BACKGROUND
grapes  are one of the world’s most economically valuable fruit crops  <cit> .
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  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  <cit> .
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  <cit>  and east asia  <cit> .
up to  <dig> species are native to a vast area in eastern asia, china, japan and java, two species across middle asia and europe, and up to  <dig> species across the eastern and southwestern us and mexico  <cit>  .
appendix  <dig> expands discussion of the biogeographic background.
the genus vitis is divided into two subgenera: muscadinia planch.
and vitis planch.
.
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  <cit>  that is shared with all rosids  <cit> .
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  <cit> .
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  <cit> .
this has led to many extraneous species names  <cit> .
the systematics of vitis is based primarily on morphology  <cit>  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  <cit> .
studies most similar in goals and pertinent to the present study were conducted by aradhya et al.
<cit> , nie et al.
<cit> , liu et al.
<cit> , and zecca et al.
<cit> .
aradhya et al.
<cit>  obtained a taxon sample similar to the present study and used ssr and aflp markers to study genetic diversity within vitis.
these markers have limited value for phylogeny reconstruction  <cit>  and dating divergences was not attempted.
nie et al.
<cit>  and liu et al.
<cit>  provided well-reasoned paleontological dates to estimate divergences in vitaceae.
these calibration points were applied in the present study.
zecca et al.’s  <cit>  chronogram is a tantalizing expansion of the vitis component of nie et al.’s  <cit>  chronogram.
their inferences were limited by the small number of markers and the limited variability available in those markers, which did not fully resolve the tip clades.
further, limited intra-specific replication  limited the ability to make species-level inferences.
adding more data can be useful for resolving difficult phylogenies that were based on a few genes  <cit> .
the present study attempted to improve on three aspects of previous phylogeographical studies of vitis by more extensive sampling of the nuclear genome, the species, and intraspecific variation.
this study developed and used  <dig> nuclear gene markers and sequenced  <dig> accessions of  <dig> vitis species, varieties, and four out-groups to: 1) reconstruct a phylogenetic hypothesis of the genus vitis, 2) date important time points in the evolution of vitis, 3) elucidate the biogeographic history of the genus, and 4) evaluate systematics of vitis within the framework of phylogeny.
RESULTS
molecular characteristics of the nuclear sequences of vitis
most vitis accessions had complete sequence or had minimal missing data .
indel sequences in the  <dig> gene markers were unambiguous and easy to align.
the starting alignment matrix for the  <dig> gene markers and all  <dig> accessions was  <dig>  bp long.
gap coding for maximum parsimony added  <dig> characters.
amino acid coding sequence accounted for  <dig>  nt, 3’ or 5’ untranslated regions for  <dig>  nt, and introns for  <dig>  nt .
because trees by new technology  does not output the number of parsimony-informative characters, we report the number of unique site patterns from bayesian evolutionary analysis by sampling trees  and randomized accelerated maximum likelihood .
the 52-otu matrix had  <dig>  unique site patterns; the 273-otu matrix had  <dig>  unique site patterns.
their distributions among the gene markers are listed in additional file  <dig>  under the uncorrelated log-normal relaxed molecular clock, estimated from three combined runs in beast and calibrated with three fossil dates, the mean rate of substitution  in the data set was  <dig> ×10- <dig> per million years  = 959) and a coefficient of variation  of  <dig>  .
dividing this rate by the size of the data set , the average rate of substitution in this data set was  <dig>  × 10- <dig> substitutions per site per million years.
extent of reticulation and network in vitis
ancestral polymorphism with subsequent lineage sorting is difficult to distinguish from reticulation based on a phylogenetic pattern  <cit> .
in our study, six  gene fragments showed significant tests for recombination based on the phi test  <cit> : fragments  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> .
when these fragments were excluded, the concatenated matrix still showed recombination, as expected , representing independent assortment of markers.
lanier and knowles  <cit>  found that, in species-tree estimation, the gain of accuracy from sampling additional loci and/or individuals always exceeded inaccuracies related to recombination.
thus, in the present work no genes were excluded from subsequent analyses based on evidence of recombination.
we used networks to better visualize the conflicts represented by the high levels of homoplasy.
the consensus network  <cit>  of the  <dig> individual gene trees indicated that few splits were common to multiple trees .
the neighbornet  <cit>  of the concatenated 273-otu matrix  showed extensive conflict at the core of subg.
vitis representing the deeper nodes, and extensive conflict radiating outward.
estimation of divergence times in vitis
with the stem age of vitaceae constrained at  <dig>  ±  <dig>  ma, the stem age of vitis at  <dig>  ±  <dig> ma, and the divergence of v. labrusca and its closely related north american relatives at  <dig>  ±  <dig>  ma, the crown age of vitis was estimated at  <dig>  ma   <dig> ,  <dig> ), the crown age of subg.
vitis at  <dig>  ma , and the stem ages of most species fell between  <dig> and  <dig>  ma.
the ten individual runs continued for  <dig>  million to  <dig>  million steps, and estimated the crown age of subg.
vitis between  <dig>  ma and  <dig>  ma.
the bayesian divergence times in figure  <dig> were estimated from three combined, unpartitioned runs using beast.
the maximum clade credibility tree with mean estimates of divergence time for all nodes  and associated posterior probabilities  were also obtained.
phylogenetic analyses in vitis
maximum likelihood
ml analyses of  <dig> single genes analyzed independently without missing data yielded  <dig> very poorly resolved trees.
no significant conflict was observed under ml, thus the sequences were concatenated.
the  <dig> identical  partitioned rapid ml runs using the 273-otu matrix produced a range of maximum likelihood values of -42511to - <dig>  the final search on the same matrix following the bootstrap search yielded the highest likelihood value: - <dig>  this tree was used in comparisons with results from other methods using a cartoon, i.e., a simplified version with collapsed terminal clades .
bootstrap values are reported for selected nodes  and all nodes .
bayesian
the two runs timed out at  <dig> mil and  <dig> mil generations with good effective sample size , but they did not converge on the exact same phylogenetic hypothesis.
both trees were highly concordant for the species-level clades but differed on the specific placement of some clades .
