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A phylogenetic analysis of the grape genus (Vitis L.) reveals broad reticulation and concurrent diversification during neogene and quaternary climate change
Yizhen Wan1†, Heidi R Schwaninger2*†, Angela M Baldo2,3, Joanne A Labate2, Gan-Yuan Zhong2,3 and Charles J Simon2
Background: Grapes are one of the most economically important fruit crops. There are about 60 species in the genus Vitis. The phylogenetic relationships among these species are of keen interest for the conservation and use of this germplasm. We selected 309 accessions from 48 Vitis species,varieties, and outgroups, examined ~11 kb (~3.4 Mb total) of aligned nuclear DNA sequences from 27 unlinked genes in a phylogenetic context, and estimated divergence times based on fossil calibrations. Results: Vitis formed a strongly supported clade. There was substantial support for species and less for the higher-level groupings (series). As estimated from extant taxa, the crown age of Vitis was 28 Ma and the divergence of subgenera ( Vitis and Muscadinia) occurred at ~18 Ma. Higher clades in subgenus Vitis diverged 16 – 5 Ma with overlapping confidence intervals, and ongoing divergence formed extant species at 12 – 1.3 Ma. Several species had species-specific SNPs. NeighborNet analysis showed extensive reticulation at the core of subgenus Vitis representing the deeper nodes, with extensive reticulation radiating outward. Fitch Parsimony identified North America as the origin of the most recent common ancestor of extant Vitis species. Conclusions: Phylogenetic patterns suggested origination of the genus in North America, fragmentation of an ancestral range during the Miocene, formation of extant species in the late Miocene-Pleistocene, and differentiation of species in the context of Pliocene-Quaternary tectonic and climatic change. Nuclear SNPs effectively resolved relationships at and below the species level in grapes and rectified several misclassifications of accessions in the repositories. Our results challenge current higher-level classifications, reveal the abundance of genetic diversity in the genus that is potentially available for crop improvement, and provide a valuable resource for species delineation, germplasm conservation and use. Keywords: Biogeography, Divergence time estimate, Grapevine, Molecular phylogeny, Network, Northern hemisphere, Plant disjunction, Reticulation, SNP, Vitis
* Correspondence: Heidi.Schwaninger@ars.usda.gov † Equal contributors 2 US Department of Agriculture, Agriculture Research Service, Plant Genetic Resources Unit, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, USA Full list of author information is available at the end of the article
© 2013 Wan et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Wan et al. BMC Evolutionary Biology 2013, 13:141 http://www.biomedcentral.com/1471-2148/13/141
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Background Grapes (Vitis spp.) are one of the world’s most economically valuable fruit crops . They are widely used for wine, table grapes, raisins, juice, and spirits; recent trends have also focused on antioxidants and healthful products derived from grapes. Vitis vinifera L. subsp. vinifera (referred to as V. vinifera hereafter) is the most widely cultivated grape species but its productivity was historically limited due to its susceptibility to pests, diseases, and abiotic stress such as cold . Genes from wild grape germplasm have been used to improve biotic and abiotic tolerance and resistance in cultivated grapes. Centers of grapevine diversity are found in the southeastern US [3-5] and East Asia [4,6]. Up to 30 species are native to a vast area in eastern Asia, China, Japan and Java, two species across middle Asia and Europe, and up to 28 species across the eastern and southwestern US and Mexico  (Figure 1). Appendix 1 expands discussion of the biogeographic background. The genus Vitis is divided into two subgenera: Muscadinia Planch. (2n = 40, one or two species) and Vitis Planch. (2n = 38, the remaining species). Additional divisions within Vitis are “series” which are subgeneric groupings that have been used historically in the systematics of Vitis.
They rank below “sections” that are more commonly used in plant systematics for groups of species. Although all wild species are considered diploid, there is evidence of hexaploidization in their distant past [7-9] that is shared with all rosids . The two subgenera are nearly reproductively isolated while the species within subgenus Vitis are interfertile. All species are dioecious except V. vinifera which has hermaphroditic flowers, and V. rotundifolia Michx. which segregates for this trait. Many species have overlapping distributions, thus natural hybridization would occur were it not for ecological and phenological barriers [3,10,11]. Not surprisingly, the classification of Vitis is confused in part due to the lack of agreement among systematic botanists as to what constitutes a true species and because of extreme morphological variation within the species [2,3,7]. This has led to many extraneous species names [7,12]. The systematics of Vitis is based primarily on morphology  and molecular methods have only recently been used to study this taxonomic problem. Most previous molecular studies on the evolutionary history of Vitis were limited in taxonomic scope or marker choice [15-32]. Studies most similar in goals and pertinent to the present study were conducted by
American-Pacific Refuge V. californica V. girdiana
V. acerifolia V. aestivalis V. arizonica V. bloodworthiana V. cinerea V. labrusca V. monticola V. mustangensis V. palmata V. riparia V. rotundifolia V. rupestris V. shuttleworthii V. treleasii V. vulpina V. x champinii V. x doaniana
V. Vinifera subsp.sylvestris V. Vinifera subsp.vinifera
V. adenoclata V. adstricta V. amurensis V. bashanica V. bellula V. betulifolia V. coignetiae V. davidii V. ficifolia V. flexuosa V. hancockii V. liubanensis V. piasezkii V. pseudoreticulata V. qinlingensis V. quinquangularis V. romanetti V. shenxiensis V. wilsoniae V. yenshanensis
Southeastern Refuge V.V. tilifolia tiliifolia
East AsianPacific Refuge
Mediterranean Refuge V. blancoii V. popenoii V. nesbittiana V. biformis Armenian-Persian Refuge
Figure 1 Native geographic distribution of the genus Vitis (grey shading1) and geographic regions of origin of Vitis species used in this study. Dashed lines indicate southern borders of the polar ice cap during the most recent ice age2. Dash-dot lines indicate ice age refugia of the forest flora2. Areas labeled 1 through 4 were used in ancestral area optimization (reversible parsimony, Additional file 14). Redrawn from 1 Alleweldt et al. , 2Reinig .
3) elucidate the biogeographic history of the genus. The NeighborNet  of the concatenated 273-OTU matrix (Figure 2) showed extensive conflict at the core of subg. Phylogenetic analyses in Vitis Maximum likelihood ML analyses of 26 single genes analyzed independently without missing data yielded 26 very poorly resolved trees. 2) date important time points in the evolution of Vitis. Lanier and Knowles  found that.0 Ma. Aradhya et al. BMC Evolutionary Biology 2013. Under the uncorrelated log-normal relaxed molecular clock. Nie et al.14).3 Ma. 13:141 http://www. The consensus network  of the 26 individual gene trees indicated that few splits were common to multiple trees (Additional file 3).2 × 10-8 substitutions per site per million years. in species-tree estimation.  obtained a taxon sample similar to the present study and used SSR and AFLP markers to study genetic diversity within Vitis. and intraspecific variation. we report the number of unique site patterns from Bayesian Evolutionary Analysis by Sampling Trees (BEAST) and Randomized Accelerated Maximum Likelihood (RAxML). representing independent assortment of markers. the species. Thus.7 ± 1.10. which did not fully resolve the tip clades. the crown age of subg. 1973 (P = 0. and 4) evaluate systematics of Vitis within the framework of phylogeny. The starting alignment matrix for the 27 gene markers and all 309 accessions was 11. Indel sequences in the 27 gene markers were unambiguous and easy to align.896 (ESS = 1284).23). and Zecca et al.249×10-4 per million years (Effective Sample Size (ESS) = 959) and a coefficient of variation (CV) of 0. Their distributions among the gene markers are listed in Additional file 2. Vitis at 17.’s  chronogram is a tantalizing expansion of the Vitis component of Nie et al. thus the sequences were concatenated.019). No significant conflict was observed under ML.5 Ma.238 Ma.510 unique site patterns. and the stem ages of most species fell between 11 and 1. Gap coding for Maximum Parsimony added 304 characters. estimated from three combined runs in BEAST and calibrated with three fossil dates. limited intra-specific replication (sampling) limited the ability to make species-level inferences. 7022 (P = 0. and introns for 1.006).75 ± 0. 3’ or 5’ untranslated regions for 4. Estimation of divergence times in Vitis Results Molecular characteristics of the nuclear sequences of Vitis Most Vitis accessions had complete sequence or had minimal missing data (Additional file 1). Their inferences were limited by the small number of markers and the limited variability available in those markers.biomedcentral. Because Trees by New Technology (TNT) does not output the number of parsimony-informative characters.5 ± 5 Ma. The 52-OTU matrix had 1.033). . . Amino acid coding sequence accounted for 5. Extent of reticulation and network in Vitis Ancestral polymorphism with subsequent lineage sorting is difficult to distinguish from reticulation based on a phylogenetic pattern [38-42]. .21 million steps.’s  chronogram. Zecca et al. Liu et al. 2415 (P = 0. and four out-groups to: 1) reconstruct a phylogenetic hypothesis of the genus Vitis. and extensive conflict radiating outward.690 nt.32 Ma (95% Highest Posterior Density (HPD) 41.047). The present study attempted to improve on three aspects of previous phylogeographical studies of Vitis by more extensive sampling of the nuclear genome. and estimated the crown age of subg. These calibration points were applied in the present study.036 nt (Additional file 2). the concatenated matrix still showed recombination. labrusca and its closely related North American relatives at 5. the crown age of Vitis was estimated at 28.82 Ma (95% HPD 26. the 273-OTU matrix had 2. Vitis representing the deeper nodes.285 Ma and 18. The ten individual runs continued for 61.31 million to 89. and the divergence of V.000). Vitis between 17.  provided well-reasoned paleontological dates to estimate divergences in Vitaceae. varieties. 5069 (P = 0. six (22%) gene fragments showed significant tests for recombination based on the Phi Test : fragments 1313 (P = 0. Further.437 bp long.Wan et al. the mean rate of substitution (meanRate) in the data set was 8. in the present work no genes were excluded from subsequent analyses based on evidence of recombination. . the size of the data set (11.  and Liu et al. Dividing this rate by With the stem age of Vitaceae constrained at 90. These markers have limited value for phylogeny reconstruction  and dating divergences was not attempted. The maximum clade credibility tree with mean estimates of divergence time for all nodes (Additional file 4) and associated posterior probabilities (Additional file 5) were also obtained.71. The Bayesian divergence times in Figure 3 were estimated from three combined.com/1471-2148/13/141 Page 3 of 20 Aradhya et al. Adding more data can be useful for resolving difficult phylogenies that were based on a few genes . the stem age of Vitis at 58.033). 7029 (P = 0.074 nt. Nie et al. In our study. as expected (P = 0.437 nt).25. This study developed and used 27 nuclear gene markers and sequenced 309 accessions of 48 Vitis species.033). When these fragments were excluded. unpartitioned runs using BEAST.855 unique site patterns. the average rate of substitution in this data set was 7. 16. We used networks to better visualize the conflicts represented by the high levels of homoplasy. . the gain of accuracy from sampling additional loci and/or individuals always exceeded inaccuracies related to recombination.
