Poecile gambeli). Analysis of nuclear data identified two geographically-structured genetic groups, largely concordant with results from a previous mitochondrial DNA (mtDNA) study. Additionally, the estimated multilocus divergence times indicate a Pleistocene divergence, and are highly concordant with mtDNA. The previous mtDNA study showed a paucity of sympatry between clades, while nuclear patterns of gene flow show highly varied patterns between populations. The observed pattern of gene flow, from coalescent-based analyses, indicates southern populations in both clades exhibit little gene flow within or between clades, while northern populations are experiencing higher gene flow within and between clades. If this pattern were to persist, it is possible the historical legacy of Pleistocene divergence may be preserved in the southern populations only, and the northern populations would become a genetically diverse hybrid species.The Pleistocene glacial cycles left a genetic legacy on taxa throughout the world; however, the persistence of genetic lineages that diverged during these cycles is dependent upon levels of gene flow and introgression. The consequences of secondary contact among taxa may reveal new insights into the history of the Pleistocene’s genetic legacy. Here, we use phylogeographic methods, using 20 nuclear loci from regional populations, to infer the consequences of secondary contact following divergence in the Mountain Chickadee ( The Pleistocene speciation hypothesis posits that isolation of populations into separate refugia through glacial and interglacial periods was a principal driver of speciation in temperate-adapted taxa In western North America, the genetic footprint from the Pleistocene shows consistent broad scale biogeographic patterns, where forest-dwelling taxa exhibit phylogenetic splits between Rocky Mountain (RM) and pacific  regions, including both intra- and interspecific splits in birds Poecile gambeli, Dedragapus obscurus) to somewhat widespread . Because all of the aforementioned studies used mitochondrial markers, overlap of clades may indicate hybridization between distinct taxonomic units or sympatry/parapatry of non-interbreeding taxa. In birds (as well as other vertebrates), hybridization following secondary contact is common PheucticusPasserinaVermivora chrysoptera and V. pinusDendroica auduboniPasser italiaeSpecies in western North America that have RM and PAC groups show mixed levels of overlap between phylogenetic groups, ranging from local (e.g. Poecile gambeli).Although it is clear that the Pleistocene glacial cycles left a genetic “legacy” on taxa throughout the world abbreviatus (Sierra Nevada and Cascade Mountains populations), gambeli (Rocky Mountains) and inyoensis (Great Basin). A recent mitochondrial DNA (mtDNA) phylogeographic study gambeli and inyoensis) and a western clade (concordant with the geographic range of subspecies abbreviatus). The two clades are strongly geographically structured, and sampling indicated only a single, local point of contact between clades  sequenced 20 nuclear loci from the 3 morphological groups defined by The collection of all animals was conducted under the permission of applicable federal and state agencies. Permits were granted by the following institutions to J.K. and/or G.M.S.: U.S. Department of Fish and Wildlife, Arizona Game and Fish Department, California Department of Fish and Game, Colorado Division of Wildlife, Idaho Fish and Game, Nevada Department of Wildlife and the British Columbia Ministry of Forests, Lands, and Natural Resource Operations. Import and export permits were granted from the United States Department of Agriculture. Birds were collected using shotguns and frozen in the field until museum specimens could be prepared and tissues subsampled and cryogenically stored. Specimen collection was performed following review of the Institutional Animal Care and Use Committee (IACUC) at Black Hills State University (BHSU A-08-001 to GMS). Tissue samples of the specimens from the Museum of Vertebrate Zoology were obtained in accordance with the museum’s loan policy.Taeniopygia guttata) genome, or contained repetitive elements. Following these exclusions, 29 sequences were used for developing primers. Of those 29 sequences and developed primers, 14 could be broadly amplified across individuals using polymerase chain reaction (PCR) and were subsequently used in this study (See To reduce ascertainment bias in choosing genetic markers Poecile atricapillus) was used as an outgroup taxon for genetic structure and phylogenetic analyses.Tissue samples of 60 Mountain Chickadee individuals were obtained from eight populations ; howeveThe number of populations (K) estimated from the STRUCTURE analysis was k = 2  range from 0.0033 to 0.0044 between populations and have no correlation with direct geographic distance between populations (R2 = 0.0004).Summaries of genetic diversity, Tajima’s D neutrality index, and mismatch distributions are presented in IMa results are