because the  <dig> mil run  had a higher mean log likelihood of the cold chain  after burnin, the posterior probabilities from this run were used to summarize supports .
overall comparisons of relationships above the species level were facilitated using the cartoon of this tree .
maximum parsimony
tnt’s driven search function produced most parsimonious  trees of the same length in all four searches.
the individual mp trees had a score of  <dig>  consistency index  of  <dig> , and retention index  of  <dig> .
the strict consensus of all  <dig> mp trees from all four searches had a score of  <dig>  ci =  <dig> , and ri =  <dig> .
the strict consensus tree was simplified by collapsing terminal clades  for comparisons.
the full tree was annotated with support values on selected nodes of interest .
node numbers are cross referenced with additional file  <dig>  all node numbers are shown in additional file  <dig>  the mp bootstrap tree with supports >50% was illustrated .
ancestral area analysis
fitch parsimony identified eastern/southeastern north america  as the origin of the most recent common ancestor of vitis based on the strict consensus tree .
systematics of vitis spp.
it was evident that the patterns were similar among the cartoons of the strict mp consensus , the highest ml value cladogram  and the bayesian cladogram .
the clades of eurasian species were nested in north america as a monophyletic clade.
the series precoces munson  together with v. arizonica engelm.
, v. blancoii munson, v. bloodworthiana comeaux, v. xtreleasii munson ex l. h. bailey and v. girdiana munson formed the sister clade.
v. labrusca and v. aestivalis michx.
were grouped together as were v. cinerea  engelm.
ex millardet, v. palmata vahl, v. shuttleworthii house and v. mustangensis buckley.
all three analyses did not group v. monticola buckley or v. californica benth.
with other species.
the position of several clades differed among the searches.
this was a notable characteristic of this data set.
among the different ba, ml and mp trees shown, the otu composition of species clades in general was quite consistent and well supported, but a few clade or species positions were inconsistent.
correspondingly, clades above the species level were often poorly supported.
for example, v. yenshanensis j. x. chen was grouped with v. amurensis rupr.
and v. coignetiae pulliat ex planch.
in mp and ba analysis and in the network but not in ml.
similarly, the amurensis/coignetiae clade was basal to eurasia in mp and ba, but placed within the asian clade in ml and in the network.
v. nesbittiana comeaux were grouped with v. mustangensis and v. shuttleworthii in ba and ml but grouped with v. bloodworthiana and v. blancoii in mp and in the network.
to better understand clade support, we further investigated the synapomorphies defining clades of interest.
the characters supporting nodes of interest and their level of homoplasy and gene source were summarized based on mp .
summary statistics for specific nodes investigated are shown below the branches in figures  <dig> and  <dig>  many species showed good support with high bootstrap values and posterior probabilities, presence of node-specific characters and support from multiple genes : v. shuttleworthii , v. palmata , v.cinerea  , v. biformis , v. labrusca , v. californica , v. nesbittiana , v. girdiana , v. amurensis , v. qinlingensis , v. davidii  foëx, v. quinquangularis rehd.
, v. bashanica p.c.
he , v. hancockii hance , v. davidii var.
cyanocarpa  foëx , v. vinifera ssp.
sylvestris  hegi.
.
the species v. vulpina l.  and v. monticola  each formed reliable species-level clades even in the absence of a species-specific snp.
many higher level relationships were supported by a few characters and were of poor quality leading to the labile topology among major clades.
examples were the nodes defining the split between north america and asia , europe  and china , and china without v. amurensis .
these nodes had five to seven supporting characters that were frequently highly homoplasious .
the notable exception was the branch separating the muscadinia from subg.
vitis .
it was supported by  <dig> characters of which  <dig>  showed no homoplasy and represented  <dig> of the  <dig>  gene markers.
other supported higher clades were the v. cinerea-v. palmata clade  and the  clade .
both nodes had a node-specific snp, although bootstrap support and posterior probabilities were more consistent with other poorly supported higher-level clades.
the markers were informative in characterizing intra-specific variation in some species.
the mp branch lengths , mp and ml bootstrap supports and ba posterior probabilities  supported intraspecific groupings well with non-zero branch lengths present in many species such as v. shuttleworthii, v. monticola, v. californica, v. palmata, v. labrusca, v. cinerea, v. aestivalis, v. sylvestris, v. adstricta, and v. davidii.
due to space constraints these supports were not summarized in .
discussion
problems of phylogenetic study in vitis
based on mp, ml, and ba phylogenetic reconstruction methods, the nuclear dna dataset in this study had extensive variation to address genus-wide relationships in vitis.
the markers characterized intraspecific variation, defined most species, and strongly supported subg.
vitis.
however, many of the relationships and deeper nodes  were characterized by low bootstrap values and were often supported by few characters with high homoplasy.
low clade support and high homoplasy may be caused by insufficient data, parallel changes, reversals and convergences, as well as different histories of genes caused by lineage sorting and reticulation  <cit> , and different clade sizes  <cit> .
the ascertainment bias  introduced in the marker development phase may have selected markers with insufficient variation outside v. vinifera or phylogenetic depth, and differences may be distorted due to domestication.
however, we re-sequenced whole fragments, rather than genotyping a priori identified snps, thus included additional markers not restricted by our selection criteria of intermediate variability .
further, the deepest node was very well supported both in number and quality of characters, illustrating that there was sufficient phylogenetic signal available to define subg.
vitis.
similarly, several species  were well supported by multiple non-homoplasious characters.
thus, simple lack of data was not the definitive reason for poor support of deep nodes.
additional data, using the vitis9ksnp array  <cit>  or others in development, markers developed by lijavetzki et al.
<cit>  and vezzulli et al.
<cit>  or next generation sequencing  may resolve this phylogenetic problem.
however, these data will certainly add more noise .
in addition, ascertainment of homology in the data set created by ngs is very intractable because of complex paleopolyploidization and gene duplication in the grapevine genome  <cit> .
it is possible that additional data from non-recombining chloroplast or mitochondrial dna might add stable characters deeper within the tree.
however, the literature  <cit>  suggests that nucleotide substitution rates in these datasets may be too slow to add much intra-generic information.
species reticulation and incomplete lineage sorting would still present a challenge.
parallel changes, reversals, 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; these factors were further minimized by locus selection criteria.
ancestral polymorphism, reticulation and incomplete lineage sorting
homoplasy due to incomplete sorting of ancestral alleles is more likely when the time between lineage splitting is short  and the effective population size is large  <cit> .
the present estimates of divergence times showed that splitting events between the deeper clades occurred almost simultaneously within subg.
vitis.
myles et al.
