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American Journal of Botany 97(8): 1342–1353. 2010.

American Journal of Botany 97(8): 1342–1353, 2010; http://www.amjbot.org/ © 2010 Botanical Society of America

Vitaceae (the grape family) consists of ~14 genera and 900 species primarily distributed in tropical regions with a few ex-tending to temperate areas ( Wen, 2007 ; Wen et al., 2007 ). Par-thenocissus is one of the few genera of the family with an intercontinental disjunct distribution between Asia and North America ( Rossetto et al., 2001 ; Soejima and Wen, 2006 ; Wen et al., 2007 ). It contains ~12 species with approximately nine in Asia and three in North America (one extending to Central America and the Caribbean region) ( Fig. 1 ) ( Brizicky, 1965 ; Soejima and Wen, 2006 ; Chen et al., 2007 ). Several species of the genus, such as Parthenocissus tricuspidata (Sieb. & Zucc.)

Planch. (Boston ivy), P. semicordata (Wall.) Planch. (Himala-yan ivy), P. quinquefolia (L.) Planch. (Virginia creeper), and P. vitacea (Knerr) Hitchc,, are widely cultivated as climbing ornamentals.

Species of Parthenocissus are deciduous climbers with leaves usually palmately 3 – 5( – 7) foliolate, tendrils monochasially 3 – 12 branched usually with adhesive discs at the tip of tendril branches, and fi ve-merous fl owers. The infl orescence is a loose dichasium, a corymbose cyme, or an umbel, without tendrils at the base. The fl oral discs are inconspicuous and fused with the base of the ovary ( Brizicky, 1965 ; Li, 1998 ; Chen et al., 2007 ). Tendril branching and infl orescence morphology are important characters in the genus ( Wilson and Posluszny, 2003a , b ). Yua C. L. Li was recently segregated from Parthenocissus based on its two-branched tendrils and its compound dichasia opposite to leaves ( Li, 1990 ). Yua includes 2 – 3 species from central and southern China, Vietnam, Nepal, and northern India with Yua thomsonii (M. A. Lawson) C. L. Li (= Parthenocissus thom-sonii Lawson) as the type ( Li, 1990 , 1998 ; Chen et al., 2007 ; Wen, 2007 ).

Infrageneric classifi cation of Parthenocissus has been prob-lematic. Different classifi cations were proposed based on its geographic distribution or various morphological characters. The genus was divided into two groups, i.e., the Asian group and the American group according to their distribution ( Sues-senguth, 1953 ) or three series (ser. Tricuspidatae Galet, ser.

1 Manuscript received 1 March 2010; revision accepted 21 May 2010. The authors thank Li-Min Lu for critically reading earlier versions of the

manuscript. This study was supported by grants from the National Basic Research Program of China (973 Program, grant 2007CB411601), the National Science Foundation (grant DEB 0743474 to S.R.M. and J.W.), the Natural Science Foundation of China (grant NSFC 30625004 and 40771073 to H.S.), and the John D. and Catherine T. MacArthur Foundation to J.W., R. Ree, and G. Mueller. Laboratory work was done at and partially sup-ported by the Laboratory of Analytical Biology of the National Museum of Natural History, Smithsonian Institution. Fieldwork in North America was supported by the Small Grants Program of the National Museum of Natural History, the Smithsonian Institution.

7 Author for correspondence (e-mail: wenj@si.edu)

doi:10.3732/ajb.1000085

MOLECULAR PHYLOGENY AND BIOGEOGRAPHIC DIVERSIFICATION OF PARTHENOCISSUS (VITACEAE) DISJUNCT

BETWEEN ASIA AND NORTH AMERICA 1

Ze-Long Nie 2,3 , Hang Sun 2 , Zhi-Duan Chen 4 , Ying Meng 2 , Steven R. Manchester 5 , and Jun Wen 2,4,6,7

2 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204, P. R. China; 3 Graduate School of Chinese Academy of Sciences, Beijing 100039, P. R. China; 4 Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Xiangshan, Beijing

100093, P. R. China; 5 Department of Botany, University of Florida, Gainesville, Florida 32611 USA; and 6 Department of Botany, National Museum of Natural History, MRC 166, Smithsonian Institution, Washington, D.C. 20013-7012 USA

• Premise of the study : Parthenocissus is a genus of the grape family Vitaceae and has a disjunct distribution in Asia and North America with members in both tropical and temperate regions. The monophyly of Parthenocissus has not yet been tested, and the species relationships and the evolution of its intercontinental disjunction have not been investigated with extensive sam-pling and molecular phylogenetic methods.

• Methods : Plastid ( trnL-F , rps16 , and atpB-rbcL ) and nuclear GAI1 sequences of 56 accessions representing all 12 Parthenocis-sus species were analyzed with parsimony, likelihood, and Bayesian inference. Divergence times of disjunct lineages were estimated with relaxed Bayesian dating. Evolution of the leafl et number was assessed by tracing this character onto Bayesian trees using the Trace Character Over Trees option in the program Mesquite.

• Key results : Parthenocissus is monophyletic and sister to the newly described segregate genus Yua . Two major clades within Parthenocissus are recognizable corresponding to their distribution in Asia and North America. The disjunction between the two continents is estimated to be at 21.64 (95% higher posterior densities 10.23 – 34.89) million years ago.

• Conclusions : Parthenocissus is likely to have derived from the Eocene boreotropical element. Its current Asian – North Ameri-can disjunction is dated to the early Miocene, congruent with fossil and paleoclimatic evidence. The tropical species is nested within the temperate clade and is inferred to have dispersed from the adjacent temperate regions. Parthenocissus and Yua are best treated as distinct genera. Leafl et number in this genus has a complex history and cannot be used as a character for infrage-neric classifi cation.

Key words: Asia; boreotropical element; disjunction; North America; Parthenocissus ; Vitaceae.