summarized in Effective migration (2 Nm) was, in general, low 2 Nm <2;  between ENMs of contemporary and LGM conditions are shown in Contemporary ENMs have little overlap between lineages (Schoener’s D = 0.132), while LGM ENMs have relatively higher overlap (D = 0.226). The models with an applied threshold identify two regions of overlap between models: 1) in Mono County, California (population with mixed haplotypes in mtDNA study), and 2) near the Siskiyou County, California population sampled in this study, the population with the weakest nuclear genetic structure. Through glacial cycles, the RM lineage has a high degree of geographic displacement (D = 0.248) while the PAC lineage is more stable (D = 0.565). Size of potential niche space is more stable in the RM lineage (contemporary/LGM space = 1.114) than the PAC lineage (0.257) through time.Certhia americana  from the northern California population. This time period corresponds with the isolation of southern California mountain ranges by the expansion of the Sonoran and Mohave desert vegetation Following divergence in the Pleistocene, the two main lineages of Mountain Chickadee have experienced ongoing gene flow The observed pattern of gene flow among populations within and between clades creates a unique pattern of population connectivity. Populations of Mountain Chickadee from the southern extremes of the species range  are not exchanging genes with populations from different clades or populations from the same clade. Alternatively, northern populations appear to be exchanging genes with populations from both clades, which may indicate an unsampled hybrid zone between lineages of the Mountain Chickadee. If this pattern were to persist, it is possible the historical legacy of Pleistocene divergence may be preserved in the southern populations only and the northern populations would become a genetically diverse hybrid species The frequency of gene flow between populations from the two clades was somewhat surprising given the paucity of sympatry observed in the mtDNA data Calocitta FormosaPerisoreus infaustusST values in the range of the autosomal markers. Because of these results, although only based on two loci, we feel male-mediated gene flow is unlikely between lineages of Poecile gambeli.Male-mediated gene flow has been documented in birds; however, in general, males tend to be more philopatric than females Alternatively, nuclear gene flow with a lack of mtDNA introgession may be observed because of reduced “hybrid” fitness in the females  This study brings further evidence to the consequences of Pleistocene effects on biogeographic patterns of birds. Two major reviews attributed the majority of avian intraspecific phylogeographic breaks to Pleistocene events Supporting Info S1File including all supplementary tables and figures.(PDF)Click here for additional data file.Sequences S1Zip file of sequences too short for GenBank.(ZIP)Click here for additional data file.


Coffea arabica and the origin of all Arabica coffee cultivated worldwide. This wild gene pool is potentially threatened by forest fragmentation and degradation, and by introgressive hybridization with locally improved coffee varieties. We genotyped 703 coffee shrubs from unmanaged and managed coffee populations, using 24 microsatellite loci. Additionally, we genotyped 90 individuals representing 23 Ethiopian cultivars resistant to coffee berry disease (CBD). We determined population genetic diversity, genetic structure, and admixture of cultivar alleles in the in situ gene pool. We found strong genetic differentiation between managed and unmanaged coffee populations, but without significant differences in within-population genetic diversity. The widespread planting of coffee seedlings including CBD-resistant cultivars most likely offsets losses of genetic variation attributable to genetic drift and inbreeding. Mixing cultivars with original coffee genotypes, however, leaves ample opportunity for hybridization and replacement of the original coffee gene pool, which already shows signs of admixture. In situ conservation of the wild gene pool of C. arabica must therefore focus on limiting coffee production in the remaining wild populations, as intensification threatens the genetic integrity of the gene pool by exposing wild genotypes to cultivars.The montane rainforests of SW Ethiopia are the primary centre of diversity of  First, as elsewhere in the tropics  Is there genetic erosion of the wild arabica gene pool in fragmented forests managed for coffee production, compared to continuous nonmanaged forests? (ii) Is the introduction of CBD-resistant genotypes posing a threat to the integrity of the wild arabica gene pool? The answers to these questions should help the in situ conservation of arabica coffee genetic resources.The general aim of this study was to provide the first thorough assessment of population genetic diversity within the wild gene pool of Coffea arabica L. (family Rubiaceae) is the only Coffea species occurring in Ethiopia and is geographically isolated from all other species in the genus  ha−1 in the FC system compared to 18 500 individuals ha−1 in the SFC system , formed by relatively recent natural hybridization between C. canophora and C. eugenioides . Genomic DNA was extracted from 20 mg homogenized leaf material using the NucleoSpin® Plant II kit , with slight modifications of the standard CTAB protocol .