mustangensis V. and RI = 0. monticola V. 6: Occidentales (Munson). helleri V. vinifera V. ficifolia V. vulpina Euvitis Figure 2 The NeighborNet of 273 accessions based on 27 concatenated nuclear gene fragments. 13:141 http://www.Wan et al. The strict consensus tree was simplified by collapsing terminal clades (Figure 6) for comparisons.346. popenoii V.com/1471-2148/13/141 Page 4 of 20 Europe V. Aestivalis var. i. See also Additional file 15.360. jacquemontii V. bashanica V. girdiana V. aestivalis var aestivalis V. a simplified version with collapsed terminal clades (Additional file 6). 3: Cordifoliae (Munson). arizonica V. 8: Flexuosae (Galet). quinquangularis V. acerifolia V. sylvestris 7 7 8+9 8 8 V. Maximum parsimony The two runs timed out at 48 mil and 50 mil generations with good effective sample size (ESS.. BMC Evolutionary Biology 2013. pseudoreticulata V. shuttleworthii 4 5 3 5 V. munsoniana V. The final search on the same matrix following the bootstrap search yielded the highest likelihood value: -42358. V. rupestris V. consistency index (CI) of 0. 5: Ripariae (Munson). see figure legend). cyanocarpa romaneti betulifolia V. davidii var.790. and retention index (RI) of 0. Overall comparisons of relationships above the species level were facilitated using the cartoon of this tree (Additional file 10). blancoii V.biomedcentral. palmata V. Numbers indicate the series to which species have been recognized 1: Aestivales (Planchon).776. Bayesian mil run (Additional file 8) had a higher mean log likelihood of the cold chain (LnL) after burnin. amurensis piasezki V. The individual MP trees had a score of 4398. labrusca V. californica 3 North America V. This tree was used in comparisons with results from other methods using a cartoon. liubanensis V. Aestivalis var. yenjshanensis Asia Muscadinia V. The strict consensus of all 282 MP trees from all four searches had a score of 4580. V. CI = 0. bicolor V. 7: Viniferae (Planchon). but they did not converge on the exact same phylogenetic hypothesis. Node numbers are . V. rontundifolia V. 2: Cinerascentes (Planchon). Bootstrap values are reported for selected nodes (Figures 4 and 5) and all nodes (Additional file 7). lincecumii 1 4 V. 4: Labruscae (Planchon). biformis 3 6 3 6 V. Both trees were highly concordant for the species-level clades but differed on the specific placement of some clades (Additional files 8 and 9). rotundifolia var.e. acerifolia V. riparia some V. hancockii 9 V. davidii V. nesbittiana V. The 20 identical (except for the starting seed) partitioned rapid ML runs using the 273-OTU matrix produced a range of maximum likelihood values of -42511to -42380. The full tree was annotated with support values on selected nodes of interest (Figures 4 and 5. cinerea var. adstricta V. coignetiae V. cinerea V. the posterior probabilities from this run were used to summarize supports (Figures 4 and 5). Because the 48 TNT’s driven search function produced most parsimonious (MP) trees of the same length in all four searches. qinlingensis V. > > 200). Bloodworthiana V. 9: Spinosae (Galet).
californica Benth.32 Ma [41. cross referenced with Additional file 11.0 70.12 Ma [16. The series Precoces Munson (containing V. 16. but a few clade or species positions were inconsistent. acerifolia Raf. monticola Buckley or V.0 Paleocene 60.71. yenshanensis J.82 Ma [26. rupestris Scheele) together with V.36 Ma [9. Chen was . V.) Engelm. This was a notable characteristic of this data set. ex Millardet. V. For example.14] Vitis diversification 28. 13:141 http://www. All node numbers are shown in Additional file 12. shuttleworthii House and V.0 Miocene 10. aestivalis Michx.25. were grouped together as were V. V. (belonging to the series Occidentales Munson. V.0 80. ML and MP trees shown.58. V.8. Additional files 4 and 5 show nodal ages and posterior probabilities for all nodes in this tree.36] Divergence of Eurasia from North America 11. The position of several clades differed among the searches.23] Cretaceous 90. X. Significant evolutionary events are indicated with black diamonds. labrusca and V.0 Oligocene 30. H. palmata Vahl. Among the different BA. It was evident that the patterns were similar among the cartoons of the strict MP consensus (Figure 6). with other species.com/1471-2148/13/141 Page 5 of 20 * Divergence of Europe from Asia 6. Correspondingly. 3. cinerea (Engelm.. girdiana Munson formed the sister clade. Xtreleasii Munson ex L. bloodworthiana Comeaux . V. Ancestral area analysis Fitch parsimony identified Eastern/Southeastern North America (that also included Mexico and the Caribbean) as the origin of the most recent common ancestor of Vitis based on the strict consensus tree (Additional file 14). V. Asterisk indicates inclusion of a clonally propagated cultivar that may affect the local divergence estimate.biomedcentral. blancoii Munson. 10.0 Plio 0. 6. Calibration points are indicated with filled circles.0 20. V. BMC Evolutionary Biology 2013. arizonica Engelm. V. Additional file 15). Bailey and V. mustangensis Buckley. the highest ML value cladogram (Additional file 6) and the Bayesian cladogram (Additional file 10).Wan et al.0 Eocene 40. The MP bootstrap tree with supports >50% was illustrated (Additional file 13). Grey bars represent the 95% Highest Posterior Density (HPD) intervals of nodal age in million years. clades above the species level were often poorly supported. Systematics of Vitis spp. riparia Michaux. The clades of Eurasian species were nested in North America as a monophyletic clade.59] Diversification of 38-chromosome ingroup 17.0 50. the OTU composition of species clades in general was quite consistent and well supported.0 Ma Figure 3 Chronogram of Bayesian divergence time estimates of Vitis diversification based on 27 concatenated nuclear gene fragments inferred using the BEAST software. All three analyses did not group V.