1343August 2010] Nie et al. — Molecular phylogeny and biogeography of PARTHENOCISSUS

Magnolia L. ( Azuma et al., 2001 ; Nie et al., 2008 ), and Toxico-dendron Mill. ( Nie et al., 2009 ) indicate relatively high levels of molecular sequence divergence and relatively deep phyloge-netic differentiation among the intercontinental thermophilic disjuncts. Although divergence times of the thermophilic line-ages need to be rigorously estimated with more taxa, results so far indicate a more ancient origin of the thermophilic disjunct groups relative to more temperate-adapted groups ( Wen, 2001 ). Parthenocissus contains both temperate and thermophilic spe-cies ( Wen, 1999 , 2001 ; Wu et al., 2003 ) with a distribution from temperate to tropical regions in both Asia and North America ( Wen et al., 2007 ). It is thus important to employ Par-thenocissus to further elucidate biogeographic diversifi cation in the northern hemisphere involving both temperate and tropical elements.

The aims of this study were to (1) test the monophyly of Par-thenocissus , (2) infer the phylogenetic relationships within the genus using the nuclear ( GAI1 ) and plastid (the trnL-F region, the rps16 intron, and the atpB-rbcL spacer) sequences, and (3) reconstruct the biogeographic history of the genus involving tropical to temperate disjunct elements between Asia and North America.

MATERIALS AND METHODS

Sampling, DNA isolation, and sequencing — The study sampled 56 acces-sions representing all the 12 Parthenocissus together with two Yua species (Ap-pendix 1). The sampling covered the biogeographic range from tropical to temperate areas of Asia and North America. Vitis L., Rhoicissus Planch., and Ampelopsi s Michx. are considered to be close relatives of Parthenocissus

Trifoliolae Galet, and ser. Quinquefoliolae Galet) based pri-marily on the number of leafl ets ( Galet, 1967 ). Li (1998) recog-nized three sections (sect. Parthenocissus , sect. Margaritaceae C. L. Li, and sect. Tuberculiformes C. L. Li) with several series based on shape of the young tendril apex, infl orescence mor-phology, and leaf forms ( Table 1 ).

Molecular phylogenetic studies have not been conducted on this genus. Soejima and Wen (2006) included representatives of the genus in their broad analysis of Vitaceae. In the plastid data, three species of Parthenocissus were sampled, and they formed a clade with Yua austro-orientalis in the strict consensus tree with moderate bootstrap support of 76% ( Soejima and Wen, 2006 ). With more sampling of the genus, the monophyly of Parthenocissus is supported with bootstrap value of 74% by the nuclear GAI1 sequences, but phylogenetic relationships within the genus have remained unclear ( Wen et al., 2007 ).

The boreotropical fl ora was hypothesized to span the north-ern hemisphere during the climatically warm periods of the Pa-leocene. It has often been used to explain biogeographic disjunctions in the northern hemisphere ( Wolfe, 1975 ; Dono-ghue and Smith, 2004 ). The fl ora was hypothesized to have contained many thermophilic tropical and subtropical taxa with diffuse origins ( Wolfe, 1975 ; Tiffney, 1985 ). However, our un-derstanding of the phylogenetic and biogeographic relation-ships within these thermophilic groups is still preliminary ( Wen, 2001 ) with most biogeographic studies so far focused on temperate taxa in the northern hemisphere ( Wen, 1999 ; Dono-ghue et al., 2001 ). A few recent analyses of thermophilic sub-tropical/tropical elements such as Gordonia J. Ellis ( Prince and Parks, 2001 ), Illicium L. ( Hao et al., 2000 ; Morris et al., 2007 ),

Fig. 1. Geographic distribution of Parthenocissus showing its disjunction between Asia and North America.

1344 American Journal of Botany [Vol. 97

Ancestral state reconstruction of leafl et number — The leafl et number has been used as an important taxonomic character in classifying Parthenocissus (e.g., Galet, 1967 ; Li, 1998 ). We assessed phylogenetic signal for this leafl et character by optimizing this character under a maximum parsimony criterion onto the 50% majority rule Bayesian consensus tree by using the program Mes-quite version 2.72 ( Maddison and Maddison, 2009 ). The character states of the leafl et numbers were scored as unordered multistate traits (simple, three-leafl et, fi ve-leafl et, and seven-leafl et). To account for topological uncertainty, the op-tion Trace Character Over Trees was used to summarize ancestral state recon-structions of 8000 Bayesian trees sampled at stationarity based on a combined plastid and nuclear data set of 14 taxa representing all species of Parthenocis-sus and its segregate genus Yua .

Bayesian dating — To estimate the divergence time of disjunct lineages in the genus, we used the combined plastid ( trnL-F , rps16 , and atpB-rbcL ) and nuclear GAI1 sequences. Representatives from the entire grape family (Appen-dix S1, see Supplemental Data with online version of this article) were sampled to help date the ages of Parthenocissus with fossil calibrations. The program BEAST version 1.5.4 ( Drummond and Rambaut, 2007 ) was used to estimate divergence times. This software uses a “ relaxed phylogenetic ” model, where topology and branch lengths are estimated simultaneously from the data.

The partitioned input fi le was created with the program BEAUti version 1.5.4 ( Drummond and Rambaut, 2007 ) and edited manually to allow parame-ters to be estimated independently among data partitions. The data set was par-titioned into two sets with unlinked substitution models as in the Bayesian analysis. A likelihood ratio test ( Felsenstein, 1988 ) ruled out a global molecular clock ( P < 0.05) of our data sets, so we implemented the relaxed Bayesian clock with rates for each branch drawn independently from a lognormal distribution ( Drummond et al., 2006 ). A birth-death prior was set for the branch lengths and the mean.Rate parameter had a uniform prior between 0 and 0.1. The coeffi cient of variation had a uniform prior between 0 and 1.0 and the covariance prior was uniform between − 1.0 and 1.0. Runs were initiated on random starting trees. Initially, several short BEAST runs were performed to examine the MCMC performance ( Drummond et al., 2007 ). After optimal operator adjustment as suggested by the output diagnostics, two fi nal BEAST runs (each 20 million iterations) were performed. Convergence was assessed as for MrBayes using Tracer version 1.5 ( Drummond and Rambaut, 2007 ). After discarding the fi rst 2 million samples as burn-in, the trees and parameter estimates from the two runs were combined. The samples from the posterior were summarized on the maximum clade credibility tree, which is the tree that has the maximum sum of posterior probabilities on its internal nodes ( Drummond et al., 2007 ) using the program TreeAnnotator version 1.5.4 ( Drummond and Rambaut, 2007 ) with posterior probability limit set to 0.5 and summarizing mean node heights. These were visualized using the program Figtree version 1.2 ( Drummond et al., 2007 ). Means and 95% higher posterior densities (HPD) of age estimates were ob-tained from the combined outputs using Tracer version 1.5.