® PCR System 9700 thermal cycler  and a total sample volume of 10 μL containing 5 μL Qiagen® Multiplex PCR Master Mix , 2 μL sample/template DNA and 0.2 μL of each primer  in the multiplex combination complemented with RNase-free Milli-Q water. The multiplexes had equal thermocycling profiles with an initial Taq DNA polymerase heat-activation step at 95°C for 15 min; 25 cycles of 30 s at 94°C (denaturation step), 90 s at 57°C  and 60 s at 72°C (extension step); and a final extension step of 30 min at 60°C. Then, 1 μL of the PCR was added to a solution of 8.8 μL formamide and 0.2 μL of the Applied Biosystems GeneScan™ 500 LIZ® size standard. Sized fragments were scored using GeneMapper® v4.0 (Applied Biosystems).Twenty-four simple sequence repeats  were amplified in six multiplex PCRs  using a Coffea arabica genome limits the flexibility of the data analysis. We adopted two parallel approaches, one based on the codominantly scored data and allowing allele copy number ambiguity, and a second based on the scoring of each individual allele as present or absent, resulting in a dominantly scored data set comparable to the output of an amplified fragment length polymorphism (AFLP) marker approach. We used the R package polysat (Clark and Jasieniuk ® software and converting the data. To assess the resolution of the microsatellite marker set we discriminated distinct multilocus genotypes (MLGs).The allotetraploid nature of the HE,C), and the molecular variance (MV) based on the within-population sum of squares (SSWP) and calculated as SSWP×(n − 1)−1. Among-population genetic differentiation (ΦPT) was calculated based on Euclidian genetic distances  approach on the dominantly scored data set as performed in GenAlEx 6.41  as the regional grouping variable. Genetic differentiation was further assessed using principal coordinates analyses (PCoA) calculated in GenAlEx on the pairwise ΦPT matrix. Effects of coffee forest management intensity (FC vs SFC) on population genetic diversity were analysed using Wilcoxon–Mann–Whitney U-tests. To test whether the mean genetic diversities recorded in FC and SFC populations differed from the diversity in the cultivar population, we used one-sample t-tests. Finally, also the average pairwise genetic differentiation (ΦPT) between populations in FC stands was compared with the differentiation between populations in SFC stands, using a Wilcoxon–Mann–Whitney U test. Statistical analyses were performed in SPSS 15.0 .Population genetic diversity was quantified using the expected heterozygosity corrected for sample size  iterations. Correlated allele frequencies and admixture were assumed. The degree of admixture was inferred from the data using an initial value of α = 1.0 and a maximum of 10. The value of K that best fitted our data was selected using the estimated log probability of data Pr(X|K) and the derived ΔK statistic  and the molecular variance (MV) did not differ significantly between FC  and SFC populations  . The sample of CBD-resistant varieties was genetically more diverse  than both the FC and SFC populations, but this difference was only significant for HE,C . Overall among-population genetic differentiation was high , with a genetic differentiation of 0.033 (ΦRT) between production systems  than for the FC  populations . Populations from FC and SFC clustered at opposite ends in the PCoA with some overlap among clusters   .K but was less pronounced when K > 2. This, together with the fact that the ΔK statistic reached its maximum at K = 2  . These results indicate that alleles from the cultivar gene pool are more prevalent in the SFC than in the FC populations, and that most individuals in the SFC populations have more alleles from the cultivar gene pool than alleles from the wild gene pool in their genome.The admixture coefficients or hybrid indices varied between 0 (pure ‘wild’) and 1 (pure ‘cultivar’). In the SFC populations, hybrid indices were predominantly high, with  samples . The meaHE,C, MV). This was surprising because negative effects on effective population size and genetic diversity of C. arabica populations could have been expected. Changes in forest microclimate and in availability of nesting sites for insect pollinators, for instance, may have negatively affected pollinator diversity  among SFC populations than among FC populations. The conspicuous presence of alleles from the pool of introduced CBD-resistant genotypes in almost all SFC populations , this higher genetic differentiation is unlikely to be caused through isolation-by-distance or by greater environmental variation among SFC populations. Second, gene flow through pollen or