Wan et al.82 Ma F G T H I J K W B ~11 Ma Figure 4 (See legend on next page. BMC Evolutionary Biology 2013.com/1471-2148/13/141 Page 6 of 20 U D Y E X A ~17. 13:141 http://www.biomedcentral.) .
we resequenced whole fragments. V. V. V. nesbittiana Comeaux were grouped with V. Many higher level relationships were supported by a few characters and were of poor quality leading to the labile topology among major clades. The markers characterized intraspecific variation. labrusca (H). girdiana (K). amurensis (V). monticola. It was supported by 93 characters of which 57 (61%) showed no homoplasy and represented 20 of the 27 (74%) gene markers.C. as well as different histories of genes caused by lineage sorting and reticulation [40.Wan et al. and different clade sizes . and China without V. the nuclear DNA dataset in this study had extensive variation to address genus-wide relationships in Vitis. quinquangularis Rehd. shuttleworthii in BA and ML but grouped with V. The MP branch lengths (Additional file 16). Below branches: (node number: branch length/No unique characters/No genes in support of node). Summary statistics for specific nodes investigated are shown below the branches in Figures 4 and 5. palmata clade (X in Figure 4) and the (V. vulpina L. Gmel. shuttleworthii. bootstrap supports. The species V. 13. To better understand clade support. Caill. sylvestris. palmata (E). several . These nodes had five to seven supporting characters that were frequently highly homoplasious (Figure 4. V. V. and posterior probabilities for all available branches are shown in Additional files 7. V. and 6 genes contributed changes. mustangensis. reversals and convergences. V. (O). Above branches: (Maximum Likelihood BS/ Bayesian PP/ Maximum Parsimony BS). However. V. and V. V. vinifera or phylogenetic depth. V. Figure 4 continues in Figure 5. V.8. shuttleworthii (D). V. “na” indicates absence of a value in the specific support category. BMC Evolutionary Biology 2013. The annotation “319: 8/2/6” means that node number 319 has 8 character changes of which 2 are unique to that node. davidii (Rom. C. mustangensis and V. cinerea-V. Nodes labeled A-Y are discussed in the text. Values can range from 1-100 in ML. cyanocarpa (Rom. V. Vitis) were characterized by low bootstrap values and were often supported by few characters with high homoplasy. V. davidii. the deepest node was very well supported both in number and quality of characters. cinerea. MP and ML bootstrap supports and BA posterior probabilities (Additional files 7. Examples were the nodes defining the split between North America and Asia (B). thus included additional markers not restricted by our selection criteria of intermediate variability (see Methods). and V. sylvestris (C. Strict consensus of 282 most parsimonious trees from four driven searches with support values for selected nodes. and differences may be distorted due to domestication. aestivalis. vinifera ssp. The notable exception was the branch separating the Muscadinia from subg. Other supported higher clades were the V. Vitis (Figure 4 node A). Similarly. californica (I). V.) Foëx (R. many of the relationships and deeper nodes (above species within subg. 50-100 in MP. Europe (with V.) Foëx(N). cinerea with V. hancockii Hance (Q). davidii var. V. V.46]. 16. bashanica P. V. ML. V. amurensis Rupr. rather than genotyping a priori identified SNPs.com/1471-2148/13/141 Page 7 of 20 (See figure on previous page. V. palmata. parallel changes. (referred to as V.cinerea (all varieties and including V. blancoii in MP and in the network. californica. North America. Discussion Problems of phylogenetic study in Vitis Based on MP. in MP and BA analysis and in the network but not in ML. 13:141 http://www. V. but placed within the Asian clade in ML and in the network. V. (T) and V. V. V.13) supported intraspecific groupings well with nonzero branch lengths present in many species such as V.biomedcentral. amurensis (L). Low clade support and high homoplasy may be caused by insufficient data.) Figure 4 Hypothesis of phylogenetic relationships among Vitis species. palmata. jacquemontii R. sylvestris hereafter) (S). presence of node-specific characters and support from multiple genes (letters in parentheses refer to marked nodes in Figures 4 and 5): V. we further investigated the synapomorphies defining clades of interest. the amurensis/coignetiae clade was basal to Eurasia in MP and BA. davidii).) Hegi. V. coignetiae Pulliat ex Planch. The characters supporting nodes of interest and their level of homoplasy and gene source were summarized based on MP (Additional file 11). Parker) and China (C). V. Vitis. and strongly supported subg. V. monticola (U) each formed reliable species-level clades even in the absence of a species-specific SNP. Similarly. 8. grouped with V. Many species showed good support with high bootstrap values and posterior probabilities. Due to space constraints these supports were not summarized in (Figures 4 and 5). Branch lengths. defined most species. Further. biformis Rose) (F). The markers were informative in characterizing intraspecific variation in some species. and not grouping with V. adstricta. qinlingensis (M). The ascertainment bias (the systematic distortion in measuring the true frequency of SNPs due to sampling) introduced in the marker development phase may have selected markers with insufficient variation outside V. bloodworthiana and V. However. although bootstrap support and posterior probabilities were more consistent with other poorly supported higher-level clades. Caill. biformis (G). 0-1 in BA. and BA phylogenetic reconstruction methods. nesbittiana (J). biformis) clade (Y). shuttleworthii. Additional file 11). Both nodes had a node-specific SNP. Vitis. He (P). labrusca. illustrating that there was sufficient phylogenetic signal available to define subg.
Additional data. BMC Evolutionary Biology 2013.com/1471-2148/13/141 Page 8 of 20 L M N O P V Q R C ~6.  and Vezzulli et al. shuttleworthii. V. Thus.g.36 Ma S Figure 5 Hypothesis of phylogenetic relationships among Vitis species. quinquangularis) were well supported by multiple non-homoplasious characters.Wan et al. labrusca. V. V. Eurasia. 13:141 http://www. hancockii. simple lack of data was not the definitive reason for poor support of deep nodes. Continuation of Figure 4. species (e. using the Vitis9KSNP array  or others in development. V. V.  or next generation sequencing (NGS) may resolve this phylogenetic problem.biomedcentral. these data will certainly add more noise (homoplasious characters). In . palmata. However. markers developed by Lijavetzki et al.
. Red = European accessions. Blue = North and Central American accessions.biomedcentral. 13:141 http://www. Green = Asian accessions. BMC Evolutionary Biology 2013. For comparison. Additional files 6 and 10 represent cartoons of the ML and BA trees. respectively.Wan et al.com/1471-2148/13/141 Page 9 of 20 Figure 6 Simplified version (cartoon) of the MP strict consensus tree.
The present estimate of 28. compared to the ~11.8 Ma –3. It is possible that additional data from non-recombining chloroplast or mitochondrial DNA might add stable characters deeper within the tree. In Zecca et al. recombination and horizontal gene transfer .07 Ma .82 Ma (26. V. Additional file 4) and .9. The inclusion of additional more closely related outgroups could improve the accuracy of the inferred dates .  who assessed it at about 12 Ma (~22 Ma – 6 Ma). the conflict in the NeighborNet (Figure 2) can be interpreted as evidence of shared ancestral polymorphisms mixed with reticulation and lineage sorting. ascertainment of homology in the data set created by NGS is very intractable because of complex paleopolyploidization and gene duplication in the grapevine genome . reversals. The present estimates of divergence times showed that splitting events between the deeper clades occurred almost simultaneously within subg. these factors were further minimized by locus selection criteria.31 or 6. We conclude that extensive reticulation deep in the tree and incomplete lineage sorting are the likely reason for the lack of support at higher level nodes.5 Ma) by Liu et al.55. There was significant recombination in several genes and in the concatenated dataset.34 Ma .28 Ma.98 Ma to <0. a separation that was estimated by Zecca et al. Xiang et al. Finally.8 Ma) [53. the linkage disequilibrium in V. 13:141 http://www.Wan et al. The stem of Vitis did reach back that far (Figure 3).  at 4.32 Ma appears a reasonable age considering that the estimate was associated with a large CI that reached back to 41 Ma and the oldest reliable Vitis seed date to the Paleocene (65. representing 11 diverse genera of flowering plants at an average of 4.96 Ma. Recombination in the present data possibly caused the estimate of the height of the tree to be greater. reticulation and incomplete lineage sorting Homoplasy due to incomplete sorting of ancestral alleles is more likely when the time between lineage splitting is short (short branch especially when deep in the tree ) and the effective population size is large .2. Our molecular dating is close to fossil estimates and distributional inferences that place the origination of Vitis into the Paleogene. thus resulting in more ancient age estimates .  with an estimated mean age of 18.North American disjunction.5 Ma.05 Ma (29. The first two were likely major factors in the evolution of Vitis . indicating significant historical recombination within the species . piasezkii. with the radiating splits representing progressive lineage sorting and reticulation within the lineages.5 Ma -3. we estimated 9.com/1471-2148/13/141 Page 10 of 20 addition. and convergence are likely minor contributing factors to the observed homoplasy due to the shallow phylogenetic depth of this study involving moderate levels of evolutionary time.25 Ma . Using sequence data of a single chloroplast gene (rbcL) and no fossil calibrations. and 54 parsimony informative sites respectively. The diversification of subg.5 Ma (about 10 Ma . and 6.31 Ma or 4. or Zecca et al.58 Ma –6.16.25. This was earlier than estimated by Nie et al.68 Ma (V. Estimates by all three [34-36] were predominantly based on (non-recombining) chloroplast sequence with 116. Parallel changes.  this split coincided in timing with our Node A.36. The shared ancestral polymorphisms may be the cause of the central knot of conflict (an ancestral ocean of polymorphisms and reticulation) represented by the tight central mass of splits that represent incompatible and ambiguous signals in the data set . The use of the distant outgroup Leea may have caused problems in dating because of missing data and multiple substitutions.32 Ma (95% HPD 41.2 Ma).78 Ma to 5. Vitis (Node A) was presently estimated at 17.60 Ma (28.60 Ma -2. two studies [34.36] had one nuclear marker with 135 and 41 parsimony informative sites respectively. node B in Figure 4) was 11.48] suggests that nucleotide substitution rates in these datasets may be too slow to add much intra-generic information.54]. or Liu et al.31 Ma.55 Ma (9. Time frame of Vitis diversification Ancestral polymorphism.59 Ma).4. Reticulating events include hybridization. Further. depending on the details of their analyses.14 Ma). 9.440 nt of the present study. nesbittiana) and 1.75 Ma -8. while horizonatal gene transfer was an unlikely mechanism.3 Ma (V.5 Ma .36 Ma (9. The beginning of diversification among the extant taxa (crown age) in our tree was estimated at 28. .50 Ma) or 19. Higher level intra-North American divergences fell between 15. We estimated species-level divergences between 11.47 Ma (6. suggesting that grapevine species maintained large effective population sizes since their geographic isolation millions of years ago. 6. Thus.71 Ma 10. betulifolia.biomedcentral. vinifera is very low and haplotype blocks are very short [20-22.  found significant degrees of shared polymorphisms between North American wild grapevine species and European cultivated species.98 Ma and ranging from 5. .79 Ma .51].5 Ma) by Nie et al.10. and each combined data set was <6000 nt.  who assessed it at about 8 Ma with a large 95% HPD.  and about 7 Ma (11 Ma .23.28 Ma for higher level divergences among the extant taxa in Asia. the literature [19. Species reticulation and incomplete lineage sorting would still present a challenge. BMC Evolutionary Biology 2013.29.71 Ma – 2.12 Ma (16.50 Ma) as node B in Zecca et al.36 Ma) for the separation of Europe and the Near East from Asia (Node C in Figure 5). Myles et al.61 Ma). Vitis.  estimated the divergence time of 11 East Asia-North American disjunct species. a node that was dated at 6.23 Ma). 1258. However. The divergence of Eurasia from North and Central America (the Asian. about 5.