<cit>  found significant degrees of shared polymorphisms between north american wild grapevine species and european cultivated species, suggesting that grapevine species maintained large effective population sizes since their geographic isolation millions of years ago.
further, the linkage disequilibrium in v. vinifera is very low and haplotype blocks are very short  <cit> , indicating significant historical recombination within the species  <cit> .
there was significant recombination in several genes and in the concatenated dataset.
thus, the conflict in the neighbornet  can be interpreted as evidence of shared ancestral polymorphisms mixed with reticulation and lineage sorting.
the shared ancestral polymorphisms may be the cause of the central knot of conflict  represented by the tight central mass of splits that represent incompatible and ambiguous signals in the data set  <cit> , with the radiating splits representing progressive lineage sorting and reticulation within the lineages.
reticulating events include hybridization, recombination and horizontal gene transfer  <cit> .
the first two were likely major factors in the evolution of vitis, while horizonatal gene transfer was an unlikely mechanism.
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.
time frame of vitis diversification
our molecular dating is close to fossil estimates and distributional inferences that place the origination of vitis into the paleogene.
the beginning of diversification among the extant taxa  in our tree was estimated at  <dig>  ma .
this was earlier than estimated by nie et al.
<cit>  who assessed it at about  <dig> ma with a large 95% hpd, or liu et al.
<cit>  who assessed it at about  <dig> ma , or zecca et al.
<cit>  with an estimated mean age of  <dig>  ma  or  <dig>  ma , depending on the details of their analyses.
estimates by all three  <cit>  were predominantly based on  chloroplast sequence with  <dig>   <dig>  and  <dig> parsimony informative sites respectively, two studies  <cit>  had one nuclear marker with  <dig> and  <dig> parsimony informative sites respectively, and each combined data set was < <dig> nt, compared to the ~ <dig>  nt of the present study.
recombination in the present data possibly caused the estimate of the height of the tree to be greater, thus resulting in more ancient age estimates  <cit> .
the use of the distant outgroup leea may have caused problems in dating because of missing data and multiple substitutions.
the inclusion of additional more closely related outgroups could improve the accuracy of the inferred dates  <cit> .
the present estimate of  <dig>  ma appears a reasonable age considering that the estimate was associated with a large ci that reached back to  <dig> ma and the oldest reliable vitis seed date to the paleocene   <cit> .
the stem of vitis did reach back that far .
the diversification of subg.
vitis  was presently estimated at  <dig>  ma , a node that was dated at  <dig>  or  <dig>  ma  as node b in zecca et al.
<cit> , about  <dig>  ma  by nie et al.
<cit>  and about  <dig> ma  by liu et al.
<cit> .
higher level intra-north american divergences fell between  <dig>  ma - <dig>  ma.
the divergence of eurasia from north and central america  was  <dig>  ma .
in zecca et al.
<cit>  this split coincided in timing with our node a. using sequence data of a single chloroplast gene  and no fossil calibrations, xiang et al.
<cit>  estimated the divergence time of  <dig> east asia-north american disjunct species, representing  <dig> diverse genera of flowering plants at an average of  <dig>  ma and ranging from  <dig>  ma to < <dig>  ma.
finally, we estimated  <dig>  ma to  <dig>  ma for higher level divergences among the extant taxa in asia, and  <dig>  ma  for the separation of europe and the near east from asia , a separation that was estimated by zecca et al.
<cit>  at  <dig>  ma or  <dig>  ma .
we estimated species-level divergences between  <dig>  ma  and  <dig>  ma  and intraspecific divergences shown more fully in figures  <dig> and  <dig> extended into more recent times.
overall, the timeframe estimated by the present study was more ancient than the estimates by other vitis-specific molecular studies .
1for nodes "higher level intra north america", "higher level intra asia, "species stem" the range contains several clades with different estimates, see figure  <dig> and additional file  <dig> for details, and the 95% hpd included multiple hdp associated with the several clades, see figure  <dig>  2one chloroplast gene,  <dig> diverse angiosperms;  <dig> nt ;  <dig> nt ; 5estimates from analyses that either constrained the stem  or the crown of subgenus vitis;  <dig> nt cpdna; <dig>  nt,  <dig> nuclear gene fragments; 8dash: information is not applicable; 9wikström et al.
<cit>  used a 3-gene data set, rate smoothing, and a single calibration point to estimate the stem age of vitaceae at  <dig> -  <dig> ma from which the prior distribution was derived; 10estimate from magallón and castillo  <cit>  because liu et al.
<cit>  agree with arguments made by nie et al.
<cit>  that the previous vitaceae stem age estimate  <cit>  was inaccurate; 11stem age of the vitis-ampelocissus clade, located in the paleocene to which the oldest reliable vitaceae fossils date, including ampelopsis, ampelocissus and vitis <cit> ; 12prior based on estimates by nie et al.
<cit> ; 13minimum age constraint based on fossil seeds described by gong et al.
<cit> ; 14based on fossil relatives of v. labrusca described by gong et al.
<cit> .
continental origin, dispersal and diversification of vitis
the phylogenetic relationships and network of grapevines reflect the northern hemisphere cenozoic history.
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, isolation, and differentiation.
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  <cit> .
however, péros et al.
<cit>  concluded that their analysis may support an asian origin of vitis.
fossils of vitaceae have been found frequently in western north american eocene deposits  and have not yet been found in southeastern localities  <cit> .
fossils of vitis seed were found in deposits of the rocky mountains and great plains of north america  <cit>  and in central europe  <cit> .
these findings assigned the oldest age of vitis to the paleocene .
at that time the supercontinent laurasia had only begun dividing into north america and eurasia  <cit>  and the climate was considerably warmer in the northern latitudes  <cit> .
these factors facilitated dispersal of warm-temperate terrestrial organisms in the northern hemisphere.