seed between SFC populations may have been impeded due to the more fragmented nature of the agricultural landscape near Jimma town, increasing the among-population genetic differentiation. If the latter was the case, however, we expect genetic differentiation to be paralleled by loss of genetic diversity through genetic drift, which was not observed in the SFC populations.The introduction of genetically diverse coffee genotypes from elsewhere into SFC populations by coffee farmers most likely represents a farmer-mediated evolutionary force. This is supported by the higher genetic differentiation  and cotton (Gossypium hirsutum) , which becomes assimilated into the cultivar. This process has already increased the risk of extinction of the wild relatives of two of the world's 13 most important crops, rice  in SFC populations. Although we cannot provide direct evidence for hybridization and introgression, the practice of large scale planting of CBD-resistant genotypes by local farmers in SFC systems, intimately mixed with original coffee genotypes, offers ample opportunity for the exchange of alleles and the replacement of the original coffee gene pool.in situ conservation of arabica coffee genetic resources in Ethiopia, we recommend to: (i) avoid establishing plantations with foreign coffee cultivars in the centre of origin of C. arabica; (ii) allow only the use of Ethiopian cultivars that did not undergo a very strict process of selection in the SFC systems; and (iii) protect a sufficiently large area of low-intensity FC systems. The latter can probably only be realized by establishing buffer zones of SFC surrounding more strict reserves of FC in the last remaining large forest blocks in the region.Only the few remaining large forests with a FC cultivation system seem to be safe from the introduction of CBD-resistant cultivars so far. To ensure the Data for this study are available at Dryad. DOI: 10.5061/dryad.37r9p.


ST = 0.0005 between provinces) was stronger than that in many Central European populations, although smaller than what has been observed for instance in Finland; importantly, it is of the magnitude that may hamper association studies with a moderate number of markers if cases and controls are not properly matched geographically. Overall, our results underline the potential of genome-wide data in analyzing substructure in populations that might otherwise appear relatively homogeneous, such as the Swedes.The use of genome-wide single nucleotide polymorphism (SNP) data has recently proven useful in the study of human population structure. We have studied the internal genetic structure of the Swedish population using more than 350,000 SNPs from 1525 Swedes from all over the country genotyped on the Illumina HumanHap550 array. We have also compared them to 3212 worldwide reference samples, including Finns, northern Germans, British and Russians, based on the more than 29,000 SNPs that overlap between the Illumina and Affymetrix 250K Sty arrays. The Swedes - especially southern Swedes - were genetically close to the Germans and British, while their genetic distance to Finns was substantially longer. The overall structure within Sweden appeared clinal, and the substructure in the southern and middle parts was subtle. In contrast, the northern part of Sweden, Norrland, exhibited pronounced genetic differences both within the area and relative to the rest of the country. These distinctive genetic features of Norrland probably result mainly from isolation by distance and genetic drift caused by low population density. The internal structure within Sweden (F The recent flood of genome-wide association studies (GWAS) for common diseases has created an upsurge also in studies of population structure based on genome-wide autosomal single nucleotide polymorphism (SNP) array data. This is not only due to the availability of these novel datasets but also due to an increased interest into population structure as a potential confounding factor in the association studies. As a result, this new type of data has already complemented the ones classically used in population genetics. Several studies have shown a general correspondence between genetic and geographic distances within Europe The first inhabitants to the area of present-day Sweden came after the ice age from Central Europe. For millennia, the country was sparsely inhabited by hunter-gatherer populations until the slow adoption of agriculture and ceramics that began around 4000 BC in southern Sweden Genetically the Swedes have appeared relatively similar to their neighboring populations - for example the Norwegians, Danish, Germans, Dutch and British - both in a classical study based on a small number of autosomal markers The internal genetic structure of the Swedish population has been mostly studied with the Y chromosome and mtDNA. These studies have shown haplogroup frequency differences within the country In this study, we have analyzed the current autosomal population structure within Sweden using 1525 