36 4. 20124.60. labrusca described by Gong et al.50.36 9.31.34-4.  that the previous Vitaceae stem age estimate  was inaccurate.440 nt.96 multiple _ _ _ _ _ _ _ _ 9.0 _ 21.12 16. "Higher level intra Asia.75 ± 0. 8dash: information is not applicable.50 ~7 ~11-5 17.5 ± 5.71-10.5 6. . 12prior based on estimates by Nie et al.60-2. Fossils of Vitaceae have been found frequently in Western North American Eocene deposits (55. 20136  Present study7 ~8 ~16-4 ~12 ~22-6 28.07-10. 5estimates from analyses that either constrained the stem (first number) or the crown of subgenus Vitis. dispersal and diversification of Vitis The phylogenetic relationships and network of grapevines reflect the Northern hemisphere Cenozoic history.32 41.36]. 14based on fossil relatives of V.8 to 33. However. 13:141 http://www. and the 95% HPD included multiple HDP associated with the several clades.34-4.0 _ _ 11 Xiang et al. .59 _ _ _ _ _ _ _ _ 15. and differentiation.28 multiple _ _ _ _ _ _ _ _ 90.54].98 (5. .71-2. located in the Paleocene to which the oldest reliable Vitaceae fossils date.79-9.75 ± 0. Vitis crown Estimate (Ma) 95% HPD (Ma) Europe/Near East .711. including Ampelopsis.5 ± 5.3 multiple 85 ± 4.50 _ _ 11.09 58. 13minimum age constraint based on fossil seeds described by Gong et al. The extensive ancestral reticulations revealed by the network and analysis of individual genes suggested well connected ancestral populations and species throughout the distribution followed by increasing range-wide fragmentation.50.< 0. rate smoothing.31.biomedcentral.28) _ _ _ 6.412 4.80-3.055 28.47 6. 9.50. see Figure 3 and Additional file 4 for details.0 _ 5.5  18. 9Wikström et al.  used a 3-gene data set.92 Ma from which the prior distribution was derived.23 _ _ ~5. Continental origin.50-3.50-3.31.Wan et al. BMC Evolutionary Biology 2013.29.Asia Estimate (Ma) 95% HPD (Ma) Eurasia-NC America Estimate (Ma) 95% HPD (Ma) Higher level intra North America Estimate (Ma) 95% HPD (Ma) Higher level intra Asia Estimate (Ma) 95% HPD (Ma) Species stem Estimate (Ma) 95% HPD (Ma) Fossil calibrations (Ma).33.0 58.  agree with arguments made by Nie et al. the timeframe estimated by the present study was more ancient than the estimates by other Vitis-specific molecular studies (Table 1).  concluded that their analysis may support an Asian origin of Vitis. .Leea Vitis -Parthenocissus Old World . 2one chloroplast gene. 6.0 _ 5.514 13 11. 65471 nt cpDNA. 29. 43583 nt (2701 cpDNA + 882 nDNA).9 Ma) and have not yet been found in southeastern localities .25-16. 9. 4. 19.7 ± 1. 20103  Zecca et al.7 ± 1.5 1 For nodes "Higher level intra North America".78-5. 6.68-1. 11 diverse Angiosperms.5 ± 5. 27 nuclear gene fragments. 34226 nt (2794 cpDNA + 1432 nDNA).010 58.New world Parthenocissus Within Vitis _ _ _ _ 85 ± 4. The ancestral area analysis and the recurring distributional trend of American paraphyly with Eurasia in this study suggested a progression from North America to Asia to Europe consistent with previous studies [17. and a single calibration point to estimate the stem age of Vitaceae at 78 . "Species stem" the range contains several clades with different estimates.53] and in central Europe [34.58-6. Priors used Vitaceae . 20002  _8 _ Nie et al. see Figure 3.55 9.82 26. Ampelocissus and Vitis .31. Péros et al.14 _ _ _ _ 4. 6. 11stem age of the Vitis-Ampelocissus clade.5 ~10-2. Fossils of Vitis seed were found in deposits of the Rocky Mountains and Great Plains of North America [34.55 9. two or three fossil calibration points Node1 Vitis crown Estimate (Ma) 95% HPD (Ma) subg.75-8.5 90. These findings assigned the oldest age of Table 1 Comparison of divergence estimates in Vitis among five studies that analyzed SNP data and used zero.98. Overall.6 ± 6. 10 estimate from Magallón and Castillo  because Liu et al.2 Liu et al. isolation.61 _ _ 6.com/1471-2148/13/141 Page 11 of 20 intraspecific divergences shown more fully in Figures 4 and 5 extended into more recent times.
The area of the Bering and Chukchi seas lay above sea level for most of the last 50 to 60 M years  and was suitable for exchanges of temperate plants  until the establishment of the Bering Seaway 3. The present study found very low support for all series that included more than one species except for the Munson/Moore series Precoces/Ripariae (Figure 4. shuttleworthii. Vitis in the early Miocene. Wen et al. and diversifications in many groups of organisms . V. Molecular phylogenetic analyses of several disjunct genera suggested a progression from East Asia to Eastern and Western North America [65. This may be an artifact of the tenuous nature of most higher-level relationships revealed in this study. BMC Evolutionary Biology 2013. nesbittiana. V. The derived position of V. Local adaptations in heterogeneous environments likely lead to smaller population sizes and thus more rapid loss or fixation of novel characters . node A) and genus Vitis. riparia.75] or subgenera [5. These factors facilitated dispersal of warm-temperate terrestrial organisms in the northern hemisphere. These caused the primary divisions within Vitis as well as species-level and some intra-specific divisions . Phylogenetically-based Vitis systematics The systematics of Vitis is a challenging area of taxonomy. vinifera cultivars included in the present study have been . The underlying evolutionary scenario for Vitis is consistent with origin in the Eocene. palmata. At that time the supercontinent Laurasia had only begun dividing into North America and Eurasia  and the climate was considerably warmer in the northern latitudes . and V. our phylogenetic trees suggested isolation of some North American and Asian species during the Plio. Large range expansions with post glacial warming were also promoted by the physiographic homogeneity of Eastern North America . the rise of subg. bashanica. The North American species V. After fragmentation of a Paleo/Neogene range. hancockii. These species must have expanded to their large present ranges after the glacial period. blancoii have smaller ranges and multiple species-specific SNP character changes. However. V. sylvestris was unexpected. This was followed by diversification in the mid-Oligocene. girdiana. and ecological adaptation. The apparent species-specific SNPs are good candidates to apply in species delineation investigations of grapes. V. leaving Beringia as the major route for potential gene flow. Nie et al.5-5 Ma . Similarly. labrusca. Much of the current Eastern North American range of V. permitting genetic exchange at least until late Miocene to which the disjunction was timed. suggesting past hybridization with V. yenshanensis as these species in some analyses grouped firmly within Asia (as opposed to Figure 5 where they are basal to Eurasia). The most comprehensive treatment of Chinese Vitis  did not apply a series-level classification. Only Galet  assigned Asian species to series. Our findings confirmed the tenuous nature of many grapevine species and especially higher groupings such as series. supporting the division of the genus Vitis into two sections [4.76]. Additional file 15 lists a synopsis of the major Vitis classifications. bloodworthiana and V. Most Chinese species could be assigned to one series if series were to be used (Figure 5). coignetiae and V. post glacial range expansions.com/1471-2148/13/141 Page 12 of 20 Vitis to the Paleocene (65. vinifera.70] and may have no longer existed when Vitis arose. quinquangularis. V. V. the balance of grape-specific information tends to support our findings of a North American origin for the most recent common ancestor of Vitis. V.5-58. aestivalis.Wan et al. node W). Fragmentation and local adaptations were evident in physiographically heterogeneous western North America and temperate eastern Asia. Vitis (Figure 4. It could also be a result of the nature of selection and clonal propagation that all V. physiographically diverse eastern Asia  had three species with multiple speciesspecific SNPs: V. sylvestris is the suggested progenitor of V. vinifera  while the phylogenetic position suggests that V. the North American and Asian disjunction in the late Miocene. The phylogenetic position intermediate between the Asian and Eurasian species and the well-defined split revealed in the network (Figure 2) supported this conclusion. jacquemontii should be assigned to the series Viniferae. amurensis.  argue for a North American origin of Ampelopsis (Vitaceae). survival in refugia. cinerea was unsuitable for Vitis during the Wisconsinan glaciations due to coverage by the polar ice sheet and harsh conditions along its southern edge (Figure 1). our accessions had perfect flowers. V. Other well supported higher-level groupings were subg.8 Ma). V. This may not include V. V.66]. range restriction and fragmentation and speciation during the Pliocene and Pleistocene cooling cycles. vinifera (Figure 5).biomedcentral. It appears as if V.74. 13:141 http://www.  found many more lineages with North American origins and migration to Asia than vice versa . a time of maximum development of temperate Paleo/Neogene forests. This study shows clearly that Vitis was also a part of this great biogeographic phenomenon. sylvestris was derived from V. Most East Asia–North American disjuncts from diverse families have had longer histories in North America than in Asia: of nine woody East Asian–East North American disjunct genera  all appeared earlier in the fossil record of North America than in that of Asia .and Pleistocene cooling cycles. Thus. both on land and in the sea. The Pleiocene/Pleistocene cooling cycles are well known to have caused range restrictions. The North Atlantic land bridge was present in the early Paleogene [69.