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  <cit>  all appeared earlier in the fossil record of north america than in that of asia  <cit> .
wen et al.
<cit>  found many more lineages with north american origins and migration to asia than vice versa <cit> .
nie et al.
<cit>  argue for a north american origin of ampelopsis .
molecular phylogenetic analyses of several disjunct genera suggested a progression from east asia to eastern and western north america  <cit> .
thus, the balance of grape-specific information tends to support our findings of a north american origin for the most recent common ancestor of vitis.
after fragmentation of a paleo/neogene range, our phylogenetic trees suggested isolation of some north american and asian species during the plio- and pleistocene cooling cycles, post glacial range expansions, and ecological adaptation.
much of the current eastern north american range of v. riparia, v. labrusca, v. aestivalis, and v. cinerea was unsuitable for vitis during the wisconsinan glaciations due to coverage by the polar ice sheet and harsh conditions along its southern edge .
these species must have expanded to their large present ranges after the glacial period.
large range expansions with post glacial warming were also promoted by the physiographic homogeneity of eastern north america  <cit> .
fragmentation and local adaptations were evident in physiographically heterogeneous western north america and temperate eastern asia.
the north american species v. shuttleworthii, v. nesbittiana, v. girdiana, v. palmata, v. bloodworthiana and v. blancoii have smaller ranges and multiple species-specific snp character changes.
similarly, physiographically diverse eastern asia  <cit>  had three species with multiple species-specific snps: v. bashanica, v. hancockii, v. quinquangularis.
local adaptations in heterogeneous environments likely lead to smaller population sizes and thus more rapid loss or fixation of novel characters  <cit> .
the underlying evolutionary scenario for vitis is consistent with origin in the eocene, a time of maximum development of temperate paleo/neogene forests.
this was followed by diversification in the mid-oligocene, the rise of subg.
vitis in the early miocene, the north american and asian disjunction in the late miocene, range restriction and fragmentation and speciation during the pliocene and pleistocene cooling cycles.
these caused the primary divisions within vitis as well as species-level and some intra-specific divisions  <cit> .
the north atlantic land bridge was present in the early paleogene  <cit>  and may have no longer existed when vitis arose, leaving beringia as the major route for potential gene flow.
the area of the bering and chukchi seas lay above sea level for most of the last  <dig> to  <dig> m years  <cit>  and was suitable for exchanges of temperate plants  <cit>  until the establishment of the bering seaway  <dig> - <dig> ma  <cit> , permitting genetic exchange at least until late miocene to which the disjunction was timed.
the pleiocene/pleistocene cooling cycles are well known to have caused range restrictions, survival in refugia, and diversifications in many groups of organisms  <cit> , both on land and in the sea.
this study shows clearly that vitis was also a part of this great biogeographic phenomenon.
phylogenetically-based vitis systematics
the systematics of vitis is a challenging area of taxonomy.
our findings confirmed the tenuous nature of many grapevine species and especially higher groupings such as series.
the apparent species-specific snps are good candidates to apply in species delineation investigations of grapes.
the present study found very low support for all series that included more than one species except for the munson/moore series precoces/ripariae .
other well supported higher-level groupings were subg.
vitis  and genus vitis, supporting the division of the genus vitis into two sections  <cit>  or subgenera  <cit> .
additional file  <dig> lists a synopsis of the major vitis classifications.
only galet  <cit>  assigned asian species to series.
the most comprehensive treatment of chinese vitis <cit>  did not apply a series-level classification.
most chinese species could be assigned to one series if series were to be used .
this may not include v. amurensis, v. coignetiae and v. yenshanensis as these species in some analyses grouped firmly within asia .
it appears as if v. jacquemontii should be assigned to the series viniferae.
however, our accessions had perfect flowers, suggesting past hybridization with v. vinifera.
the phylogenetic position intermediate between the asian and eurasian species and the well-defined split revealed in the network  supported this conclusion.
the derived position of v. sylvestris was unexpected.
v. sylvestris is the suggested progenitor of v. vinifera <cit>  while the phylogenetic position suggests that v. sylvestris was derived from v. vinifera .
this may be an artifact of the tenuous nature of most higher-level relationships revealed in this study.
it could also be a result of the nature of selection and clonal propagation that all v. vinifera cultivars included in the present study have been subjected to, some of them potentially for thousands of years  <cit> .
evolution is arrested by clonal propagation, leaving the naturally evolving wild species to appear more derived.
myles et al.
<cit>  concluded that current commercial v. vinifera varieties are only one or two generations removed from the wild v. sylvestris.
mullins et al.
<cit>  hypothesized asian/north american sister species pairs for v. coignetiae/v.
labrusca and for v. jaquemontii/v.
tiliifolia .
our results did not support sister pair relationships for v. coignetiae /v.
labrusca as these species placed solidly into well separated asian and north american clades, respectively.
our results are inconclusive with respect to the v. jacquemontii/v.
tiliifolia pair due to the possible hybrid nature of v. tiliifolia accessions in general and the dispersed positions of v. tiliifolia samples.
v. girdiana has been considered to be a variety of v. arizonica <cit> , a variety of v. californica <cit>  and its own separate species  <cit> .
our results preliminarily identified v. girdiana as a well supported independent species  with five species-specific snps.
more samples need to be investigated to assess the discriminatory power of these snps.
wada  <cit>  also identified a monophyletic v. girdiana cluster, although it had poor bootstrap support.
samples 080- <dig> came to us as v. cinerea  engelm.
ex millardet var.
floridana  but placed solidly into the v. aestivalis clade.
this highlighted confusion in the past related to the synonym vitis simpsonii that has been claimed for two different species as described in comeaux  <cit> , one belonging to aestivales and the other to cinerescentes.
the synonym v. rufotomentosa has the same problem.
our study showed conclusively that these accessions belong to v. aestivalis.
several additional accessions were identified as misnamed and others were recognized as hybrids .
finally, two accessions, 111v.
flexuosa dvit <dig> and 304v.
wilsoniae wangmaiputao, were of asian origin yet grouped with north american accessions and remain anomalies that could not be resolved.