Vitaceae fossils and calibrations — Fossils of Vitaceae date back to the Pa-leocene and the early Eocene ( Chen and Manchester, 2007 ). Leaf impressions and pollen grains from the Cretaceous and the Tertiary of Europe, North Amer-ica, and Asia have commonly been assigned to this family, but questions have been raised concerning the validity of many of these assignments ( Kirchheimer, 1939 ). Seeds of Vitaceae are easily recognized based on a suite of unique and distinctive morphological characters (particularly a pair of ventral infolds and a dorsal chalazal scar) and are relatively common in Tertiary fl oras ( Kirchheimer, 1939 ; Tiffney and Barghoorn, 1976 ; Chen and Manchester, 2007 ). The oldest reliable records of vitaceous seeds are from the Paleocene and have been at-tributed to Vitis ( Brown, 1962 ; Mai, 1987 ), Ampelopsis ( Mai, 1987 ), and Ampe-locissus Planch. ( Chen and Manchester, 2007 ). The best preserved of these is A. parvisemina Chen & Manchester from the Beicegal Creek locality in North Dakota ( Chen and Manchester, 2007 ), which is considered to be late Paleo-cene (Tiffanian) based on mammal correlations and fl oristic composition ( Manchester et al., 2004 ). A close relationship among Ampelocissus and Vitis has been well supported by various authors based on morphology ( Brizicky, 1965 ; Chen and Manchester, 2007 ) and molecular analyses ( Soejima and Wen, 2006 ; Wen et al., 2007 ). With the possible nonmonophyly of Ampelocissus (J. Wen, unpublished data), we ran two analyses constraining the stem or the crown Ampelocissus - Vitis clade, respectively, with a normal prior distribution of 58.5 ± 5.0 million years ago (Ma) corresponding to the Tiffanian stage of the Paleo-cene, approximately ranging from 56.8 to 62.0 Ma ( Gradstein et al., 2004 ).

The distinctive seeds of Vitaceae have not been detected in the pre-Tertiary fossil record. However, as sister to the rosids, the family is predicted to have a Cretaceous history based on the presence of Cretaceous rosid fossils and molecular

( Soejima and Wen, 2006 ; Wen et al., 2007 ), and eight representative taxa of these genera (Appendix 1) were selected as outgroups in the phylogenetic analyses.

Total DNAs were extracted from silica-gel-dried leaves by using a modifi ed CTAB method ( Doyle and Doyle, 1987 ) or using the DNeasy Plant Mini Kit (Qiagen, Crawley, UK). Amplifi cation and sequencing followed Soejima and Wen (2006) for the plastid sequences ( trnL-F , rps16 , and atpB-rbcL ) and Wen et al. (2007) for the nuclear GAI1 gene. DNA sequences were assembled using the program Sequencher v4.1.4 (Gene Codes Corp., Ann Arbor, Michigan, USA).

Sequence alignment and phylogenetic analyses — Sequence alignment was initially performed using the program CLUSTAL X version 1.81 ( Thompson et al., 1997 ) in the multiple alignment routine using default settings and the ac-curate search option followed by manual adjustment in the program Se-Al ver-sion 2.0a11 ( Rambaut, 2002 ).

Phylogenetic trees were reconstructed using maximum parsimony (MP) ( Fitch, 1971 ), maximum likelihood (ML), and Bayesian inference (BI). The parsimony analyses were conducted under the option heuristic search with 10 random stepwise additions and tree-bisection-reconnection (TBR) branch swapping with PAUP* version 4.0 b10 ( Swofford, 2003 ). Zero-length branches were collapsed and gaps were treated as missing data. Subsequently, parsimony bootstrap (BP) analyses ( Felsenstein, 1985 ) with 1000 replicates were per-formed under the option fast and stepwise addition to evaluate the robustness of the MP trees.

Before the model-based analytical approaches, the model of DNA evolution that best fi ts the sequence data were explored. A hierarchical likelihood ratio test as implemented in the software MrModeltest ( Nylander, 2004 ) suggests the generalized time reversible model (GTR + I + G) as the best-fi t model of se-quence evolution for the nuclear GAI1 as well as the plastid data sets. In the following ML and BI analysis the substitution models and parameters were adjusted according to the estimates of MrModeltest. Maximum likelihood anal-yses were performed in Garli version 0.96 beta ( Zwickl, 2006 ).

Bayesian inference was used to estimate the posterior probabilities of phy-logenetic trees by employing a 5 million generations Metropolis-coupled Mar-kov chain Monte Carlo (MCMC) with MrBayes version 3.1.2 ( Huelsenbeck and Ronquist, 2001 ). Different sequences were partitioned with unlinked sub-stitution models as estimated before. Sampling rate of the trees was 1000 gen-erations. The Bayesian trees sampled for the last 4 million generations were used to construct a 50%-majority rule consensus cladogram. The proportion of bifurcations found in this consensus tree was given as posterior clade probabili-ties (PP) as an estimator of the robustness of the BI trees.

To evaluate the congruence of the plastid and nuclear data sets, we fi rst employed the incongruence length difference (ILD) test ( Farris et al., 1994 ). The ILD test was conducted with 1000 replicates of heuristic search using TBR branch-swapping with 10 random sequence additions in PAUP*.

Table 1. Synopsis of Li ’ s (1998) classifi cation system of Parthenocissus .

Section Series Species

Parthenocissus Trifoliolae P. cuspidifera (Miq.) Planch. var. pubifolia C. L. Li P. feddei (L é vl.) C. L. Li P. semicordata (Wall.) Planch. P. chinensis C. L. Li

Parthenocissus P. quinquefolia (L.) Planch. P. heptaphylla (Buckl.) Britt. ex Small P. vitacea (Knerr) A. S. Hitchc.

