101 Cayratia debilis C3459 Cayratia debilis C4136 Cayratia gracilis 5828 Cyphostemma sp. L11552 Cyphostemma sp. RG6814 Cyphostemma duparquetii L11534 Cyphostemma dysocarpum L11457 Cyphostemma kilimandscharicum L11469 Cyphostemma kibweziense L11481 Cyphostemma vogelli 4127 Cyphostemma sp. RG6878 Cyphostemma cyphopetalum L11451 Cyphostemma heterotrichum Lovett4027 Cyphostemma kirkianum L11473 Cyphostemma maranguense L11468 Cyphostemma thomasii L11448 Cyphostemma zimmermannii L11476 Cyphostemma montagnacii W6672 Cissus sp. NW53919 Cissus erosa W8574 Cissus sp. W8738 Cissus incisa W7287 Cissus sicyoides W8734 Cissus assamica NM362 Cissus assamica W9406 Cissus discolor 20061111 Cissus cornifolia L11452 Cissus producta L11528 Cissus trothae L11537 Cissus albiporcata L11456 Cissus phymatocarpa L11474 Cissus quadrangularis W7368 Cissus subtetragona R55110 Cissus rotundifolia L11478 Cissus rotundifolia L11458 Cissus aralioides 19870062 Cissus sciaphila L11477 Fig. 1. Strict consensus tree of 208 equally most parsimonious trees from the combined analysis of the plastid trnC-petN, trnH-psbA, and trnL-F (1549 steps; CI = 0.74; RI = 0.93). A, Four-merous clade including the core Cissus clade and the Cayratia-Tetrastigma-Cyphostemma clade; B, fivemerous taxa including the Ampelocissus-Vitis-Nothocissus-Pterisanthes-Parthenocissus-Yua clade and the weakly supported Ampelopsis-Rhoicissus-Cissus striata clade. Numbers above branches indicate bootstrap values, and numbers below branches are Bayesian posterior probability values. 632 TAXON 60 (3) • June 2011: 629–637 Ren & al. • Plastid phylogeny of Vitaceae 75 0.98 <50 0.90 57 1.00 92 1.00 88 1.00 98 1.00 88 1.00 88 1.00 100 1.00 75 1.00 96 1.00 88 1.00 100 1.00 100 1.00 63 1.00 100 1.00 <50 0.99 97 1.00 82 0.99 <50 0.81 92 1.00 100 1.00 100 1.00 85 0.95 79 0.70 91 1.00 90 0.99 Ampelocissus acapulcensis W8696 Ampelocissus erdwendbergii W8702 Nothocissus spicifera W8384 Pterisanthes stonei W8346 Vitis betulifolia W9308 Vitis lanata W9184 Vitis lanata W9197 Vitis mengziensis NM415 Vitis chunganensis W9305 Vitis heyneana W9042 Vitis heyneana W9378 Vitis riparia W8658 Vitis tilifolia W8713 Vitis thunbergii W9446 Vitis sp. NM372 Vitis popenoei W8724 Vitis rotundifolia W11087 Parthenocissus chinensis NM455 Parthenocissus chinensis W6530 Parthenocissus dalzielii W9325 Parthenocissus dalzielii W9372 Parthenocissus suberosa NM358 Parthenocissus tricuspidata NM355 Parthenocissus henryana NM359 Parthenocissus laetevirens W9379 Parthenocissus quinquefolia W8684 Parthenocissus vitacea W7234 Yua austro-orientalis S1313 Yua thomsoni NM469 Ampelopsis bodinieri W8017 Ampelopsis bodinieri R55193 Ampelopsis delavayana W9377 Ampelopsis glandulosa W9380 Ampelopsis glandulosa var. kulingensis Ampelopsis glandulosa var. hancei Ampelopsis japonica R55207 Ampelopsis cordata W7141 Ampelopsis denudata W8699 Ampelopsis cantoniensis W9381 Ampelopsis grossedentata R55072 Ampelopsis chaffanjonii W9359 Ampelopsis rubifolia W9285 Cissus striata W7355 Rhoicissus tridentata L11453 Rhoicissus tomentosa 19656252 Leea aequata W8382 Leea indica W8341 Leea rubra 05-716 Leea guineensis W8250 Leea guineensis W9408 Leea macrophylla R55105 Subg. Vitis Africa Asia New World 5-merous taxa Subg. Muscadinia <50 1.00 3-foliolate Simple or 