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.
the genome-wide sampling of snps provided insight into the evolutionary history of the grape genus and supported previous notions of paleogene origins, range fragmentation, and recent nature of the species, joining vitis with the large group of organisms whose extant species differentiated in response to pliocene and quarternary climate change  <cit> .
we found that the most recent common ancestor of vitis was north american.
the major clades formed throughout the native distribution at 23- <dig> ma , suggesting that vicariance  in conjunction with local adaptation, 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.
many species were well supported, 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.
most higher-level relationships within the genus suffered from weak support.
the genus itself was extremely well supported.
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.
two of the most important forces, probably acting concurrently or alternating, are incomplete lineage sorting of ancestral polymorphism and reticulation.
broad reticulation across many species probably prevented the ancestral gene pool from diverging during the neogene forest stage, maintained reproductive compatibility, and is still acting today as evidenced by the prevalence of hybrids found in the wild and in repository collections.
however, climatic oscillations during the pliocene and quaternary, coupled with physiographic heterogeneity, provided enough recent barriers to gene flow to facilitate evolutionary divergence.
in light of the recency of divergence and diffuse genetic boundaries, higher-level taxonomic groupings, such as series, may be misleading.

 grapes 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 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 and east asia. up to species are native to a vast area in eastern asia, china, japan and java, two species across middle asia and europe, and up to species across the eastern and southwestern us and mexico. appendix expands discussion of the biogeographic background. the genus vitis is divided into two subgenera: muscadinia planch. and vitis planch.. 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 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. 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. this has led to many extraneous species names. 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. studies most similar in goals and pertinent to the present study were conducted by aradhya et al., nie et al., liu et al., and zecca et al.. aradhya et al. obtained a taxon sample similar to the present study and used ssr and aflp markers to study genetic diversity within vitis. these markers have limited value for phylogeny reconstruction and dating divergences was not attempted. nie et al. and liu et al. provided well-reasoned paleontological dates to estimate divergences in vitaceae. these calibration points were applied in the present study. zecca et al.’s chronogram is a tantalizing expansion of the vitis component of nie et al.’s chronogram. their inferences were limited by the small number of markers and the limited variability available in those markers, which did not fully resolve the tip clades. further, limited intra-specific replication limited the ability to make species-level inferences. adding more data can be useful for resolving difficult phylogenies that were based on a few genes. the present study attempted to improve on three aspects of previous phylogeographical studies of vitis by more extensive sampling of the nuclear genome, the species, and intraspecific variation. this study developed and used nuclear gene markers and sequenced accessions of vitis species, varieties, and four out-groups to: 1) reconstruct a phylogenetic hypothesis of the genus vitis, 2) date important time points in the evolution of vitis, 3) elucidate the biogeographic history of the genus, and 4) evaluate systematics of vitis within the framework of phylogeny.  molecular characteristics of the nuclear sequences of vitis most vitis accessions had complete sequence or had minimal missing data. indel sequences in the gene markers were unambiguous and easy to align. the starting alignment matrix for the gene markers and all accessions was bp long. gap coding for maximum parsimony added characters. amino acid coding sequence accounted for nt, 3’ or 5’ untranslated regions for nt, and introns for nt. because trees by new technology does not output the number of parsimony-informative characters, we report the number of unique site patterns from bayesian evolutionary analysis by sampling trees and randomized accelerated maximum likelihood. the 52-otu matrix had unique site patterns; the 273-otu matrix had unique site patterns. their distributions among the gene markers are listed in additional file under the uncorrelated log-normal relaxed molecular clock, estimated from three combined runs in beast and calibrated with three fossil dates, the mean rate of substitution in the data set was ×10- per million years  = 959) and a coefficient of variation of. dividing this rate by the size of the data set, the average rate of substitution in this data set was × 10- substitutions per site per million years. extent of reticulation and network in vitis ancestral polymorphism with subsequent lineage sorting is difficult to distinguish from reticulation based on a phylogenetic pattern. in our study, six gene fragments showed significant tests for recombination based on the phi test: fragments,,,,,. when these fragments were excluded, the concatenated matrix still showed recombination, as expected, representing independent assortment of markers. lanier and knowles found that, in species-tree estimation, the gain of accuracy from sampling additional loci and/or individuals always exceeded inaccuracies related to recombination. thus, in the present work no genes were excluded from subsequent analyses based on evidence of recombination. we used networks to better visualize the conflicts represented by the high levels of homoplasy. the consensus network of the individual gene trees indicated that few splits were common to multiple trees. the neighbornet of the concatenated 273-otu matrix showed extensive conflict at the core of subg. vitis representing the deeper nodes, and extensive conflict radiating outward. estimation of divergence times in vitis with the stem age of vitaceae constrained at  ±  ma, the stem age of vitis at  ±  ma, and the divergence of v. labrusca and its closely related north american relatives at  ±  ma, the crown age of vitis was estimated at ma, ), the crown age of subg. vitis at ma, and the stem ages of most species fell between and ma. the ten individual runs continued for million to million steps, and estimated the crown age of subg. vitis between ma and ma. the bayesian divergence times in figure  were estimated from three combined, unpartitioned runs using beast. the maximum clade credibility tree with mean estimates of divergence time for all nodes and associated posterior probabilities were also obtained. phylogenetic analyses in vitis maximum likelihood ml analyses of single genes analyzed independently without missing data yielded very poorly resolved trees. no significant conflict was observed under ml, thus the sequences were concatenated. the identical partitioned rapid ml runs using the 273-otu matrix produced a range of maximum likelihood values of -42511to - the final search on the same matrix following the bootstrap search yielded the highest likelihood value: - this tree was used in comparisons with results from other methods using a cartoon, i.e., a simplified version with collapsed terminal clades. bootstrap values are reported for selected nodes and all nodes. bayesian the two runs timed out at mil and mil generations with good effective sample size, but they did not converge on the exact same phylogenetic hypothesis. both trees were highly concordant for the species-level clades but differed on the specific placement of some clades. because the mil run