Margaritaceae Tricuspidatae P. tricuspidata (Sieb. & Zucc.) Planch. P. suberosa Hand.-Mazz.

Heterophyllae P. dalzielii Gagnep. Tuberculiformes P. laetevirens Rehd.

P. henryana (Hemsl.) Diels & Gilg

1345August 2010] Nie et al. — Molecular phylogeny and biogeography of PARTHENOCISSUS

Fig. 2. The strict consensus tree of the > 100 000 most parsimonious trees based on combined plastid ( trnL-F , rps16 , and atpB-rbcL ) and nuclear GAI1 sequences (tree length = 533 steps, CI = 0.89, RI = 0.95, and RC = 0.85). The bootstrap values are shown above the branches and the Bayesian posterior probabilities > 95% are below. Samples from the New World are in boldface.

1346 American Journal of Botany [Vol. 97

and the New World and the Old World clades ( Fig. 3 ). Within the New World clade, the seven-leafl et character was inferred to have arisen from the fi ve-leafl et state ( Fig. 3 ). In the Old World clade, the three-leafl et state seems to have evolved from the ancestral fi ve-leafl et condition. The state with both simple and three-leafl et leaves might also have derived from the fi ve-leafl et state ( Fig. 3 ).

Bayesian estimation of divergence times — When the stem or the crown of the Vitis-Ampelocissus clade was constrained at 58.5 ± 5.0 Ma, the divergence time of Parthenocissus between Asia and North America was estimated at 21.64 (95% HPD 10.23 – 34.89) Ma or 27.57 (95% HPD 13.63 – 44.23) Ma, respec-tively. We are also interested in the evolution of tropical and tem-perate elements in Parthenocissus . The divergence time between the tropical Asian P. heterophylla and the southern Himalayan P. semicordata was estimated to be 6.87 (2.11 – 12.34) Ma or 9.00 (2.68 – 16.37) Ma, depending on the two different constraining strategies as described earlier. It has been argued in favor of us-ing a fossil constraint on a stem lineage more conservatively than on a crown group ( Doyle and Donoghue, 1993 ; Magall ó n and Sanderson, 2001 ; Near and Sanderson, 2004 ; Gandolfo et al., 2008 ). Therefore, we emphasized the results based on the cali-bration using the Vitis-Ampelocissus stem line age in the present study, and the corresponding chronogram is presented in Fig. 4 .

DISCUSSION

Monophyly of Parthenocissus and its sister relationship with Yua — With only three species of Parthenocissus sampled, the plastid sequence data supported the Parthenocissus - Yua clade,

divergence time estimates on the rosids ( Wang et al., 2009 ) and the angio-sperms ( Wikstr ö m et al., 2001 ). The divergence time between Vitaceae and Leea was estimated to be 78 – 92 Ma by Wikstr ö m et al. (2001) . We used this estimate as the basis to set the normal prior distribution of 85 ± 4.0 Ma for the stem age of Vitaceae (roughly matching 78 – 92 Ma).

Although fossil seeds of Parthenocissus have been reported in the literature as early as the middle Eocene (ca. 47 Ma) based in part on long, divergent, ventral infolds (e.g., Manchester, 1994 ), Chen (2009) observed morphological differences, particularly the lack of a pronounced apical groove, that shed doubt on the generic assignment. Chen (2009) noted that some other fossil seeds sim-ilar to Parthenocissus are present in the early Eocene, but have been assigned to other genera, e.g., Tetrastigma sheppeyensis Chandler ( Chandler, 1978 ). Our estimates thus did not use these fossils as calibrating points, and we prefer to treat them as an independent data source for comparison with results of our analyses calibrated on other more securely placed fossils.

RESULTS

Molecular analysis — The aligned sequences of the trnL-F region generated a data matrix of 990 base pairs (bp) with 45 parsimony-informative sites. The rps16 data set had 863 aligned bp, 38 of which were parsimony-informative. The aligned posi-tions of the atpB-rbcL data set were 941 bp in length, with 33 parsimony-informative sites. Because there is no recombina-tion in the chloroplast DNA, we combined all the plastid data in our analysis. The aligned matrix of combined cpDNA data had 2794 characters including 116 parsimony informative sites. The aligned data matrix of the GAI1 comprised 1432 bp, 135 of which were parsimony-informative. Results of the ILD test showed no signifi cant incongruence was found between cp-DNA and GAI1 data sets ( P = 0.12). Hence, the two data sets were then combined for the phylogenetic analysis. The matrix of the combined plastid and nuclear data set had 4226 charac-ters including 251 parsimony-informative sites.

Trees generated with different analytical methods (MP, ML, and BI) were consistent with respect to various clades in Parthenocissus . Therefore, only the MP strict consensus clado-gram with bootstrap and Bayesian support is shown in Fig. 2 ( > 100 000 equally parsimonious trees, 533 steps, consistency index CI = 0.89, retention index RI = 0.95, and rescaled consis-tency index RC = 0.85).

The monophyly of Parthenocissus was well supported with BP = 95% and PP = 100%, with two clades recognized corre-sponding to their biogeographic distribution in Asia and North America ( Fig. 2 ). The combined data strongly supported the North American clade (BP = 100% and PP = 100%). Parthenocissus vitacea and P. heptaphylla (Buckl.) Britton ex Small form a clade with moderate support (BP = 77% and PP = 100%). Three groups were recognized within the Old World clade ( Fig. 2 ). One group includes the fi ve-leafl et P. laetevirens and P. henryana (Hemsl.) Graebn. ex Diels & Gilg. The second is composed of species with simple and less common three-leafl et leaves ( P. suberosa , P. tri-cuspidata , and P. dalzielii ) except for P. dalzielii , which has both simple and three-leafl et leaves. The third clade includes all the three-leafl et species: Parthenocissus heterophylla (Bl.) Merr. from tropical Asia, P. semicordata from Nepal and southern Tibet of China, P. feddei from central China, and those broadly defi ned as P. chinensis C. L. Li from western to central China.