78]. both European species. joining Vitis with the large group of organisms whose extant species differentiated in response to Pliocene and A total of 309 accessions of 48 species or varieties (~80% of the approximately 60 known species of the genus) and outgroups were sampled in this study: 21 species from Asia. were of Asian origin yet grouped with North American accessions and remain anomalies that could not be resolved. Cayratia japonica Thunb. The genus itself was extremely well supported. BMC Evolutionary Biology 2013.32. labrusca as these species placed solidly into well separated Asian and North American clades. Planch.34] were chosen as outgroups in the dating of divergences using BEAST: Parthenocissus spp. China. Wada  also identified a monophyletic V. CA. USA. Many species were well supported. range fragmentation. some of them potentially for thousands of years [77. Geneva. NY. one belonging to Aestivales and the other to Cinerescentes. The genome-wide sampling of SNPs provided insight into the evolutionary history of the grape genus and supported previous notions of Paleogene origins. Two of the most important forces. The major clades formed throughout the native distribution at 23-8 Ma (broad range due to large HPDs). aestivalis. This suggested that the phylogenetic signal was too weak to overcome the level of noise created by evolutionary forces acting within the Vitis gene pool. sylvestris. More samples need to be investigated to assess the discriminatory power of these SNPs. Most higher-level relationships within the genus suffered from weak support. flexuosa DVIT1385 and 304V. Additional file 1). Samples 080-084 came to us as V. Two of the . 2) USDA-ARS. Our results preliminarily identified V. Quarternary climate change . girdiana has been considered to be a variety of V. maintained reproductive compatibility. tiliifolia pair due to the possible hybrid nature of V. This highlighted confusion in the past related to the synonym Vitis simpsonii that has been claimed for two different species as described in Comeaux . jacquemontii/V. National Clonal Germplasm Repository (NCGR). Finally. jaquemontii/V. cinerea (Engelm. coupled with physiographic heterogeneity. arizonica . and the markers with low homoplasy defining those lineages will likely be useful in species delineation and assessing the reliability of different morphological taxonomic characters. tiliifolia accessions in general and the dispersed positions of V. such as series. Shaanxi Province. Ampelopsis glandulosa (Wall. lanata and V.  concluded that current commercial V. caribaea in ). labrusca and for V. tiliifolia samples. and 25 species and varieties from North America (Figure 1. aestivalis clade. Evolution is arrested by clonal propagation. higher-level taxonomic groupings.  hypothesized Asian/North American sister species pairs for V. californica  and its own separate species . brevipedunculata (Maxim. Broad reticulation across many species probably prevented the ancestral gene pool from diverging during the Neogene forest stage. Davis. Mullins et al. Our study showed conclusively that these accessions belong to V. probably acting concurrently or alternating. respectively. are incomplete lineage sorting of ancestral polymorphism and reticulation. USA. Myles et al. Our results did not support sister pair relationships for V. These samples were obtained from: 1) the Grape Germplasm Collection at the Northwest A&F University (NAFU). Four closely related genera based on chloroplast and nuclear markers [19. Methods Plant materials Conclusions This is the first study to apply sequences of a large number of nuclear loci combined with extensive species and intraspecific sampling to the phylogeny and biogeographic history of Vitis and the problem of Vitis systematics. ‘Rubra’. rufotomentosa has the same problem. However. girdiana as a well supported independent species (using the general lineage concept  and diagnosability ) with five species-specific SNPs.biomedcentral. V. and 3) USDA-ARS. a variety of V. (DNA). and is still acting today as evidenced by the prevalence of hybrids found in the wild and in repository collections. 13:141 http://www. two accessions. coignetiae/V. Several additional accessions were identified as misnamed and others were recognized as hybrids (Additional file 1). was a dominant force in structuring genetic diversity of extant Vitis spp. We demonstrated that genome-wide nuclear SNPs were a productive approach to address questions at and below the species level in grapes. provided enough recent barriers to gene flow to facilitate evolutionary divergence.Wan et al. The synonym V. var.28.) Engelm. We found that the most recent common ancestor of Vitis was North American. climatic oscillations during the Pliocene and Quaternary. Plant Genetic Resources Unit (PGRU).) Momiy. Leea coccinea Planch. Our results are inconclusive with respect to the V. may be misleading. suggesting that vicariance (the fragmentation of a large Paleo/Neogene Northern hemisphere distribution) in conjunction with local adaptation. vinifera varieties are only one or two generations removed from the wild V.) Momiy. floridana (Munson) but placed solidly into the V. 111V. and recent nature of the species. Yangling.com/1471-2148/13/141 Page 13 of 20 subjected to. although it had poor bootstrap support. leaving the naturally evolving wild species to appear more derived. In light of the recency of divergence and diffuse genetic boundaries. tiliifolia (V. coignetiae /V. wilsoniae Wangmaiputao. girdiana cluster. ex Millardet var.