had a higher mean log likelihood of the cold chain after burnin, the posterior probabilities from this run were used to summarize supports. overall comparisons of relationships above the species level were facilitated using the cartoon of this tree. maximum parsimony tnt’s driven search function produced most parsimonious trees of the same length in all four searches. the individual mp trees had a score of consistency index of, and retention index of. the strict consensus of all mp trees from all four searches had a score of ci = , and ri = . the strict consensus tree was simplified by collapsing terminal clades for comparisons. the full tree was annotated with support values on selected nodes of interest. node numbers are cross referenced with additional file all node numbers are shown in additional file the mp bootstrap tree with supports >50% was illustrated. ancestral area analysis fitch parsimony identified eastern/southeastern north america as the origin of the most recent common ancestor of vitis based on the strict consensus tree. systematics of vitis spp. it was evident that the patterns were similar among the cartoons of the strict mp consensus, the highest ml value cladogram and the bayesian cladogram. the clades of eurasian species were nested in north america as a monophyletic clade. the series precoces munson together with v. arizonica engelm., v. blancoii munson, v. bloodworthiana comeaux, v. xtreleasii munson ex l. h. bailey and v. girdiana munson formed the sister clade. v. labrusca and v. aestivalis michx. were grouped together as were v. cinerea engelm. ex millardet, v. palmata vahl, v. shuttleworthii house and v. mustangensis buckley. all three analyses did not group v. monticola buckley or v. californica benth. with other species. the position of several clades differed among the searches. this was a notable characteristic of this data set. among the different ba, ml and mp trees shown, the otu composition of species clades in general was quite consistent and well supported, but a few clade or species positions were inconsistent. correspondingly, clades above the species level were often poorly supported. for example, v. yenshanensis j. x. chen was grouped with v. amurensis rupr. and v. coignetiae pulliat ex planch. in mp and ba analysis and in the network but not in ml. similarly, the amurensis/coignetiae clade was basal to eurasia in mp and ba, but placed within the asian clade in ml and in the network. v. nesbittiana comeaux were grouped with v. mustangensis and v. shuttleworthii in ba and ml but grouped with v. bloodworthiana and v. blancoii in mp and in the network. to better understand clade support, we further investigated the synapomorphies defining clades of interest. the characters supporting nodes of interest and their level of homoplasy and gene source were summarized based on mp. summary statistics for specific nodes investigated are shown below the branches in figures  and many species showed good support with high bootstrap values and posterior probabilities, presence of node-specific characters and support from multiple genes: v. shuttleworthii, v. palmata, v.cinerea, v. biformis, v. labrusca, v. californica, v. nesbittiana, v. girdiana, v. amurensis, v. qinlingensis, v. davidii foëx, v. quinquangularis rehd., v. bashanica p.c. he, v. hancockii hance, v. davidii var. cyanocarpa foëx, v. vinifera ssp. sylvestris hegi.. the species v. vulpina l. and v. monticola each formed reliable species-level clades even in the absence of a species-specific snp. many higher level relationships were supported by a few characters and were of poor quality leading to the labile topology among major clades. examples were the nodes defining the split between north america and asia, europe and china, and china without v. amurensis. these nodes had five to seven supporting characters that were frequently highly homoplasious. the notable exception was the branch separating the muscadinia from subg. vitis. it was supported by characters of which showed no homoplasy and represented of the gene markers. other supported higher clades were the v. cinerea-v. palmata clade and the clade. both nodes had a node-specific snp, although bootstrap support and posterior probabilities were more consistent with other poorly supported higher-level clades. the markers were informative in characterizing intra-specific variation in some species. the mp branch lengths, mp and ml bootstrap supports and ba posterior probabilities supported intraspecific groupings well with non-zero branch lengths present in many species such as v. shuttleworthii, v. monticola, v. californica, v. palmata, v. labrusca, v. cinerea, v. aestivalis, v. sylvestris, v. adstricta, and v. davidii. due to space constraints these supports were not summarized in. discussion problems of phylogenetic study in vitis based on mp, ml, and ba phylogenetic reconstruction methods, the nuclear dna dataset in this study had extensive variation to address genus-wide relationships in vitis. the markers characterized intraspecific variation, defined most species, and strongly supported subg. vitis. however, many of the relationships and deeper nodes were characterized by low bootstrap values and were often supported by few characters with high homoplasy. low clade support and high homoplasy may be caused by insufficient data, parallel changes, reversals and convergences, as well as different histories of genes caused by lineage sorting and reticulation, and different clade sizes. the ascertainment bias introduced in the marker development phase may have selected markers with insufficient variation outside v. vinifera or phylogenetic depth, and differences may be distorted due to domestication. however, we re-sequenced whole fragments, rather than genotyping a priori identified snps, thus included additional markers not restricted by our selection criteria of intermediate variability. further, the deepest node was very well supported both in number and quality of characters, illustrating that there was sufficient phylogenetic signal available to define subg. vitis. similarly, several species were well supported by multiple non-homoplasious characters. thus, simple lack of data was not the definitive reason for poor support of deep nodes. additional data, using the vitis9ksnp array or others in development, markers developed by lijavetzki et al. and vezzulli et al. or next generation sequencing may resolve this phylogenetic problem. however, these data will certainly add more noise. in addition, ascertainment of homology in the data set created by ngs is very intractable because of complex paleopolyploidization and gene duplication in the grapevine genome. it is possible that additional data from non-recombining chloroplast or mitochondrial dna might add stable characters deeper within the tree. however, the literature suggests that nucleotide substitution rates in these datasets may be too slow to add much intra-generic information. species reticulation and incomplete lineage sorting would still present a challenge. parallel changes, reversals, 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; these factors were further minimized by locus selection criteria. ancestral polymorphism, reticulation and incomplete lineage sorting homoplasy due to incomplete sorting of ancestral alleles is more likely when the time between lineage splitting is short and the effective population size is large. the present estimates of divergence times showed that splitting events between the deeper clades occurred almost simultaneously within subg. vitis. myles et al. found significant degrees of shared polymorphisms between north american wild grapevine species and european cultivated species, suggesting that grapevine species maintained large effective population sizes since their geographic isolation millions of years ago. further, the linkage disequilibrium in v. vinifera is very low and haplotype blocks are very short, indicating significant historical recombination within the species. there was significant recombination in several genes and in the concatenated dataset. thus, the conflict in the neighbornet can be interpreted as evidence of shared ancestral polymorphisms mixed with reticulation and lineage sorting. the shared ancestral polymorphisms may be the cause of the central knot of conflict represented by the tight central mass of splits that represent incompatible and ambiguous signals in the data set, with the radiating splits representing progressive lineage sorting and reticulation within the lineages. reticulating events include hybridization, recombination and horizontal gene transfer. the first two were likely major factors in the evolution of vitis, while horizonatal gene transfer was an unlikely mechanism. 