Character optimization — The character of leafl et number was traced on the Bayesian consensus tree of 14 species using 8000 Bayesian trees. The consensus tree is the same as the strict consensus tree (results not shown). The fi ve-leafl et state was inferred to be ancestral for the stem lineages of Parthenocissus

Fig. 3. Mapping of leafl et number onto the Bayesian consensus tree generated from the combined plastid and nuclear data set of 14 species from Parthenocissus and Yua .

1347August 2010] Nie et al. — Molecular phylogeny and biogeography of PARTHENOCISSUS

Fig. 4. Chronogram of Parthenocissus and its relatives from Vitaceae based on combined plastid ( trnL-F , rps16 , and atpB-rbcL ) and nuclear GAI1 sequences inferred from BEAST. Samples from the New World are shown in boldface. Gray bars represent the 95% high posterior density credibility in-terval for node ages. Calibration points are indicated with black asterisks.

1348 American Journal of Botany [Vol. 97

evolutionary diversifi cation of Parthenocissus in the New World.

All Asian samples form a clade with BP = 95% and PP = 100% ( Fig. 2 ). This clade covers the morphological diversity of the entire genus, with the number of leafl ets varying from sim-ple to fi ve and the tendril apex showing a high level of varia-tion. Three groups are well recognized within the Asian clade, which are consistent with morphology. The two Asian fi ve-leafl et species ( P. laetevirens and P. henryana ) are well sup-ported to be close to each other ( Fig. 2 ). Species of P. tricuspidata , P. suberosa , and P. dalzielii form a well-supported clade ( Fig. 2 ). Parthenocissus dalzielii is unusual with two types of leaves: simple and small ones on long branches and three-leafl et leaves on short branches. Parthenocissus tricuspidata has mainly sim-ple leaves usually with three lobes, rarely three-leafl et ones on the lower 2 – 4 short shoots. Their close relationship is also supported by their similarity in leaf texture, the more or less glaucous lower leaf surface, and infl orescence morphology. Parthenocissus suberosa is morphologically similar to P. tri-cuspidata in leaf and infl orescence morphology. The two spe-cies differ in that P. suberosa mostly has woody wings (vs. without wings in P. tricuspidata ) on old branches and is densely ferruginously pilose (vs. glabrous in P. tricuspidata ) on branch-lets, petioles, and leaf surfaces.

The three-leafl et clade falls into three subgroups ( Fig. 2 ): one subgroup consisting of P. heterophylla from southeastern Asia and P. semicordata from southern Himalaya (Nepal, northern India, and southern Tibet), the second including only P. feddei from central China, and the third subgroup composed of P. chinensis mainly from China. Parthenocissus chinensis was initially described as an endemic species to dry or semidry valleys of central-west China ( Li, 1990 ). It is similar to P. semicordata in its three-leafl et leaves, yet differs in its leafl et margin with only 3 – 5 obtuse teeth on each side and acuminate leafl et apex. Our analysis indicates that “ P. semicordata ” from southern China to the eastern Himalaya is distinct from the typical P. semicordata from southern Himalaya ( Fig. 2 ). We thus propose a broadly circumscribed P. chinensis with the in-clusion of the previously identifi ed P. semicordata from east-ern Tibet, southwestern Yunnan, and Sichuan to Chongqing of China as in Li (1998) ( Fig. 2 ). As it is delimited now, P. chin-ensis is broadly distributed in southwestern to central China. Morphologically, the leafl ets of P. semicordata are larger (9 – 14 cm long), with more lateral veins (8 – 9) and the sepals rounded at the tip, while the broadly defi ned P. chinensis has smaller (3 – 7 cm long) leafl ets, fewer (4 – 6) lateral veins, and acute sepal tips.

We traced the character evolution of leafl et numbers on the Bayesian consensus tree from the combined plastid and nuclear data of 12 Parthenocissus and two Yua species ( Fig. 3 ). The optimization suggests the fi ve-leafl et state as ancestral in Par-thenocissus . The leafl et number increased once from fi ve to seven in the New World clade, whereas a complicated evolu-tionary pattern was inferred in the Old World clade. Both the simple and the three-leafl et states were inferred to have derived from the ancestral fi ve-leafl et condition independently ( Fig. 3 ). The evolution of simple leaves needs to be further explored in the genus because the simple leaf condition may occur with the three-leafl et condition in P. tricuspidata and P. suberosa . In Parthenocissus dalzielii , three-leafl et leaves are more common with simple leaves occurring only at the tip of branches. Leafl et number in this genus has a complex history and cannot be used as a character for infrageneric classifi cation.

but the relationship between Yua and taxa of Parthenocissus was unresolved( Soejima and Wen, 2006 ). The nuclear GAI1 data supported the monophyly of Parthenocissus with moder-ate support ( Wen et al., 2007 ). In the present study with all species of Parthenocissus sampled ( Fig. 2 ), the combined plas-tid and nuclear data strongly support the monophyly of the genus (BP = 95%, PP = 100%). The synapomorphies of Par-thenocissus include the highly branched tendrils, and usually expanded tendril tips as suckers ( Brizicky, 1965 ; Chen et al., 2007 ; Wen, 2007 ).

Members of the small genus of Yua were previously included in Parthenocissus ( Planchon, 1887 ; Rehder, 1945 ). Both gen-era have members with digitately palmate leaves, inconspicu-ous fl oral disks, and fi ve-merous fl owers ( Wen, 2007 ). However, Li (1990) argued that Yua differs in their tendril and infl ores-cence morphology and should be separated as a distinct genus. Yua possesses bifurcate (vs. 3 – 12 branched in Parthenocissus ) tendrils and leaf-opposed (vs. terminal or pseudoterminal in Parthenocissus ) infl orescence ( Li, 1990 ; Wen, 2007 ; Wen et al., 2007 ). The GAI1 data weakly supported the monophyly of Parthenocissus with bootstrap support (BS) = 74% and poste-rior probability (PP) = 93% ( Wen et al., 2007 ). Our combined plastid and GAI1 data strongly support the monophyly of Par-thenocissus with BP = 95% and PP = 100% and a clear separa-tion of Yua from Parthenocissus ( Fig. 2 ). We prefer to treat Yua as a separate genus distinct from Parthenocissus based on mor-phology and our phylogenetic data, even though Parthenocis-sus and Yua are also strongly supported as a clade (BP = 99% and PP = 100% in Fig. 2 ).