When the V. data sets. 13:141 http://www. accessions that placed in unexpected positions or had very weak support on preliminary phylogenetic trees were submitted to a taxonomic expert (Dr. Sequence alignment. cuttings) can be requested from the clonally maintained vines at these sites. Muscadinia was used as the outgroup in analyzing subg. and Mutation Surveyor (Soft Genetics) was used for base calling. were resequenced in a total of 309 accessions (Additional file 1). mol.com/1471-2148/13/141 Page 14 of 20 outgroup genera (five species of Cissus and Cayratia japonica) were obtained from a research collection (Dr. were included except for V. romanetii (Pingli7). One to 27 accessions or genotypes were sampled per species. rotundifolia as the sister species to subgenus Vitis and it is consistent with other studies e. Exact geographic coordinates of origin were not available for most accessions. Based on preliminary analyses. vinifera genome sequence became available . ‘Jiangxi2’). All available varieties (not cultivars) of a species were sampled. a predicted SNP may not be verifiable. vinifera ssp. rotundifolia. vinifera for which no wild accessions are known . BMC Evolutionary Biology 2013.792 clusters.7%).biomedcentral. Because this study was intended to survey broadly across the entire genus. Additional file 17 provides the detailed conditions. vinifera ‘Rotberger. Europe (V. quinquangularis (Weinan3). presently E & J Gallo Winery). SNP discovery and selection DNA isolation and re-sequencing DNA was isolated from fresh or frozen young leaves and apical meristems using a modified CTAB (cetyltrimethylammonium bromide. Cousins. vinifera sequences which formed 3. The exploratory sequencing was performed in-house at PGRU on ABI-3100xl Genetic Analyzer. romanetii Rom. davidii (Xuefeng). Sigma H6292) protocol [84. V. Sigma PVP40) in the extraction buffer to remove secondary compounds. Pairs of PCR primers were designed using the program ‘Primer 3’  for 281 gene fragments of 400-600 base pairs (bp) containing moderate polymorphism. V. The sequences of the 27 final primer pairs and supporting information are listed in Additional file 2. Then 96 primer pairs with robust single bands were chosen for resequencing to test sequence quality using eight species (V. single bands were obtained for 201 of 281 primer pairs (71. P. Inc (New Haven CT. (PI588194). California). The 62 variablysized EST libraries and additional grape mRNAs included 108. When the final dataset was assembled. coding of gaps ProSeq  was used for editing sequence based on trace files. Predicted genes were identified in comparison with the NCBI non-redundant protein sequence database. Thirty of the most consistently amplifiable gene fragments. two chloroform purifications to remove proteins and a NaCl and ethanol precipitation to remove polysaccharides. The outgroup Leea coccinea “Rubra” was grown from seeds obtained from Carter Seeds (Vista. hancockii (Lingye_F) and V. ’ DVIT2339) and North America (V. the primer sequences and gene fragments were BLASTed  against this genome to determine their chromosome locations and confirm their homology and identity. The accessions located in the US repositories can be requested through the Genetic Resources Information Network (GRIN)  and plant materials (leaves. both within Vitis and outgroups.429 V. V. USA). widely distributed species were more extensively sampled to include potential geographic differentiation. 50 μL PCR volumes were cleaned for sequencing. Cousins) for an independent assessment of species identity. gene markers that were predicted to be monomorphic among V. To mitigate long-branch attraction.000. Primer screening was performed in 25 μL PCR volumes. Because EST data are often based on one sequencing pass and are not filtered for error. and suspected duplicate loci (two markers). V. amurensis (Zuoshan1).Wan et al. Vitis. Similarly. three gene markers were excluded because of unalignable indels (one marker). yenshanensis (Yanshan_F). Robust. wt. The amplifications were tested using three DNA samples. No cultivars of Vitis spp. labrusca L.85] with 2-5% PVP (Polyvinylpyrrolidone.33-36]. This was justified by all preliminary analyses on the complete data set that identified V. V. cinerea (PI588575). [23. concentrated and used in 12 μL cycle sequencing reactions. one each from Asia (V. the 40 chromosome Vitis rotundifolia Michx. V. A few sibling groups were also included to test the ability of the markers to place or distinguish those accessions. DVIT1689). Markers with extreme levels of polymorphism were also excluded to minimize possible selection of duplicated loci.29. Caill. vinifera were discarded. but without indicating the nature of the conflict. Both strands were separately sequenced using the PCR forward or reverse primer. The labels in the figures and tables indicate the corrected names unless otherwise indicated. Accessions and pertinent details are listed in Additional file 1. The highthroughput sequencing (30 gene fragments for 309 accessions) was performed by Genaissance Pharmaceuticals. P. USDA/ARS. 40. with suitable polymorphisms and only minor sequence length variation in the eight tested. subg. Additional file 1 lists the results of all assessments. Heterozygotes were manually edited to use the IUPAC-IUB symbols for nucleotide nomenclature . Expressed Sequence Tags (ESTs)  of Vitis vinifera and grape mRNAs in NCBI in 2004 were sub-clustered and surveyed to predict SNPs using an in-house pipeline as described by Labate and Baldo . The results from ProSeq and Mutation .g.
9 (splits present in 90% of the trees). Gaps were treated as characters using ‘simple indel coding' (SIC)  and implemented in SeqState 1.103] was used to visualize conflict in the 273-OTU matrix with 27 concatenated gene fragments. The final .4 [95.biomedcentral. Marker-specific and whole-dataset-specific substitution models were determined in Findmodel  and are listed in Additional file 2. 19. the number of ingroup OTUs was reduced to one accession per species and variety for efficient calculations  and to satisfy the Yule model of speciation. met general quality requirements outlined in Additional file 24. Following the reasoning of Nie et al.100]. with partitions listed in Additional file 21) and subsets of OTUs revealed known and new hybrids which were excluded in the final analysis because phylogenetic trees can be strongly influenced by hybrid taxa . ML and BA treated gaps as missing data. The conditions for the partitioned runs were: 27 unlinked partitions with individual substitution models. network analyses 0. Calculation of divergence time The Phi Test  implemented in Splitstree4  was used to test for recombination in each gene fragment. 096_Leea coccinea ‘Rubra’. dev. an uncorrelated relaxed clock with log normally distributed uncorrelated rates between branches . estimated frequencies of nucleotides.5 Ma (st. Discrepancies were resolved by examining trace files manually.04 (all splits present in at least one tree.3. a random starting tree. NeighborNet with uncorrected P distance in SplitsTree4 [102. 20). and parameters were sampled every 10. and 2) fix the stem age of Vitaceae with a normal prior distribution of 90.4.  was used in this study. 129_Cayratia japonica . 0. Five runs of the partitioned dataset were combined after removing a . Gap information was used for parsimony analysis only. = 0. Liu et al. Combinability of DNA partitions was ascertained using Wien’s  method: existence of corresponding but incongruent clades with bootstrap support greater than 70% are seen as support for not concatenating data sets. Test for recombination. and nearly 100% agreement was found. default operators modified based on preliminary runs.7 Ma (st.Wan et al. Vitis at 5. Leea and Cayratia had substantial amounts of missing data (Additional file 1).1 . Operators were optimized automatically.75 Ma (st. Trees were visualized in Figtree V1. = 1 Ma). Additional file 1 summarizes the members of each analysis.xml file were combined if they shared the same trace. Findmodel uses Weighbor . 23) were run ten times using the maximum time available at the Computational Biology Service Unit (CBSU) BioHPC computer cluster at Cornell University.1 . BMC Evolutionary Biology 2013.000 steps. 13:141 http://www.7. dev. Many preliminary runs were conducted on the partitioned and unpartitioned data file to explore parameters. 20.’s  reasoning was adopted to 1) place the second calibration point of V.106] software were used to estimate divergence dates using Markov Chain Monte Carlo (MCMC) sampling. Simple gap coding was chosen because it is a preferred coding method for empirical studies [99. labrusca and closely related North American relatives in the subg. Bayesian (BA) estimates in the BEAST V1. 2) With the presence of multiple individuals per species dating is a more complex issue and would preferably apply coalescence methods. and partitioned and unpartitioned concatenated sets for a total evidence dataset . Extensive preliminary phylogenetic and PCA  (Additional file 18) analyses using all (Additional files 19. These modifications resulted in the 52OTU dataset (Additional file 22). The term ‘Tertiary’ was replaced by Paleogene and Neogene . 0.  a normal prior was used with a mean of 58. and if the addition increased the ESS of key parameters. auto-optimization turned on. Preliminary ML analyses were conducted on all single gene fragments. The final phylogenetic data set contained 273 OTU composed of subgenera Vitis and Muscadinia (Additional files 1. This does not guarantee that the final analyses were devoid of hybrids as they are not always identifiable based on morphology. Inclusion of gaps in phylogenetic analyses is limited by the optimality criterion used for phylogenetic inference. The three unpartitioned runs with the highest (almost identical) likelihoods were combined after 10% burnin was removed to produce the chronogram in Figure 3. 247_Parthenocissus spp. 0. 1/26).08 (splits present in at least two of the trees).com/1471-2148/13/141 Page 15 of 20 Surveyor were compared for accuracy. Sequences were aligned manually and also aligned using Clustal W  with default parameters.5 (splits present in half the trees). : 060_Ampelopsis brevipedunculata. The conditions for the unpartitioned runs were identical except that there was only one partition and one substitution model. This dataset was modified for dating divergences using BEAST: 1) Four outgroup taxa were added to match calibration points in Nei et al. = 5 Ma) for the stem age of Vitis. Unpartitioned runs were used because the combined partitioned runs did not have sufficient support for important parameters. dev. PAML  and methods in Modeltest  to determine substitution models.. Thus. The best single gene tree from each locus was combined into a file from which a consensus network was constructed in Splitstree4 .5 Ma). Thresholds used were The geologic time scale of Gradstein et al.xml files (Additional files 22. Yule model of speciation (a pure birth process). Runs from the same . Preliminary analyses indicated that the present data set was not sufficiently informative to allow a well-supported coalescent analysis.