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. time frame of vitis diversification our molecular dating is close to fossil estimates and distributional inferences that place the origination of vitis into the paleogene. the beginning of diversification among the extant taxa in our tree was estimated at ma. this was earlier than estimated by nie et al. who assessed it at about ma with a large 95% hpd, or liu et al. who assessed it at about ma, or zecca et al. with an estimated mean age of ma or ma, depending on the details of their analyses. estimates by all three were predominantly based on chloroplast sequence with and parsimony informative sites respectively, two studies had one nuclear marker with and parsimony informative sites respectively, and each combined data set was < nt, compared to the ~ nt of the present study. recombination in the present data possibly caused the estimate of the height of the tree to be greater, thus resulting in more ancient age estimates. the use of the distant outgroup leea may have caused problems in dating because of missing data and multiple substitutions. the inclusion of additional more closely related outgroups could improve the accuracy of the inferred dates. the present estimate of ma appears a reasonable age considering that the estimate was associated with a large ci that reached back to ma and the oldest reliable vitis seed date to the paleocene. the stem of vitis did reach back that far. the diversification of subg. vitis was presently estimated at ma, a node that was dated at or ma as node b in zecca et al., about ma by nie et al. and about ma by liu et al.. higher level intra-north american divergences fell between ma - ma. the divergence of eurasia from north and central america was ma. in zecca et al. this split coincided in timing with our node a. using sequence data of a single chloroplast gene and no fossil calibrations, xiang et al. estimated the divergence time of east asia-north american disjunct species, representing diverse genera of flowering plants at an average of ma and ranging from ma to < ma. finally, we estimated ma to ma for higher level divergences among the extant taxa in asia, and ma for the separation of europe and the near east from asia, a separation that was estimated by zecca et al. at ma or ma. we estimated species-level divergences between ma and ma and intraspecific divergences shown more fully in figures  and extended into more recent times. overall, the timeframe estimated by the present study was more ancient than the estimates by other vitis-specific molecular studies. 1for nodes "higher level intra north america", "higher level intra asia, "species stem" the range contains several clades with different estimates, see figure  and additional file for details, and the 95% hpd included multiple hdp associated with the several clades, see figure  2one chloroplast gene, diverse angiosperms; nt; nt; 5estimates from analyses that either constrained the stem or the crown of subgenus vitis; nt cpdna; nt, nuclear gene fragments; 8dash: information is not applicable; 9wikström et al. used a 3-gene data set, rate smoothing, and a single calibration point to estimate the stem age of vitaceae at - ma from which the prior distribution was derived; 10estimate from magallón and castillo because liu et al. agree with arguments made by nie et al. that the previous vitaceae stem age estimate was inaccurate; 11stem age of the vitis-ampelocissus clade, located in the paleocene to which the oldest reliable vitaceae fossils date, including ampelopsis, ampelocissus and vitis; 12prior based on estimates by nie et al.; 13minimum age constraint based on fossil seeds described by gong et al.; 14based on fossil relatives of v. labrusca described by gong et al.. continental origin, dispersal and diversification of vitis the phylogenetic relationships and network of grapevines reflect the northern hemisphere cenozoic history. 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, isolation, and differentiation. 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. however, péros et al. concluded that their analysis may support an asian origin of vitis. fossils of vitaceae have been found frequently in western north american eocene deposits and have not yet been found in southeastern localities. fossils of vitis seed were found in deposits of the rocky mountains and great plains of north america and in central europe. these findings assigned the oldest age of vitis to the paleocene. 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. these factors facilitated dispersal of warm-temperate terrestrial organisms in the northern hemisphere. 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. wen et al. found many more lineages with north american origins and migration to asia than vice versa. nie et al. argue for a north american origin of ampelopsis. molecular phylogenetic analyses of several disjunct genera suggested a progression from east asia to eastern and western north america. thus, the balance of grape-specific information tends to support our findings of a north american origin for the most recent common ancestor of vitis. after fragmentation of a paleo/neogene range, our phylogenetic trees suggested isolation of some north american and asian species during the plio- and pleistocene cooling cycles, post glacial range expansions, and ecological adaptation. much of the current eastern north american range of v. riparia, v. labrusca, v. aestivalis, and v. cinerea was unsuitable for vitis during the wisconsinan glaciations due to coverage by the polar ice sheet and harsh conditions along its southern edge. these species must have expanded to their large present ranges after the glacial period. large range expansions with post glacial warming were also promoted by the physiographic homogeneity of eastern north america. fragmentation and local adaptations were evident in physiographically heterogeneous western north america and temperate eastern asia. the north american species v. shuttleworthii, v. nesbittiana, v. girdiana, v. palmata, v. bloodworthiana and v. blancoii have smaller ranges and multiple species-specific snp character changes. similarly, physiographically diverse eastern asia had three species with multiple species-specific snps: v. bashanica, v. hancockii, v. quinquangularis. local adaptations in heterogeneous environments likely lead to smaller population sizes and thus more rapid loss or fixation of novel characters. the underlying evolutionary scenario for vitis is consistent with origin in the eocene, a time of maximum development of temperate paleo/neogene forests. this was followed by diversification in the mid-oligocene, the rise of subg. vitis in the early miocene, the north american and asian disjunction in the late miocene, range restriction and fragmentation and speciation during the pliocene and pleistocene cooling cycles. these caused the primary divisions within vitis as well as species-level and some intra-specific divisions. the north atlantic land bridge was present in the early paleogene and may have no longer existed when vitis arose, leaving beringia as the major route for potential gene flow. the area of the bering and chukchi seas lay above sea level for most of the last to m years and was suitable for exchanges of temperate plants until the establishment of the bering seaway - ma, permitting genetic exchange