individuals genotyped on the Illumina HumanHap550 SNP array, and compared the Swedes also to Finns, Germans, Russians and other reference populations. We observed that the Southern Swedes were genetically close to northern Central Europeans and exhibited subtle genetic substructure, whereas the northern part of Sweden, Norrland, clearly differed from the rest of the country and showed significant internal structure.We used genome-wide SNP genotypes of 1525 Swedes and 3212 worldwide reference individuals to study the autosomal population structure within Sweden and relative to neighboring populations  showed cST distances between European populations  but not within Svealand (p = 0.19). .) When FST was analyzed between the three regions and the provinces simultanously, differences both among the regions (FST = 0.0004) and among the provinces within the regions (FST = 0.0003) were significant (p<0.0002 for both). Pairwise FST values between the Swedish provinces , and by limiting the analyses to the SNPs that have been genotyped in all the populations, instead of using imputed data.ST = 0.0012) is likely caused by a difference in the geographical sampling distributions. Notably, the datasets also behaved rather differently in the FST comparisons to Central Europeans. This nicely illustrates how differences in geographical sampling between studies could affect quite a lot the way that Swedes appear in comparisons with other populations.An issue of bigger concern are the effects of the sampling scheme, both in terms of ancestry ascertainment and geographic distribution. For instance, although we observed a much more subtle internal structure in Sweden than in Finland, it is difficult to estimate to which degree the difference was caused by the differential ascertainment . Nevertheless, the substructure within Sweden was significant, stronger than between Northern and Southern Germany ST analyses. Furthermore, LD in Norrland was stronger than in the rest of Sweden. Together, these patterns of variation could be partly explained by migration. While the influence from Finland seemed moderate, at the most, we unfortunately lacked the reference samples necessary for analyzing possible Sami and Norwegian contributions. However, earlier mitochondrial DNA and Y-chromosomal studies have indicated influence from the Sami and/or Finns in Northern Sweden, as well as decreased genetic diversity The northern part of Sweden, Norrland, showed a particularly strong population structure, which could be explained by genetic drift in this sparsely inhabited region. However, this hypothesis was challenged by the genetic diversity within Norrland that was not consistently reduced: in fact, Norrland showed significantly lower IBS similarity than Götaland. This could suggest the presence of several isolates within Norrland, and indeed, signs of such were detected in the MDS and FInterestingly, the Finnish province genetically closest to Norrland was not the neighboring Northern Ostrobothnia, but the Swedish-speaking Ostrobothnia and Southwestern Finland hundreds of kilometers further south. Although this pattern might first seem surprising, it is consistent with the history of Northern Ostrobothnia, where the current population is largely derived from a 16th-century migration that originated from the province of Southern Savo Among our Finnish sample, genetically closest to Swedes were the Swedish-speaking Finns of coastal Ostrobothnia. This agrees well with the history of the Swedish-speakers, who arrived into the western and southern coastal areas of Finland in the beginning of the second millennium In our earlier study Several studies have now shown a general correspondence between geographic and autosomal genetic distances between European populations Population substructure can be a crucial issue in association studies, where population differences between cases and controls can cause spurious association signals. In GWAS, it is possible to correct for population stratification by using the bulk of data that is not assumed to correlate with the phenotype of interest, but in replication or candidate gene-based association studies that involve a more limited number of markers, such corrections are not possible. The amount of allele frequency differences we detected within Sweden warrants caution when matching controls for cases geographically, especially if individuals with descent from the northern part of Sweden are involved: for example in a study with cases from Norrland and controls from Götaland, a random SNP would have a substantially inflated chance of showing a chi-square p<0.05 due to the population structure alone - even in our moderately sized dataset of less than 1000 individuals, the chance was 13%. As the observed structure within Sweden is mostly caused by random forces such as drift, the differing SNPs are scattered throughout the genome, and there is no means of recognizing them without prior population data. Thus, especially with phenotypes where cases are likely to be