Phylogenetic relationships within Parthenocissus — Two clades are supported within Parthenocissus corresponding to their distribution in the Old and the New World ( Fig. 2 ). This result is consistent with the infrageneric classifi cation of Sues-senguth (1953) based on distribution. However, species rela-tionships within the genus do not support the classifi cations of Galet (1967) and Li (1998) , who emphasized leaf morphology. For instance, Galet (1967) used leafl et number in his classifi ca-tion of the genus, but our results suggest more complex evo-lution of leafl et numbers ( Figs. 2, 3 ). Furthermore, a close relationship between the Asian three-leafl et species (e.g., P. semicordata and P. chinensis ) and the North American fi ve-leafl et species (e.g., P. quinquefolia and P. vitacea ) is not sup-ported by our data, although these species were placed in the same sect. Parthenocissus by Li (1998) based on their young apex of tendrils being expanded and hooked (see Table 1 ).

The three species from the New World form a robust clade with BP = 100% and PP = 100% ( Fig. 2 ). Morphologically, this clade is characterized by their leaves with 5 – 7 leafl ets. Acces-sions of Parthenocissus vitacea and the narrow endemic P. heptaphylla (restricted to Texas, United States) form a clade that is nested within the group of the widely distributed P. quin-quefolia ( Fig. 2 ). Parthenocissus vitacea and P. heptaphylla are morphologically similar to P. quinquefolia with overlapping distribution. The main difference between P. vitacea and P. quinquefolia is that the tendrils of P. vitacea do not have sticky pads ( Brizicky, 1965 ). Thus, Parthenocissus vitacea does not climb smooth walls, although it successfully climbs shrubs and trees. Parthenocissus heptaphylla differs from the other two by its 6 – 7 leafl ets. Together with morphological and biogeographic evidence, it seems reasonable to recognize P. vitacea and P. heptaphylla each as distinct species. Yet, we need further pop-ulational and phylogeographic analyses to better understand the

1349August 2010] Nie et al. — Molecular phylogeny and biogeography of PARTHENOCISSUS

Intercontinental disjunction and relationships of thermo-philic and temperate elements — The split of the Old and the New World Parthenocissus is estimated to be at least 21.64 (95% HPD 10.23 – 34.89) Ma. The full range spans from the late Eocene to the middle Miocene with a mean age close to the boundary between the Oligocene and the Miocene. The disjunction in the early Miocene is largely consistent with the fossil evidence. Vitaceae fossil data suggest that all major groups of the grape family were already diversifi ed in the early Eocene with various seed types such as those similar to Vitis , Ampelopsis , Parthenocissus , and Ampelocissus ( Chen and Manchester, 2007 ; Chen, 2009 ). Many seed fossils previ-ously assigned to Parthenocissus seem to be problematic ( Chen, 2009 ). Nevertheless, some seed fossils similar to Par-thenocissus and to Yua are observed by the middle Eocene of Europe and North America ( Manchester, 1994 ; Chen, 2009 ). Whether these belong to the crown group or to the stem lin-eage has not been determined. Our externally calibrated phy-logeny suggests that they may be members of the stem group.

The crown of extant Parthenocissus is dated to the early Miocene (node 1 in Fig. 4 ). Nevertheless, the stem Partheno-cissus - Yua clade may be as old as the Paleocene when it di-verged from other taxa of Vitaceae ( Fig. 4 ), suggesting that the Parthenocissus - Yua clade might have originated from an ancestral element of the boreotropical fl ora (see Wolfe, 1978 ). In addition, fossil seeds similar to Parthenocissus were found from the early Eocene London Clay fl ora in England and the middle Eocene in Oregon, United States, which may suggest a minimum age of the Parthenocissus - Yua clade at the Eocene. The boreotropical fl ora waned in the late Eocene when the global temperature declined drastically ( Wolfe, 1978 ; Zachos et al., 2001 ). The Miocene was a period with globally warm climates in comparison with those in the preceding Oligocene or the subsequent Pliocene ( Zachos et al., 2001 ). The extant species of Parthenocissus may have diversifi ed in the Mio-cene with the favorable diversifi cation conditions of temper-ate elements in the northern hemisphere ( White et al., 1997 ). Later in the Miocene, there was a distinct climatic cooling period, which may have resulted in the further range reduction of both tropical and coniferous forests, with the development of grasslands and savannas as their replacement. These Mio-cene fl oras had many elements in common with the modern mesophytic fl oras of eastern Asia and eastern North America ( Parks and Wendel, 1990 ), supporting the proposal that the modern north temperate elements diverged during that period.

It is worth noting that geographic areas outside the current range of Parthenocissus species may have been important to the divergences and diversifi cations within this clade. For ex-ample, fossil seeds similar to those of extant Parthenocissus are well represented in the Tertiary of Europe, and it is possible that the extinction of European populations due to climatic cooling of the Neogene and Pleistocene contributed to the divergence of North American and Asian clades. On the basis of seed mor-phological characters, Chen (2009) recognized several fossil occurrences consistent with extant Parthenocissus. The nomen-clature of these fossils remains to be revised, but the pertinent fossils include those called Vitis cuneata Chandler from the early Eocene Dorset Pipe Clay, England ( Chandler, 1962 : plate 14, fi gs. 45, 46), V. elegans Chandler from early Eocene Europe London Clay, England ( Chandler, 1961 : plate 24, fi gs. 35, 36), and specimens described and illustrated as V. teutonica A.

Braun from late Miocene Naumburg, Bober, Germany, by Kirchheimer (1938 , Fig. 4). The seeds called Parthenocissus boveyana Chandler from the Oligocene of Bovey Tracey, Eng-land ( Chandler, 1957 ), are included by Chen (2009) in the in-formal seed morphological category “ st- Ampelopsis -smooth. ” Seeds of this type closely resemble extant Yua ( Y. chinensis and Y. thomsonii , but not Y. austro-orientalis ), but also Ampelopsis . Additional studies of the fossils, including anatomical charac-ters are needed to resolve relationships, but the present infor-mation is consistent with the hypothesis that the evolution and extinction of former thermophilic representatives of Partheno-cissus and Yua populations in Europe contributed to the com-plexity of species now found in Asia.