Maximum Parsimony (MP) The 273-OTU matrix was analyzed under the ML criterion using RAxML  versions 7.com/1471-2148/13/141 Page 16 of 20 burnin of 10-75% to compute the evolutionary rates (gene. Five more replicates were added if the final maximum likelihood values varied extensively among replicates.1  and Figtree v1.5 million (50 mil run) and 10 million (48 mil run). meanRate) of the individual genes. Maximum likelihood Multiple short runs were performed to determine the temperature and number of chains that would support chain swap.6. Gap coding was removed. tree drifting. manually converted to Newick trees. The data were partitioned by gene fragment (Additional file 21).meanRates had large ESS. To test for conflict among genes. 13:141 http://www. TreeAnnotator v1.3.114]. The driven searches included the ratchet . Bayes  on the concatenated. Europe/near East.10. and so on until the consensus stabilized . The character values. Twenty replicate searches were run on this final data set using a rapid hill climbing algorithm and the GTRGAMMA (= GTR + Optimization of substitution rates + GAMMA model of rate heterogeneity. and the default of 25 rate categories. indicating the level of homoplasy. Additional criteria of more stringent species definitions were considered. visualized and annotated using Dendroscope V 2. where species are defined as separately evolving segments of metapopulation lineages .biomedcentral. and sample frequency every 5. Due to computational limitations.1 were used to annotate and visualize maximum credibility trees listing all posterior probabilities. the single gene analyses were performed only in RAxML at CIPRES.100]. The two final and longest runs of 48 and 50 million generations (fitting just within the 168 hr time limit) were run with 8 chains. Phylogenetic and ancestral area analyses To evaluate species we used a phylogenetically based general lineage concept. such as monophyly  and diagnosability [81. temperature of 0. Because of the sparse yield of information and low information content of most markers. this computational expense was not repeated with the 273-OTU matrix. Bootstrap support was based on 1.000 replicates of driven searches using the same search components and default parameters. Bayesian (BA) The software package TNT. assuming unordered character state transformation and equal weights (Fitch parsimony) . Incongruent clades with bootstrap support of 70% or greater were considered as support for not combining data sets [42. the alpha parameter was estimated) model of substitution as recommended by the program’s author.Wan et al. The strict consensus tree was constructed using all most parsimonious trees from all four searches. An area code was added to each accession in the 273-OTU data matrix (Additional file 20). To keep trees comparable no OTUs were excluded. BMC Evolutionary Biology 2013. were obtained in TNT to study the support at nodes of interest.4. Ancestral area analysis Bayesian analyses were performed on the 273-OTU matrix using Mr. Output was visualized in Dendroscope V2. Trees for illustrations were exported in a NEXUS format. and those of interest were reconstructed. Synapomorphies were optimized and listed. This option searched until a minimum tree length was found a certain number of times and then a consensus was estimated.000 generations. The geographic distribution was partitioned into four continental area units that correspond to broad distributional trends in Vitis: Asia. The search was repeated four times using a different random starting seed and without specifying a target score.2. as determined in Findmodel. . The rapid bootstrapping option  was chosen to generate 1. Means and 95% Highest Posterior Density (HPD) from these combined runs were computed using TreeAnnotator.2. Uninformative characters were excluded and gaps were coded. Fitch optimization (reversible parsimony)  was performed in TNT to optimize the area on the strict consensus tree . Each gene was run in 10 replicates. One-thousand bootstrap replicates were collected for each gene fragment. Hennig Society version  was used to analyze the unpartitioned 273-OTU matrix under parsimony.5  was used to evaluate the MCMC runs. The default settings were used except that the consensus was stabilized four times instead of twice. 7. After a second round of searching the new consensus was compared to the previous one. The best-scoring ML tree was obtained in the same search and bootstrap values were annotated. tree fusion and sectorial search . nonpartitioned data set using the K80 substitution model (Nst = 2. These gene.6 and HPC2 at the web portal of the Cyber Infrastructure for Phylogenetic Research (CIPRES) cluster. E.2 .2.2. Burn in was 2. All characters were included.and SENorth America (including Mexico and the Caribbean) and Western North America (Figure 1). The efficient option of “driven search” in TNT was used for the search.0. Tracer V1. a preliminary analysis was performed for each gene fragment using the same parameters as above but without a partitioned model.000 bootstrap replicates. 4 by 4) plus Gamma. Indels were treated as missing data.
Node ages (Ma) of all nodes in maximum clade credibility tree inferred with BEAST from three combined runs. and concurrent reduction in precipitation caused further partitioning of the East Asian habitats [63. Blue = Europe/Near East. Primer sequence.pdf.pdf.9). NCBI accession numbers: [Genbank: JX952227-JX960379. Support values >50% are indicated. trees and BEAST . Abbreviated uncorrected taxon labels.8 Ma) based on fossil evidence synthesized in Nei at al.126. Green = Eastern/Southeastern North America including Mexico.130-132]. Blue = North American OTUs. 48 million generations. Additional file 2 lists accession numbers by marker. followed by regions of dry continental climates [65.pdf. atpB.133]. and up to 28 species across the eastern and southwestern US and Mexico  (Figure 1). providing dispersal for these species. 50% of the trees (threshold = 0. The data sets supporting the results of this article are included within the article and its additional files.5061/dryad. and conversion to absolute time using three fossil reference time points. Maximum parsimony Bootstrap tree.54] and were not detected in the preceding Cretaceous period. Cartoon of Bayesian tree 48 million generations. doi. chromosome location. Red = Asia. burn in 10 million steps. xlsx. Communities on different continents were linked by migration across the Bering Land Bridge. Additional file 10: Cartoon Of Bayesian Tree_48MilGen. source. Additional file 3: a-d. Yellow = Western North America. not partitioned.129. gene. Additional file 7: Best ML tree with Bootstrap supports. Climatic cooling at the start of the Oligocene (33.Wan et al.pdf.pdf. The flora retreated into refugial regions in response to Pliocene cooling (5. Node numbers of MP strict consensus tree (Figure 4A-B). 13:141 http://www. For comparison with trees reconstructed with other methods. . Up to 30 species are native to a vast area in eastern Asia. Additional file 12: Node Numbers of MP strict consensus tree. linking North America and Europe particularly in the early Eocene [65. among them the early grapevines.com/1471-2148/13/141 Page 17 of 20 Appendix 1 Biogeography: the Eastern Asia-Eastern North American disjunction Supporting data The genus Vitis contributes to one of the great distributional phenomena in plant biogeography.pdf. burn in 5%.126-129].70. Additional file 1 lists the sequence accession number for all OTUs. and 3) the presence of well preserved Vitis seed at the late Neogene Gray Fossil site in Tennessee (7-4. BMC Evolutionary Biology 2013. Tectonic uplifting of mountain ranges and plateaus during the Pliocene into the Holocene. Intra-continental migration was impeded between Europe and Asia by an epicontinental seaway (CretaceousEocene) as was migration between east and west North America (upper Cretaceous). Wild grapes are a savored food of birds and some small mammals. their degree of homoplasy and marker identity. taxonomy.04). A character value of one indicates that the character changed once in the phylogeny and there is no homoplasy. Additional file 2: Fragment information. Additional file 14: Ancestral Area optimization_JacquIsAsian. List of characters supporting selected nodes in strict consensus tree (Figure 3).129. There is widespread agreement that these disjunct floras are relicts of plant communities that were distributed throughout a large part of the Northern Hemisphere during much of the Paleogene and early Neogene (formerly the Tertiary) Periods (65-15 Ma) [69.org/10. threshold = 0.pdf.134].3-2. . Posterior probabilities (0 to 1) are listed along branches. Additional file 6: Cartoon BestMLtree. EMBL: HF544510-HF544512]. not partitioned. d.129]. Green = Asian OTUs. China.8 Ma) [53. Best ML tree of 273 accessions with bootstrap supports from 1. GenBank accession numbers.134]. Supports 1-100% are listed along branches. A small number of species extend into the Tropics both in Asia and in North America [6. estimated by Magallón and Castillo  at 90. Consensus Networks.133. Fossil distributions suggest that. burn in 10 million steps. correspond to node numbers in Additional file 11. this estimate was based on a five gene data set (chloroplast rbcL.pdf.pdf. 90% of the trees (threshold = 0. Bayesian tree. Japan and Java.5 Ma) and Quaternary glaciations (2. Additional file 5: Posterior Probabilities.meanRate.08). Cartoon of best ML tree. two trees (2/26. Alignment.000 replicates. data set membership. Red = European (mostly) OTUs. As detailed in Nei et al. Consensus Network of 26 single gene trees. by the end of the Neogene. the Eastern Asia-Eastern North American disjunction among the temperate to warm temperate northern hemisphere taxa [69.pdf.5-55. Additional file 8: Bayesian Tree_48MGen.5-0 Ma) . Accession name. Additional file 4: Node Ages.xlsx. Bayesian tree.9 Ma) gave rise to the Mixed Mesophytic Forest of deciduous and evergreen trees and associated taxa that comprise the modern Paleogene/Neogene relict floras . Important estimated time points in the Vitaceae diversifications were: 1) the divergence of Vitaceae and Leeaceae (stem age of Vitaceae). Abbreviated taxon labels. substitution models used (from Findmodel).s1s75. Additional file 11: Nodes With Support And Character scores Genes Filled. b. phylogenies.0-56. and nuclear 18S and 26S nrDNA) obtained from GenBank.5). showing all splits present in at least a. one tree (1/26. linking North America and Asia beginning in the Miocene . fragment length. 50 million generations.xlsx. Additional files Additional file 1: Accession Information Table_with GenBankAccNo.82 – 90. Posterior probabilities (0 to 1) are listed along branches. individual GenBank and EMBL accession numbers. not partitioned. Germplasm accessions information. Posterior probabilities of all nodes in maximum clade credibility tree inferred with BEAST from three combined runs. the oldest reliable Vitis seeds are from the Paleocene (65. Additional file 13: MP BS tree 1000rep. A character value of 10 means that the character changed 10 times in the phylogeny. two species across middle Asia and Europe. Ancestral Area Fitch Parsimony optimization on strict consensus tree. and across the North Atlantic Land Bridge. original EST annotation.matK. Additional file 9: BayesianTree_50MilGen. the genusVitis was widely distributed in the Northern Hemisphere .xml files were deposited at http://dx. 2) the divergence of the Ampelocissus-Vitis clade in the Tiffian stage of the Paleocene (62. c. number of unique site patterns (an indicator of informative variation).65 Ma. Gene fragment information. continental origin. . threshold = 0.biomedcentral.5 Ma) . 1000 replicates.pdf.