geographically clustered, rigorous matching of controls may be needed in order to avoid effects of stratification.Genome-wide SNP datasets are quickly proving their usefulness in population genetic studies. Firstly, such datasets greatly increase the number of available loci, and they can therefore yield a more balanced picture of the diverse aspects of a population's history than for instance the uniparental markers that comprise only two loci. Secondly, the large number of individuals typically involved in a GWAS improves the resolution of population genetic analyses. Admittedly, GWAS control individuals can lack detailed ancestry information or might not represent populations with particularly interesting ancestry, which may limit their utility for population history studies. Nevertheless, studies such as ours that are based on residence information can uncover the patterns of the current population structure, which are often more important for practical applications, and still provide novel information of the population history in high precision.All the samples were analyzed anonymously. The Swedish, Finnish and German samples were collected with informed consent according to the principles of the Declaration of Helsinki, and their use for population genetics study was approved by the ethics committees of the Karolinska Institute, Umeå University, Finnish Red Cross, and the Kiel Medical Faculty. For details of the collection of the British and worldwide samples, see the references below.http://www.stanford.edu/group/morrinst/hgdp.html) http://hapmap.ncbi.nlm.nih.gov) We used genome-wide SNP genotypes from altogether 1642 Swedes, 280 Finns, 492 Germans, 740 British, and 1587 worldwide population samples to study the genetic structure in Northern Europe, especially within Sweden. The Swedes consisted of 774 case and 755 control females from a breast cancer study http://pngu.mgh.harvard.edu/purcell/plink/) −7 for each population  and minor allele frequency (MAF) 0.05 for the whole dataset. In 119 pairs of individuals with high identity by state (IBS) similarity, the individual with lower genotyping success was excluded; additionally, the whole MKK population of HapMap was excluded due to multiple cases of high IBS relatedness. In Dataset 2, also the Affymetrix genotypes  were added. Only the markers overlapping between the source datasets were used; no imputation was done. QC thresholds were the same as for Dataset 1, except for individual success 97% for the Affymetrix individuals. Nine SNPs were removed due to highly discordant frequencies (chi-square p<10−10) between comparable parts of the Affymetrix and Illumina datasets . Dataset 3 consists of the Swedish individuals of Dataset 1 supplemented by Swedish cases (774 cases and 751 controls). In addition to the same QC procedure as for Dataset 1, 41868 SNPs with p<0.1 in a chi-square test of allele frequencies between cases and controls were excluded; The genotypes used in this study originated from various platforms: the Swedish females from Illumina HumanHap550 ; the Swedish males, the Finns, the Germans and the British from Affymetrix 250K StyI ; the HGDP samples from Illumina HumanHap650K; and the HapMap samples from Affymetrix 6.0 and Illumina 1M. DNA extraction, genotyping and genotype calling had been done for the datasets separately according to manufacturers' instructions and is described in more detail in the references given above. Quality control (QC) filtering and LD-based pruning were done using Plink version 1.06 (www.geonames.org) based on both their postal code and county information, and confirmed from Google Maps in case of disparity. Coordinates for the Finnish individuals were based on the grandparental birthplaces, and a mean of these was used as a coordinate for the individual.Coordinates for the Swedish individuals were obtained from GeoNames  of Sweden . Some prST calculations were done in Arlequin 3.11 www.R-project.org) ST were visualized by classical multidimensional scaling (MDS) in R. The proportion of variance explained by each MDS dimension was calculated as the ratio of the respective eigenvalue to the sum of all eigenvalues The Fhttp://www.pantherdb.org) http://www.genome.gov/gwastudies) Allele frequency differences in pairs of populations were calculated by a chi-square test (1 degree of freedom) in Plink, and the general degree of inflation (λ) was calculated as the ratio of the means of the smallest 90% of observed vs. expected chi-square test statistics as in ST) on the geographic distance (between provinces' average sample locations). In allele frequency difference and LD analyses all populations were sampled to 115 individuals to avoid the effects of sample size. The reported significance levels for the IBS distributions, LD and inbreeding have been Bonferroni-corrected for the number of tests done within each analysis.Individuals were clustered with Structure 2.2 software Figure S1Multidimensional scaling plots of genetic distances between individuals in three dimensions. Identity by state distances in Northern Europe (left), and Sweden and Finland (right). The proportions of variance explained by the three axes are 0.64%, 0.24%, and 0.17% in Northern Europe and 1.04%, 0.26%, and 0.24% in Sweden and Finland, respectively. The animation files can be opened e.g. in most internet browsers. Abbreviations as in (GIF)Click here for additional data file.Figure S2The probabilities of different numbers of clusters (K) in the Structure analysis of Northern Europe.