3-foliolate 5-7-foliolate Sect. Ampelopsis B The Ampelopsis-Rhoicissus-Cissus striata clade was supported in previous studies (e.g., Soejima & Wen, 2006; Wen & al., 2007). This clade is present in both the strict consensus tree and the Bayesian 50% majority-rule tree with low support in the current analyses. Ampelopsis is a genus of about 25 species, which are disjunctively distributed between temperate to subtropical Asia and North and Central America (Wen, 2007a). This genus is characterized by its leaf-opposed corymbose cymes, 5-merous flowers, well-developed floral disks and M-shaped endosperm. In particular, it covers the leaf diversity of the entire family, with leaves varying from simple, trifoliate, palmate to 1- or 2-pinnate. Two sections corresponding to leaf morphology have been recognized: A. sect. Ampelopsis with simple or palmately divided (rarely palmately compound) Sect. Leeaceifoliae Asia and characterized by their three leaflets, long and curving tendrils, and a compound pseudoterminal dichasium on a short branch with two to three leaves. The close relationship between Parthenocissus henryana (Hemsl.) Graebn. ex Diels & Gilg and P. laetevirens Rehder is supported by their 5-foliolate leaves, young apex of tendrils expanding as tubercles, and a paniculate-polychasium with a well-developed main axis. The clades recognized by our analyses are generally congruent with those from the recent phylogenetic analysis of Parthenocissus by Nie & al. (2010). The monophyly of Parthenocissus has been well supported by previous molecular work (Wen & al., 2007; Nie & al., 2010), but the relationship between Parthenocissus and Yua was not resolved by the three plastid markers employed in this study. 633 Ren & al. • Plastid phylogeny of Vitaceae leaves, and A. sect. Leeaceifoliae with 1- or 2-pinnately compound leaves (Galet, 1967). Ampelopsis was shown to be paraphyletic by previous plastid and nuclear GAI1 analyses. The plastid data (Soejima & Wen, 2006) supported that the African Rhoicissus and the South American Cissus striata complex formed a clade with the simple or palmately leaved Ampelopsis, while the nuclear data (Wen & al., 2007) suggested that they were more closely related to the pinnately leaved Ampelopsis. In the current analysis, sect. Ampelopsis, sect. Leeaceifoliae, Rhoicissus and Cissus striata form a polytomy. Congruent with previous analyses, the two sections based on leaf morphology are supported as monophyletic (Fig. 1B). Beyond the differences in leaf morphology, the two sections also differ in that taxa in sect. Ampelopsis have serial accessory buds, while those in sect. Leeaceifoliae have complex axillary buds as in Vitis vinifera (Bernard, 1972–1973; Gerrath & Posluszny, 1989; Soejima & Wen, 2006; Wen & al., 2007). The Ampelopsis-Rhoicissus-Cissus striata clade is suggested to be sister to the major clade consisting of all the other taxa of Vitaceae. This result is not consistent with the previous plastid and nuclear GAI1 data and needs to be tested. The rbcL data (Ingrouille & al., 2002), however, resolved Ampelopsis as the basalmost branch of Vitaceae albeit with no support. Ingrouille & al. (2002) argued that the presence of pinnate leaves, the thick corolla, and the floral and vegetative