Edited by Janick J.TNT file used in the present study (273 OTU. Sala F: Phylogeographical structure and conservation genetics of wild grapevine. Grassi F. CA supplied tissue samples. Bouquet A. HRS conceived the ideas. Koehmstedt A. Prins BH. doi:10. Rhodes AM: A taximetric study of interspecific variation in Vitis. Bayes. Bob Nearpass provided local IT support. NCGR in Davis. management. Additional file 17: Laboratory procedures. Arnold C.edu/china/mss/volume12/Vitaceae. simple indel coding. Del Fabbro C.pdf] 7.Wan et al. PhD thesis. Cambridge: Cambridge University Press. XML file used to date divergences in BEAST (unpartitioned). Buckler ES: Genetic structure and domestication history of the grape. 12:173–222.pdf. JAL. 138:421–432. Hugueney P. In Biology of the grapevine. Labra M. Ibanez A. CJS collected or arranged for the materials. Nesbitt WB. rotundifolia. The . Noel B. Additional file 19: TNTfile_303OTU_WithSIC_uninformCharsDeact. 13. Van der Bank M. NY 14456. Reynolds A. USA. Author details College of Horticulture. Geneva. 19. 18. HRS. In Genetic resources of temperate fruit and nut crops. PDF at [http://flora. 10. and phylogeny of the genus Vitis: implications for genetic conservation. Pablo Goloboff answered many basic and operational questions about TNT.org] 2. YW. HRS analyzed the data except for PCA. This file was used for some preliminary analyses and can be opened with a text editor. Genet Res. Mullins MG. CRIS Project Number 1910-21000-020-00D. Peter Cousins supplied outgroup material and offered excellent advice on grape species identification and anything else related to grapes. 9. 2US Department of Agriculture. Camb 2003. Lijavetzky D. Mark Miller and Wayne Pfeiffer among others at the San Diego Supercomputer Center gave very effective support to RAxML and endeavored to bring BEAST online at the CIPRES portal. Anthouard V. Branch lengths for the strict consensus tree of the MP driven search. green and blue dots North American species. Alleweldt G. Stover E: Genetic structure. Vezzi A. Horner D. 7:837–845. Martinez-Zapater JM: High throughput SNPdiscovery and genotyping in grapevine (Vitis vinifera L. Comeaux BL. Vico V.M. Simon CJ: Genetic structure and differentiation in cultivated grape. 108:3530–3535. Flora of China 2007. Acta Hort 2008. Rogers DJ. 8:424. New York State Agricultural Experiment Station.pdf. in TNT format). 12.fao. Additional file 16: MP Branch lengths. USA. Bouquet A. 13:141 http://www. NY 14456. Policriti A. tnt. Edited by Moore JN. 1992:17–36. Chia J-M. Cattonaro F. Dumas V. Failla O. Billault A. Shaanxi 712100. Jaillon O. Poulain J. Casagrande A. Tome 1 Les vignes Americaines. Wageningen: Acta Hort 290. BMC Evolutionary Biology 2013. YW. TNT data file including all 303 original accessions. Vitis 1969. 42:141–159. [http:// faostat. Northwest A&F University.harvard. The hybrid V. Classifications of Vitis proposed by six major systematists between 1895 and 1991. Conserv Genet 2006. Ugarte E. Pratt C: Grapes. Anonymous reviewers provided suggestions that improved this work. 8. All authors read and approved the final submission. Plant Genetic Resources Unit. Part of this work was carried out using the resources of the Computational Biology Service Unit from Cornell University which is partially funded by Microsoft Corporation. Boyko AR. Reisch BI. 4. 16. Bustamante CD. Am J Bot 1955. Agriculture Research Service. AMB developed and performed PCA. Vitis vinifera L. Prins B. Mica E. Choisne N. Rogers CF: Systematics of North American grape species. Acknowledgements We would like to thank Ashley Egan for supplying crucial sparks and expertise at several analytical key points of this project. Fay MF. 3. YW. Vitulo N.tnt. People’s Republic of China. Bruijn ADE: Systematics of Vitaceae from the viewpoint of plastid rbcl DNA sequence data. Imazio S. Gouyvenoux M. Aradhya MK. Cornell University. 2004C103). Dehan: Montpellier. Lauren Chan provided answers to specific questions in BEAST and Mr.pdf. Aradhya MK. Moore JN. 14. BMC Genomics 2007. Aubourg S. Branch length reflects the number of character changes along a specific branch. 8:177–187. Spiegel-Roy P.pdf. AMB. AMB. Agriculture Research Service. 6. Owens C. Chen Z. Fantz PR: Taxonomy of the native grapes of North Carolina. 14:339–367. 15. Di Gaspero G. Geneva. 81:179–192. Additional file 18: PCA scatter plot. Grape Genetic Research Unit. Aradhya MK. and early analysis of datasets. Myles S. Cabezas JA. 449:463–467. Evaluation criteria for Tracer files. YW was supported by the China Scholarship Council (22861057). Circled accessions are discussed in text. McNally J. Sida 1991. Dasilva C. gap coded.pdf. Aury J-M. Wen J: Vitaceae. and the Young Scientist Foundation of NWAFU (QN2009-013). Funding This project was funded by the United States Department of Agriculture. Scienza A. PCR and cycle sequencing protocols. Additional file 22: 800mil10Klog_52taxaNoTiliWithCay_11437_ LognRelYule3CalPts_NOpartK80G.1186/1471-2164-8-424. 2nd edition. Reisch B: Grapes (Vitis). Chase MW. 799:43–49. CJS. In Fruit breeding.xml. Last character in each sequence was added for the ancestral area reconstruction. Ingrouille MJ. Ocete-Rubio R. Cramer SG. vinifera x V. Rosetto M. Gene partitions are listed in Additional file 21. Food and Agriculture Organization of the United Nations: FAO. 1984. Biol J Linn Soc 2002. 5. Jubin C. 21. Authors’ contributions YW. the Shaanxi Natural Science Foundation (No. Additional file 24: Evaluation criteria for BEAST runs. Additional file 23: 400Mil10Klog_53taxa With Cayratia_11437_Yule_ LogNrelaxedClock_partitioned3calibration Many Models. Proc Natl Acad Sci 2011. Am J Bot 2002. 52:197–215. YW wrote the paper. Segurens B. New York State Agricultural Experiment Station. Edited by Mullins MG. Brown PJ.) by combining a re-sequencing approach and SNPlex technology. Ware D. Jena: Gustav Fischer Verlag. Dangle G.huh. 89:22–28. 20. et al: The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. . black dots intercontinental hybrids. Grassi F.com/1471-2148/13/141 Page 18 of 20 Additional file 15: Vitis classifications. differentiation. Am J Enol Vitic 1978. New York: Wiley. Reinig WF: Die Holarktis. JAL developed the markers. (Vitaceae) north of Mexico. Castanea 1987. Henry RJ: The application of SSRs characterized for grape (Vitis vinifera) to conservation studies in Vitaceae. 17. Legeai F. 29:73–78. Williams LE: The grapevine and its relatives. Comeaux BL: Taxonomic studies on certain native grapes of the Eastern United States. Agricultural Research Service. Ballington JR. Pink dots represent Asian species. Additional file 20: TNTinfile_273otu_noAmpWith272_6670fixed_sic_ LastAreaChar_JacquIsAsian. Jublot D. 11. Clepet C. 1 Received: 20 November 2012 Accepted: 28 May 2013 Published: 5 July 2013 Competing interests The authors declare they have no competing interests. CJS and GYZ managed the project. Prins BH. Olmo HP: Cytogenetics of Vitis: I. Williams LE. Galet P: Cepages et vignobles de France. XML file used to estimate evolutionary rate of change per partition (27 partitions).biomedcentral. Bruyère C. Dangle GS. Meredith CP. Yangling. Bowman D. Boursiquot J. Nature 2007. Hui R. 1990:291–337. Warren Lamboy worked extensively on data accuracy. 1988. North Carolina State University: Department of Horticultural Science. Moore MO: Classification and systematics of Eastern North American Vitis L. 3US Department of Agriculture.xml. Alaux M. Cornell University. Rodriguez V. Can be opened using a text editor. Patel GI. Barrett HC. USDA is an equal opportunity provider and employer. brown dots European species. 1937. 1996:297–369. References 1. 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