Parthenocissus heterophylla is the only species extending to tropical Asia. This tropical Asian species is nested within the temperate Asian clade ( Fig. 2 ). Such a pattern of tropical – tem-perate Asian distribution is found in other thermophilic taxa, e.g., Aralia L. sect. Dimorphanthus Miq. ( Wen, 2000 ). The subtropical and tropical members of Aralia are nested within the north temperate group of Dimorphanthus ( Wen, 2000 , 2004 ). Similarly, the tropical Altingia Noronha and Semiliqui-dambar H. T. Chang of Altingiaceae are nested within the tem-perate Liquidambar L. ( Shi et al., 1998 ; Ickert-Bond and Wen, 2006 ). In Toxicodendron ( Nie et al., 2009 ) and Magnoliaceae ( Nie et al., 2008 ), the temperate and the tropical – subtropical elements are, however, clearly separated. The phylogenetic data so far suggest that the origins of thermophilic tropical and subtropical taxa seem to be complex in the context of tropical and temperate elements ( Tiffney, 1985 ; Wen, 1999 ). Even in the case of Parthenocissus , the evolutionary interactions of temperate and tropical elements may not be the most parsimo-nious, because Vitaceae is largely a tropical family. A broader, familywide biogeographic analysis should help resolve these issues. Nevertheless, the divergence between tropical and tem-perate Parthenocissus is relatively recent with the age estimated as 6.87 Ma (95% HPD of 2.11 – 12.34 Ma, node 2 in Fig. 4 ). The biogeographic interactions of the Himalaya and tropical Asia are poorly understood at present. Birds are usually observed as dispersing agents in Parthenocissus ( Harvey, 1915 ; Martin et al., 1961 ). For example, Harvey (1915) reported that seeds of Parthenocissus voided by Passer domesticus sprouted and grew rapidly. Migration via birds may explain the current distribution of the Parthenocissus heterophylla - P. semicordata clade along the corridor of the mid-elevational montane forests from south-ern Himalaya to Southeast Asia (to Java) with some disjunc-tions within Asia, especially in the intervening regions such as Thailand and Vietnam.

Conclusions — Phylogenetic analyses based on the plastid and nuclear GAI1 sequences support the monophyly of Parthe-nocissus and suggest its sister relationship to Yua . Two major clades within Parthenocissus are recognizable corresponding to its distribution in Asia and North America. Within the North American clade, P. vitacea and P. heptaphylla form a subclade with moderate support. Within the Asian clade, three subgroups are supported congruent with their leafl et numbers. The inter-continental disjunction between Asia and North America is among the temperate taxa with the tropical elements nested within the temperate Asian group. The intercontinental disjunc-tion is estimated to be in the early Miocene corresponding to the fossil and paleoclimatic evidence. The tropical P. heterophylla seems to have been recently dispersed from the adjacent tem-perate regions in eastern Asia.

1350 American Journal of Botany [Vol. 97

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1352 American Journal of Botany [Vol. 97

Appendix 1. Voucher information and GenBank accession numbers for species of Parthenocissus , Yua , and outgroup genera. Abbreviations of herbaria are as follows: KUN, Kunming Institute of Botany, Chinese Academy of Sciences; PE, Institute of Botany, Chinese Academy of Sciences; and US, the United States National Herbarium.

Taxon Voucher Locality GAI1 trnL-F rps16 atpB-rbcL

Parthenocissus chinensis C. L. Li

Nie & Meng 455 (KUN) China, Sichuan, Maoxian EF141263 HM223263 HM223320 HM223373

Wen 6538 (US) China, Yunnan HM223424 AB235034 — AB234926 Wen 6530 (US) China, Yunnan EF141266 HM223278 — HM223390 Wen 8208 (US) China, Chongqing HM223427 AB235039 HM223326 — Nie & Meng 460 (KUN) China, Sichuan, Maoxian EF141262 HM223264 HM223321 HM223374 Nie & Meng 470 (KUN) China, Sichuan,

PengzhouEF141269 HM223270 HM223327 HM223381

Nie & Meng 424 (KUN) China, Yunnan, Lijiang — HM223265 HM223322 HM223375 Nie & Meng 434 (KUN) China, Yunnan, Wenshan HM223422 HM223261 HM223318 HM223371 Nie & Meng 373 (KUN) China, Guizhou, Leishan HM223423 HM223266 — HM223376 Wen et al. (Tibet-MacArthur) 226 (US)

China, Xizang (Tibet), Linzhi

— HM223279 HM223336 HM223391

Wen 10635 (US) China, Yunnan HM223440 HM223287 HM223344 HM223399 Wen et al. (Tibet-MacArthur) 1737 (US)

China, Sichuan, Muli — HM223290 HM223347 HM223402

Wen 10645 (US) China, Yunnan HM223445 HM223293 HM223350 HM223405Nie 1567 (KUN) China, Yunnan, Lijiang HM223453 — HM223356 HM223412

Parthenocissus dalzielii Gagnep.

DWRC122 (PE) China, Fujian, Shanming HM223449 HM223300 — —

Chen 2007120501 (PE) Japan, Okinawa, Naha HM223448 HM223299 — — Fan 090820-46 (PE) China, Hunan, Xinning HM223451 HM223302 — — DQ 024 (PE) China, Chongqing,

NanchuanHM223452 HM223303 — HM223411

Parthenocissus feddei (H. L é v.) C. L. Li

ZDG 315 (KUN) China, Hunnan, Yongshun

HM223455 HM223305 — HM223414

ZDG 317 (KUN) China, Hunnan, Sangzhi HM223456 HM223306 HM223358 HM223415 ZDG 319 (KUN) China, Hunnan, Sangzhi HM223457 HM223307 HM223359 HM223416 ZDG 320 (KUN) China, Hunnan, Sangzhi HM223458 HM223308 HM223360 —

Parthenocissus henryana (Hemsl.) Graebn. ex Diels & Gilg

Nie & Meng 359 (KUN) China, Guizhou, Pingtang EF141265 HM223272 HM223329 HM223383

Fan 090825-26 (PE) China, Hunan, Xinning HM223450 HM223301 — — Parthenocissus heptaphylla (Buckl.)