development in Ampelopsis were the least-derived characters within Vitaceae as compared with those in the outgroup taxa from Leeaceae (also see Gerrath & Posluszny, 1988a). We will test the position of the Ampelopsis-Rhoicissus-Cissus striata clade in our future analyses. Four-merous taxa. — Our data have resolved the 4-merous taxa as a moderately supported clade. Beyond the 4-merous flowers, the 4-merous clade also has stomatal apparatuses that are mostly hemiparacytic, cyclocytic or staurocytic (vs. mostly anomocytic in the 5-merous taxa), and the chromosome number as n = 10, 11, 12, 13 (mostly 19 or 20 in the 5-merous species) (Ren & al., 2003; Wen, 2007a). Cissus is the largest genus in Vitaceae with about 350 species distributed throughout the tropics (Wen, 2007a) and possesses remarkable morphological diversity (Jackes, 1988; Lombardi, 2007). This genus usually has simple leaves, welldeveloped and thick floral disks, and one-seeded fruits, although there are species with compound leaves and more than one seed per fruit (Rossetto & al., 2002; Wen, 2007a; Wen & al., 2007). A core group of Cissus was supported in Rossetto & al. (2001, 2002, 2007), Soejima & Wen (2006), and Wen & al. (2007). This group contains most taxa from the Old World (Africa, Asia, Australia) and the New World (the Americas), but does not include the South American Cissus striata, C. simsiana Roem. & Schult., C. tweediana and the Australasian C. antarctica Vent., C. hypoglauca A. Gray, C. oblonga (Benth.) Planch., C. opaca and C. sterculiifolia (Benth.) Planch. Cissus opaca has been recently transferred to Clematicissus (Jackes & Rossetto, 2006). The core Cissus clade is resolved into two subclades (Fig. 1A), with one composed of taxa from the Old World only (Africa and Asia), and the other containing both the New and Old World taxa, in which the New World core 634 TAXON 60 (3) • June 2011: 629–637 Cissus sampled so far form a monophyletic group. In particular, both subclades include species from Africa and Asia, suggesting the intercontinental disjunction between Africa and Asia has evolved at least twice within Cissus. Recent phylogenetic analyses resolved Cissus as polyphyletic and recognized three distinct clades: the core Cissus clade, the Cissus striata clade and the Cissus antarctica clade (Rossetto & al., 2002, 2007; Soejima & Wen, 2006; Wen & al., 2007). The core Cissus clade contains the majority taxa of Cissus including the type of the generic name C. quadrangularis L. and covers the morphological diversity of the entire genus. In the present study, the core Cissus clade is composed of both simple-leaved (e.g., C. quadrangularis) and compound-leaved taxa (e.g., Cissus erosa Rich., C. aralioides Planch.). The Cissus striata clade includes the South American Cissus striata and its close relatives (e.g., C. simsiana, C. tweediana), which were supported as closely related to Rhoicissus and Ampelopsis by Soejima & Wen (2006) and Wen & al. (2007). Rossetto & al. (2007), however, strongly supported Cissus striata and C. tweediana as sister to Clematicissus. Wen & al. (2007) hypothesized that Clematicissus may well belong to the Ampelopsis-RhoicissusCissus striata clade. The Cissus antarctica clade includes four Australian species: C. antarctica, C. hypoglauca, C. oblonga and C. sterculiifolia, which were resolved as nested within the Vitis clade and were weakly supported as more closely related to V. rotundifolia by the combined plastid (trnL) and nuclear (ITS1) data (Rossetto & al., 2002). Rossetto & al. (2002) suggested that the four Australian Cissus species may be placed in Muscadinia or as part of a new genus distinct from Vitis. The updated phylogenetic work of Rossetto & al. (2007), however, did not replicate the sister relationships between Vitis rotundifolia and the Cissus antarctica clade. Furthermore, the comparative ontogenetic studies of Vitis riparia, V. rotundifolia, Cissus antarctica and C. quadrangularis did not support the close relationship between C. antarctica and Vitis (Gerrath & Posluszny, 1988b,c, 1994; Timmons & al., 2007a,b). Timmons & al. (2007b) proposed to segregate the four Australian species from Cissus and establish a distinct genus based on the number of chromosomes, DNA variability, presence of supernumerary buds, degree of stipule connectivity, uncommitted primordial, and type of inflorescence branching. Cissus exhibits remarkable morphological diversity, complex biogeographic distributions and variable karyotypes and chromosomal numbers (2n = 20, 22, 24, 26, 28, 32, 44, ca. 45, 48, 50, and 96) and it needs to be further analyzed with an expanded sampling scheme. Within the Cayratia-Tetrastigma-Cyphostemma clade, Cyphostemma and Tetrastigma are each monophyletic, while Cayratia is paraphyletic. The paraphyly of Cayratia has been reported previously (e.g., Ingrouille & al., 2002; Rossetto & al., 2002, 2007; Soejima & Wen, 2006; Wen & al., 2007). The analysis of Ingrouille & al (2002) indicated a weakly supported Cayratia-Tetrastigma clade. Rossetto & al. (2002) supported the close relationship between Cayratia and Tetrastigma. In addition, Cayratia japonica (Thunb.) Gagnep. and C. trifolia (L.) Domin were suggested by the ITS data to be closely related (Rossetto & al., 2002). With only four Asian Cayratia species sampled, the previous studies using plastid data and the TAXON 60 (3) • June 2011: 629–637 nuclear GAI1 data (Soejima & Wen, 2006; Wen & al., 2007) supported a close relationship between Cayratia japonica and the monophyletic Tetrastigma with the other Asian Cayratia species forming another clade (Soejima & Wen, 2006; Wen & al., 2007). Cayratia contains 63 species and is distributed mostly in tropical and subtropical Africa, Asia, Australia, and the Pacific. This genus is characterized by its axillary, or pseudo-axillary, or sometimes leaf-opposed inflorescences with bisexual, tetramerous flowers and T- or U-shaped endosperm in transverse section. With the sampling of the African Cayratia, our plastid analyses suggest that the three Cayratia from Africa form a strongly supported clade, while the Asian species sampled so far form a robust clade with the monophyletic Tetrastigma. The close relationship between Tetrastigma and the Cayratia japonica complex (e.g., C. trifolia, C. pseudotrifolia W.T. Wang) has been reported by previous studies (Soejima & Wen, 2006; Wen & al., 2007). The circumscription of Cayratia japonica, however, is very broad. With four individuals sampled from China, Vietnam, and Malaysia, respectively, Cayratia