at least until late miocene to which the disjunction was timed. the pleiocene/pleistocene cooling cycles are well known to have caused range restrictions, survival in refugia, and diversifications in many groups of organisms, both on land and in the sea. this study shows clearly that vitis was also a part of this great biogeographic phenomenon. phylogenetically-based vitis systematics the systematics of vitis is a challenging area of taxonomy. our findings confirmed the tenuous nature of many grapevine species and especially higher groupings such as series. the apparent species-specific snps are good candidates to apply in species delineation investigations of grapes. the present study found very low support for all series that included more than one species except for the munson/moore series precoces/ripariae. other well supported higher-level groupings were subg. vitis and genus vitis, supporting the division of the genus vitis into two sections or subgenera. additional file lists a synopsis of the major vitis classifications. only galet assigned asian species to series. the most comprehensive treatment of chinese vitis did not apply a series-level classification. most chinese species could be assigned to one series if series were to be used. this may not include v. amurensis, v. coignetiae and v. yenshanensis as these species in some analyses grouped firmly within asia. it appears as if v. jacquemontii should be assigned to the series viniferae. however, our accessions had perfect flowers, suggesting past hybridization with v. vinifera. the phylogenetic position intermediate between the asian and eurasian species and the well-defined split revealed in the network supported this conclusion. the derived position of v. sylvestris was unexpected. v. sylvestris is the suggested progenitor of v. vinifera while the phylogenetic position suggests that v. sylvestris was derived from v. vinifera. this may be an artifact of the tenuous nature of most higher-level relationships revealed in this study. it could also be a result of the nature of selection and clonal propagation that all v. vinifera cultivars included in the present study have been subjected to, some of them potentially for thousands of years. evolution is arrested by clonal propagation, leaving the naturally evolving wild species to appear more derived. myles et al. concluded that current commercial v. vinifera varieties are only one or two generations removed from the wild v. sylvestris. mullins et al. hypothesized asian/north american sister species pairs for v. coignetiae/v. labrusca and for v. jaquemontii/v. tiliifolia. our results did not support sister pair relationships for v. coignetiae /v. labrusca as these species placed solidly into well separated asian and north american clades, respectively. our results are inconclusive with respect to the v. jacquemontii/v. tiliifolia pair due to the possible hybrid nature of v. tiliifolia accessions in general and the dispersed positions of v. tiliifolia samples. v. girdiana has been considered to be a variety of v. arizonica, a variety of v. californica and its own separate species. our results preliminarily identified v. girdiana as a well supported independent species with five species-specific snps. more samples need to be investigated to assess the discriminatory power of these snps. wada also identified a monophyletic v. girdiana cluster, although it had poor bootstrap support. samples 080- came to us as v. cinerea engelm. ex millardet var. floridana but placed solidly into the v. aestivalis clade. this highlighted confusion in the past related to the synonym vitis simpsonii that has been claimed for two different species as described in comeaux, one belonging to aestivales and the other to cinerescentes. the synonym v. rufotomentosa has the same problem. our study showed conclusively that these accessions belong to v. aestivalis. several additional accessions were identified as misnamed and others were recognized as hybrids. finally, two accessions, 111v. flexuosa dvit and 304v. wilsoniae wangmaiputao, were of asian origin yet grouped with north american accessions and remain anomalies that could not be resolved.  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. the genome-wide sampling of snps provided insight into the evolutionary history of the grape genus and supported previous notions of paleogene origins, range fragmentation, and recent nature of the species, joining vitis with the large group of organisms whose extant species differentiated in response to pliocene and quarternary climate change. we found that the most recent common ancestor of vitis was north american. the major clades formed throughout the native distribution at 23- ma, suggesting that vicariance in conjunction with local adaptation, 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. many species were well supported, 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. most higher-level relationships within the genus suffered from weak support. the genus itself was extremely well supported. 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. two of the most important forces, probably acting concurrently or alternating, are incomplete lineage sorting of ancestral polymorphism and reticulation. broad reticulation across many species probably prevented the ancestral gene pool from diverging during the neogene forest stage, maintained reproductive compatibility, and is still acting today as evidenced by the prevalence of hybrids found in the wild and in repository collections. however, climatic oscillations during the pliocene and quaternary, coupled with physiographic heterogeneity, provided enough recent barriers to gene flow to facilitate evolutionary divergence. in light of the recency of divergence and diffuse genetic boundaries, higher-level taxonomic groupings, such as series, may be misleading. 
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. we selected accessions from vitis species,varieties, and outgroups, examined ~ kb of aligned nuclear dna sequences from unlinked genes in a phylogenetic context, and estimated divergence times based on fossil calibrations. nuclear snps effectively resolved relationships at and below the species level in grapes and rectified several misclassifications of accessions in the repositories. fitch parsimony identified north america as the origin of the most recent common ancestor of extant vitis species. higher clades in subgenus vitis diverged – ma with overlapping confidence intervals, and ongoing divergence formed extant species at – ma. neighbornet analysis showed extensive reticulation at the core of subgenus vitis representing the deeper nodes, with extensive reticulation radiating outward. as estimated from extant taxa, the crown age of vitis was ma and the divergence of subgenera occurred at ~ ma. the phylogenetic relationships among these species are of keen interest for the conservation and use of this germplasm. 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. there are about species in the genus vitis. there was substantial support for species and less for the higher-level groupings.
 grapes are one of the most economically important fruit crops. there are about 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 accessions from vitis species,varieties, and outgroups, examined ~ kb of aligned nuclear dna sequences from unlinked genes in a phylogenetic context, and estimated divergence times based on fossil calibrations.  vitis formed a strongly supported clade. there was substantial support for species and less for the higher-level groupings. as estimated from extant taxa, the crown age of vitis was ma and the divergence of subgenera occurred at ~ ma. higher clades in subgenus vitis diverged – ma with overlapping confidence intervals, and ongoing divergence formed extant species at – 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.  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. biogeographydivergence time estimategrapevinemolecular phylogenynetworknorthern hemisphereplant disjunctionreticulationsnpvitis 