Britton ex Small Wen 9770 (US) USA, Texas, Comal Co. HM223419 HM223256 HM223313 HM223366

Wen 9788 (US) USA, Texas — HM223257 HM223314 HM223367 Parthenocissus heterophyla (Bl.)

Merr. Wen 10696 (US) Indonesia, Java HM223441 HM223288 HM223345 HM223400

Parthenocissus laetevirens Rehder Wen 9376 (US) China, Hunan HM223425 HM223267 HM223323 HM223378 Parthenocissus quinquefolia (L.)

Planch. Wen 10015 (US) USA, South Carolina HM223447 HM223297 HM223354 HM223409

Wen 8662 (US) USA, Virginia EF141268 HM223269 HM223325 HM223380 Wen 8684 (US) Mexico, Oaxaca — HM223275 HM223332 HM223386 Wen 9711 (US) USA, Texas, Coryell Co. HM223420 HM223258 HM223315 HM223368 Wen 9750 (US) USA, Texas — HM223259 HM223316 HM223369 Wen 9777 (US) USA, Texas, Travis Co. HM223421 HM223260 HM223317 HM223370 Wen 10433 (US) USA, Delaware HM223433 HM223280 HM223337 HM223392 Wen 10509 (US) USA, Tennessee HM223434 HM223281 HM223338 HM223393 Wen 10773 (US) Mexico, Yucatan,

cultivatedHM223437 HM223284 HM223341 HM223396

Wen 10423 (US) USA, Delaware, Sussex Co.

HM223438 HM223285 HM223342 HM223397

Wen 10429 (US) USA, Delaware, Sussex Co.

HM223442 HM223289 HM223346 HM223401

Wen 10498 (US) USA, Tennessee, Carter Co.

HM223444 HM223292 HM223349 HM223404

Wen 9802 (US) USA, Virginia — HM223294 HM223351 HM223406 Wen 10500 (US) USA, Tennessee — HM223296 HM223353 HM223408 Wen 9971 (US) USA, Virginia — HM223298 HM223355 HM223410 Wen 11135 (US) USA, South Carolina HM223454 HM223304 HM223357 HM223413

Parthenocissus semicordata (Wall.) Planch.

Nie & Zhu 564 (KUN)

Nepal, Godwara HM223435 HM223282 HM223339 HM223394

Wen et al. (Tibet-MacArthur) 887 (US)

China, Xizang (Tibet), Nielamu

HM223428 HM223271 HM223328 HM223382

Nie & Zhu 610 (KUN) China, Xizang (Tibet), Zhangmu

HM223436 HM223283 HM223340 HM223395

Nie & Zhu 651 (KUN) Nepal, Daman HM223443 HM223291 HM223348 HM223403

1353August 2010] Nie et al. — Molecular phylogeny and biogeography of PARTHENOCISSUS

Appendix 1. Continued.

Taxon Voucher Locality GAI1 trnL-F rps16 atpB-rbcL

Parthenocissus suberosa Hand.-Mazz.

Nie & Meng 358 (KUN) China, Guizhou, Pingtang

HM223429 HM223273 HM223330 HM223384

Parthenocissus tricuspidata (Sieb. & Zucc.) Planch.

Nie & Meng 355 (KUN)

China, Guizhou, Dushan HM223430 HM223274 HM223331 HM223385

Wen 7316 (US) USA, Illinois (cult.) EF141271 AB235042 AB234964 AB234928 Parthenocissus vitacea (Knerr)

Hitchc. Nie & Meng 394 (KUN) China, Yunnan, Liuku HM223426 HM223268 HM223324 HM223379

Wen 7157 (US) USA, Illinois EF141267 AB235036 AB234963 AB234927 Wen 7312 (US) USA, Illinois EF141261 AB235035 — HM223377 Wen 9725 (US) USA, Texas, Taylor Co. — HM223262 HM223319 HM223372 Wen 10488 (US) Canada, Queb é c HM223446 HM223295 HM223352 HM223407

Rhoicissus tomentosa (Lam.) Wild & R. B. Drumm.

Wen 10064 (US) South Africa, Western Cape

HM223417 HM223251 — HM223361

Wen 10076 (US) South Africa, KwaZulu Natal

HM223418 HM223252 HM223309 HM223362

Vitis aestivalis Michx. Wen 10428 (US) USA, Delaware HM223439 HM223286 HM223343 HM223398 Vitis henryana Hemsl. Nie & Meng 405 (KUN) China, Yunnan, Dali HM223431 — HM223334 HM223388 Vitis mengziensis C. L. Li Nie & Meng 415 (KUN) China, Yunnan, Lijiang EF141295 HM223276 HM223333 HM223387 Yua austro-orientalis (F. P. Metcalf)

C. L. Li S. Ickert-Bond 1313 (US) China, Guangdong HM223432 AB235085 AB234977 —

Yua thomsonii (M. A. Lawson) C. L. Li

Nie & Meng 469 (KUN) China, Sichuan, Pengzhou

EF141298 HM223277 HM223335 HM223389

Ampelopsis delavayana Planch. ex Franch.

Nie & Meng 410 (KUN) China, Yunnan, Lijiang EF141206 HM223253 HM223310 HM223363

Ampelopsis glandulosa (Wall.) Momiy. var. kulingensis (Rehder) Momiy.

Nie & Meng 381 (KUN) China, Zhejiang, Mt. Tianmu

EF141211 HM223254 HM223311 HM223364

Ampelopsis grossedentata (Hand.-Mazz.) W. T. Wang

Nie & Meng 361 (KUN) China, Guizhou, Leishan EF141212 HM223255 HM223312 HM223365

Note: A dash indicates the region was not sequenced for the sample.