japonica is supported to be paraphyletic with C. corniculata (Benth.) Gagnep. nested within it. Thus, the delimitation of this species and the relationships within the Cayratia japonica complex need further investigation. CONCLUSIONS Our analyses provide further insights into the generic relationships within Vitaceae. Parthenocissus and Yua are supported as closely related to Vitis instead of to Ampelopsis. The basalmost position of Ampelopsis is weakly supported, which requires further test with morphological and molecular evidence. Instead of being nested within the 5-merous clade as in previous analyses, the core Cissus clade is resolved as sister to a clade composed of all other 4-merous taxa with moderate support. The relationships within the core Cissus clade were further clarified with the expanded sampling. Cayratia was conformed as paraphyletic with the monophyletic Tetrastigma nested within it. Cayratia japonica and its close relatives from Asia form a clade sister to the monophyletic Tetrastigma, whereas the two African Cayratia species we sampled form a distinct clade. ACKNOWLEDGEMENTS The study was supported by the National Science Foundation (grant DEB 0743474 to S.R. Manchester and J. Wen) and the John D. and Catherine T. MacArthur Foundation to J. Wen. Laboratory work was done at and partially supported by the Laboratory of Analytical Biology of the National Museum of Natural History, Smithsonian Institution. Fieldwork in North America and Madagascar was supported by the Small Grants Program of the National Museum of Natural History of the Smithsonian Institution, and an Endowment grant from the Office of Undersecretary of Sciences of the Smithsonian Institution. Ren & al. • Plastid phylogeny of Vitaceae LITERATURE CITED APG III (Angiosperm Phylogeny Group). 2009. 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(2010). Taxon: collector(s) and collection number; locality; GenBank accession numbers for trnC-petN; trnH-psbA; trnL-F. Ampelocissus acapulcensis (Kunth) Planch.: J. Wen 8696; Mexico, Oaxaca; JF437172; JF437058; JF437281. Ampelocissus erdwendbergii Planch.: J. Wen 8702; Mexico, Chiapas; JF437173; JF437059; JF437282. Ampelopsis bodinieri (H. Lév. & Vaniot) Rehder: H. Ren 55193; China, Guangdong; JF437175; JF437061; 636 TAXON 60 (3) • June 2011: 629–637 Ren & al. • Plastid phylogeny of Vitaceae Appendix. Continued. JF437284. Ampelopsis bodinieri (H. Lév. & Vaniot) Rehder: J. Wen 8017; China, Gansu; JF437174; JF437060; JF437283. Ampelopsis cantoniensis (Hook. & Arn.) K. Koch: J. Wen 9381; China, Guangxi; JF437176; JF437062; JF437285. Ampelopsis chaffanjonii (H. Lév.) Rehder: J. Wen 9359; Madagascar, Antsiranana; JF437177; JF437063; JF437286. Ampelopsis cordata Michx.: J. Wen 7141; U.S.A., Illinois (cult.); JF437178; JF437064; AB234997. Ampelopsis delavayana Planch. ex Franch.: J. Wen 9377; China, Guangxi; JF437179; JF437065; JF437287. Ampelopsis denudata Planch.: J. Wen 8699; Mexico, Chiapas; JF437180; JF437066; JF437288. Ampelopsis glandulosa (Wall.) Momiy. var. hancei (Planch.) Momiy.: J. Wen 9402; China, Taiwan; JF437183; JF437069; JF437291. Ampelopsis glandulosa (Wall.) Momiy. var. kulingensis(Rehder) Momiy.: J. Wen 9283; China, Hunan; JF437182; JF437068; JF437290. Ampelopsis glandulosa (Wall.) Momiy.: J. Wen 9380; China, Guangxi; JF437181; JF437067; JF437289. Ampelopsis grossedentata (Hand.-Mazz.) W.T. Wang: H. Ren 55072; China, Yunnan; JF437184; JF437070; JF437292. Ampelopsis japonica (Thunb.) Makino: H. Ren 55207; China, Guangdong; JF437185; JF437071; –. Ampelopsis rubifolia (Wall.) Planch.: J. Wen 9285; China, Hunan; JF437186; JF437072; JF437293. Cayratia corniculata (Benth.) Gagnep.: J. Wen 9461; China, Taiwan; JF437188; JF437074; –. Cayratia debilis (Baker) Suess.: Carvalho 3459; Africa; JF437189; JF437075; JF437295. Cayratia debilis (Baker) Suess.: Carvalho 4136; Africa; JF437190; JF437076; JF437296. Cayratia gracilis (Guill. & Perr.) Suess.: 5828; Africa; JF437191; JF437077; JF437297. Cayratia japonica (Thunb.) Gagnep.: J. Wen 8330; Malaysia, Selangor; JF437192; JF437078; JF437298. Cayratia japonica (Thunb.) Gagnep.: J. Wen 6140; Vietnam, Lao Cai; JF437196; JF437082; AB235009. Cayratia japonica (Thunb.) Gagnep.: J. Wen 9262; China, Sichuan; JF437197; JF437083; JF437300. Cayratia japonica (Thunb.) Gagnep.: J. Wen 9263; China, Sichuan; JF437198; JF437084; JF437301. Cayratia maritima Jackes: J. Wen 9403; China, Taiwan; JF437193; JF437079; JF437299. Cayratia trifolia (L.) Domin: H. Ren 55095; China, Yunnan; JF437194; JF437080; –. Cayratia trifolia (L.) Domin: H. Ren 55101; China, Yunnan; JF437195; JF437081; –. Cissus albiporcata Masinde & L.E. Newton: Luke & Luke 11456; Kenya, Chyulu Plains; JF437201; JF437087; JF437304. Cissus aralioides Planch.: Aplin 19870062; Belgium, National Botanical Garden (cult.); JF437202; JF437088; JF437305. Cissus assamica (M.A. Lawson) Craib: J. Wen 9406; China, Taiwan; JF437204; JF437090; JF437307. Cissus assamica (M.A. Lawson) Craib: Z.-L. Nie & Y. Meng 362; China, Guizhou; JF437203; JF437089; JF437306. Cissus cornifolia (Baker) Planch.: Luke & Luke 11452; Kenya, Nr KWS Rhino Camp.; JF437205; JF437091; JF437308. Cissus discolor Blume: 20061111; Belgium, National Botanical Garden (cult.); JF437206; JF437092; JF437309. Cissus erosa Rich.: J. Wen 8574; Peru; JF437207; JF437093; JF437310. Cissus incisa Des Moul.: J. Wen 7287; U.S.A., Texas; JF437208; JF437094; AB235014. Cissus phymatocarpa Masinde & L.E. Newton: Luke & Luke 11474; Kenya, Diani Forest; JF437209; JF437095; JF437311. Cissus producta Afzel.: Luke & al. 11528; Tanzania, Udzungwa Mountain; JF437210; JF437096; JF437312. Cissus quadrangularis L.: J. Wen 7368; Thailand, Chiangmai (cult.); JF437211; JF437097; JF437313. Cissus rotundifolia (Forssk.) Vahl: Luke & Luke 11478; Kenya, Taru; JF437212; JF437098; JF437314. Cissus rotundifolia (Forssk.) Vahl: Luke & Luke 11458; Kenya, Merueshi; JF437213; JF437099; JF437315. Cissus sciaphila Gilg: Luke & Luke 11477; Kenya, Shimba Hills; JF437214; JF437100; JF437316. Cissus sicyoides L.: J. Wen 8734; Mexico; JF437215; JF437101; JF437317. Cissus sp.: M. Nee & J. Wen 53919; Bolivia, Santa Cruz; JF437199; JF437085; JF437302. Cissus sp.: J. Wen 8738; Mexico, Chiapas; JF437200; JF437086; JF437303. Cissus striata Ruiz & Pav.: J. Wen 7355; Chile; –; JF437104; JF437319. Cissus subtetragona Planch.: H. Ren 55110; China, Yunnan; JF437216; JF437102; –. Cissus trothae Gilg & M. Brandt: Luke & al. 11537; Tanzania, Udzungwa Mountain; JF437217; JF437103; JF437318. Cyphostemma cyphopetalum (Fresen.)Wild & R.B. Drumm.: Luke & Luke 11451; Kenya, Nr KWS Rhino Camp.; JF437221; JF437108; JF437323. Cyphostemma duparquetii (Planch.) Desc.: Luke & al. 11534; Kenya, Udzungwa Mountain; JF437222; JF437109; JF437324. Cyphostemma dysocarpum (Gilg & M. Brandt) Desc.: Luke & Luke 11457; Kenya, Chyulu Plains; JF437223; JF437110; JF437325. Cyphostemma heterotrichum (Gilg & R.E. Fr.) Desc. ex Wild & R.B. Drumm.: L. Lovett 4027; Tanzania; JF437224; JF437111; JF437326. Cyphostemma kibweziense Verdc.: Luke & Luke 11481; Kenya, Mbinzau; JF437229; JF437116; JF437330. Cyphostemma kilimandscharicum (Gilg) Wild & R.B. Drumm.: Luke & Luke 11469; Kenya, Chyulu Hills; JF437225; JF437112; JF437327. Cyphostemma kirkianum (Planch.) Wild & R.B. Drumm: Luke & Luke 11473; Kenya, Diani Forest; JF437226; JF437113; JF437328. Cyphostemma maranguense (Gilg) Desc.: Luke & Luke 11468; Kenya, Chyulu Hills; JF437227; JF437114; JF437329. Cyphostemma montagnacii Desc.: J. Wen 6672; U.S.A., Missouri Botanical Garden (cult); JF437228; JF437115; AB235027. Cyphostemma sp.: R.G.6814; Africa; JF437219; JF437106; JF437321. Cyphostemma sp.: R.G. 6878; Africa; JF437220; JF437107; JF437322. Cyphostemma sp.: Luke 11552; Kenya, Nairobi; JF437218; JF437105; JF437320. Cyphostemma thomasii (Gilg & M. Brandt) Desc.: Luke & Luke 11448; Kenya, Makindu; JF437230; JF437117; JF437331. Cyphostemma vogelli (Hook.) Desc.: 4127; Africa; JF437231; JF437118; JF437332. Cyphostemma zimmermannii Verdc.: Luke & Luke 11476; Kenya, Shimba Hills; JF437232; JF437119; JF437333. Leea aequata L.: J. Wen 8382; Malaysia, Perak; JF437233; JF437120; –. Leea guineensis G. Don: J. Wen 8250; Philippines, Laguna; JF437234; JF437121; –. Leea guineensis G. Don: J. Wen 9408; China, Taiwan; JF437235; JF437122; –. Leea indica Merr.: J. Wen 8341; Malaysia; JF437236; JF437123; JF437334. Leea macrophylla Roxb. ex Hornem. & Roxb.: H. Ren 55105; China, Yunnan; JF437237; JF437124; JF437335. Leea rubra Blume: MAC 05-716; Thailand, Sai Yok; JF437238; JF437125; –. Nothocissus spicifera (Griff.) Latiff: J. Wen 8384; Malaysia, Perak; JF437239; JF437126; JF437336. Parthenocissus chinensis C.L. Li: Z.-L. Nie & Y. Meng 455; China, Sichuan; JF437240; JF437127; HM223263. Parthenocissus chinensis C.L. Li: J. Wen 6530; China, Yunnan; JF437241; JF437128; HM223278. Parthenocissus dalzielii Gagnep.: J. Wen 9325; China, Hunan; JF437242; JF437129; JF437337. Parthenocissus dalzielii Gagnep.: J. Wen 9372; China, Hunan; JF437243; JF437130; JF437338. Parthenocissus henryana (Hemsl.) Graebn. ex Diels & Gilg: Z.-L. Nie & Y. Meng 359; China, Guizhou; JF437244; JF437131; HM223272. Parthenocissus laetevirens Rehder: J. Wen 9379; China, Guangxi; JF437245; JF437132; JF437339. Parthenocissus quinquefolia (L.) Planch.: J. Wen 8684; Mexico, Oaxaca (cult.); JF437246; JF437133; HM223275. Parthenocissus suberosa Hand.-Mazz.: Z.-L. Nie & Y. Meng 358; China, Guizhou; JF437247; JF437134; HM223273. Parthenocissus tricuspidata (Sieb. & Zucc.) Planch.: Z.-L. Nie & Y. Meng 355; China, Guizhou; JF437248; JF437135; HM223274. Parthenocissus vitacea (Knerr) Hitchc.: J. Wen 7234; U.S.A., Texas; JF437249; JF437136; JF437340. Pterisanthes stonei Latiff: J. Wen 8346; Malaysia, Selangor; –; JF437137; AB235046. Rhoicissus tomentosa (Lam.) Wild & R.B. Drumm.: 19656252; Belgium, National Botanical Garden (cult.); JF437251; JF437139; JF437342. Rhoicissus tridentata (L.f.) Wild & R.B. Drumm.: Luke & Luke 11453; Kenya, Chyulu Hills; JF437250; JF437138; JF437341. Tetrastigma bioritsense (Hayata) Hsu & Kuoh: J. Wen 9451; China, Taiwan; JF437252; JF437140; HM585964. Tetrastigma erubescens Planch.: H. Ren 55116; China, Yunnan; JF437253; JF437141; JF437343. Tetrastigma garrettii Gagnep.: N.P. Pui s.n.; Thailand, Chiang Mai; JF437254; JF437142; JF437344. Tetrastigma hemsleyanum Diels & Gilg: Z.-L. Nie & Y. Meng 451; China, Sichuan; JF437255; JF437143; HM586000. Tetrastigma jinghongense C.L. Li: J. Wen 8471; China, Yunnan; JF437256; JF437144; HM586006. Tetrastigma lanyuense C.E. Chang: J. Wen 9404; China, Taiwan; JF437257; JF437145; HM586009. Tetrastigma obtectum (Wall.) Planch.: J. Wen 9121; China, Yunnan; JF437258; JF437146; JF437345. Tetrastigma obtectum (Wall.) Planch.: Z.-L. Nie & Y. Meng 454; China, Sichuan; JF437266; JF437154; HM585751. Tetrastigma pachyphyllum (Hemsl.) Chun: J. Wen 8319; Philippines, Ifugao; JF437259; JF437147; HM586032. Tetrastigma planicaule Gagnep.: H. Ren 55071; China, Yunnan; JF437260; JF437148; JF437346. Tetrastigma serrulatum Planch.: Z.-L. Nie & Y. Meng 445; China, Yunnan; JF437261; JF437149; HM586042. Tetrastigma siamense Gagnep. & Craib: 03-439; Thailand, Bahng Mah Pah District; JF437262; JF437150; JF437347. Tetrastigma sp.: J. Wen 5983; Vietnam, Lao Cai; JF437187; JF437073; JF437294. Tetrastigma sp.: J. Wen 8370; Philippines; JF437268; JF437156; JF437351. Tetrastigma triphyllum (Gagnep.) W.T. Wang: J. Wen 9051; China, Yunnan; JF437264; JF437152; JF437348. Tetrastigma triphyllum (Gagnep.) W.T. Wang: Z.-L. Nie & Y. Meng 342; China, Yunnan; JF437263; JF437151; HM586061. Tetrastigma xishuangbannaense C.L. Li: H. Ren 55108; China, Yunnan; JF437265; JF437153; –. Tetrastigma yunnanense Gagnep.: J. Wen 9143; China, Yunnan; JF437267; JF437155; JF437350. Vitis betulifolia Diels & Gilg: J. Wen 9308; China, Hunan; JF437269; JF437157; JF437352. Vitis chunganensis Hu: J. Wen 9305; China, Hunan; JF437271; JF437159; JF437353. Vitis heyneana Roem. & Schult: J. Wen 9042; China, Yunnan; JF437272; JF437160; –. Vitis heyneana Roem. & Schult: J. Wen 9378; China, Guangxi; JF437273; JF437161; JF437354. Vitis lanata Roxb.: J. Wen 9184; China, Tibet; JF437274; JF437162; JF437355. Vitis lanata Roxb.: J. Wen 9197; China, Tibet; JF437275; JF437163; JF437356. Vitis mengziensis C.L. Li: Z.-L. Nie & Y. Meng 415; China, Yunnan; JF437270; JF437158; HM223276. Vitis popenoei J.L. Fennell: J. Wen 8724; Mexico, Chiapas; JF437276; JF437164; HM586072. Vitis riparia Michx.: J. Wen 8658; U.S.A., Virginia; JF437277; JF437165; JF437357. Vitis rotundifolia Michx.: J. Wen 11087; U.S.A., Arkansas; –; JF437166; JF437358. Vitis sp.: Z.-L. Nie & Y. Meng 372; China, Guizhou; JF437280; JF437169; JF437360. Vitis thunbergii Sieb. & Zucc.: J. Wen 9446; China, Taiwan; JF437278; JF437167; AB235082. Vitis tilifolia Humb. & Bonpl.: J. Wen 8713; Mexico, Chiapas; JF437279; JF437168; JF437359. Yua austro-orientalis (F.P. Metcalf) C.L. Li: S. Ickert-Bond1313; China, Guangdong; –; JF437170; AB235085. Yua thomsoni (M.A. Lawson) C.L. Li: Z.-L. Nie & Y. Meng 469; China, Sichuan; –; JF437171; HM223277. 637 View publication stats