(TIF)Click here for additional data file.Figure S3ST distances visualized by multidimensional scaling.F Pairwise distances between European populations (a), Swedish and Finnish provinces (b), and Swedish provinces (c). The corresponding FST values can be found in (TIF)Click here for additional data file.Figure S4Distributions of pairwise identities by state (IBS). IBS distributions between six populations and Russians (a) and within the six populations (b). The location of distribution medians is denoted by triangles of corresponding color. In (a), Western Finland differed significantly from all other populations except Germany, and Germany from Norrland and Eastern Finland (p < 0.031 after a Bonferroni correction). In (b), Eastern Finland differed significantly from all other populations, Norrland from Götaland and Western Finland, and Western Finland from Svealand (p < 0.034 after a Bonferroni correction). Abbreviations as in (TIF)Click here for additional data file.Figure S5Multidimensional scaling plots of genetic distances between individuals in Svealand and Götaland. The genetic distance used is based on identity by state (IBS). Abbreviations as in (TIF)Click here for additional data file.Figure S6Clustering of Swedish individuals by the Structure software. Each individual is represented by a thin vertical line, and its proportional ancestry in the two inferred clusters is denoted by colors. The individuals are sorted according to latitude from south to north.(TIF)Click here for additional data file.Figure S7Six zones of strong genetic change inferred by the Barrier software. The borders (in red) are numbered in decreasing order of strength, and they are based on FST distances that have been corrected for the geographical distance between provinces. Note that the FST values differ significantly from zero only for the first two borders.(TIF)Click here for additional data file.Figure S8Linkage disequilibrium as a function of distance between markers. Median D' in overlapping 40 kb windows at 10 kb intervals is plotted for each population using 67620 marker pairs. All distributions differed significantly (p < 0.002) except Germany vs. Great Britain and Svealand vs. Götaland. Abbreviations as in (TIF)Click here for additional data file.Figure S9Genomic locations of SNPs whose allele frequency differs in Norrland. The p value from a chi-square test of allele frequencies between Norrland (n  =  115) and the rest of Sweden (n  =  635) is indicated for each SNP. The most differing SNPs are listed in (TIF)Click here for additional data file.Figure S10Differences between cases and controls in Dataset 3. A quantile-quantile plot of observed vs. expected test statistics (in blue) from a chi-square test of allele frequency differences between cases and controls for the SNPs that remain in Dataset 3 after quality control. Lambda denotes the overdispersion factor of observed vs. expected chi-square statistics.(TIF)Click here for additional data file.Table S1Names, abbreviations and sample sizes for the Swedish and Finnish provinces.(XLS)Click here for additional data file.Table S2ST values  between European populations.Pairwise F(XLS)Click here for additional data file.Table S3ST values  between Swedish and Finnish provinces.Pairwise F(XLS)Click here for additional data file.Table S4ST values  between Swedish provinces.Pairwise F(XLS)Click here for additional data file.Table S5Number of SNPs with geographical differentiation within Sweden located in genome areas associated with eight phenotypes.(XLS)Click here for additional data file.Table S6The SNPs with largest allele frequency difference between Norrland and the rest of Sweden, and their nearest genes (within 200 kb).(XLS)Click here for additional data file.Table S7−3).A list of the SNPs whose frequency differs between Norrland and the rest of Sweden (chi-square p<10(XLS)Click here for additional data file.Table S8The numbers of SNPs and individuals in the different datasets after quality control.(XLS)Click here for additional data file.Table S9The datasets used in each analysis.(XLS)Click here for additional data file.


Calotes versicolor) is one of the few non-gekkonid lizards that are geographically widespread in the tropics. We investigated its population dynamics on Hainan Island and the adjacent mainland of China and Vietnam, focusing on the impact of cyclic upheaval and submergence of land bridges during the Pleistocene. Our Bayesian phylogenetic analysis reveals two mitochondrial lineages, A and B, which are estimated to have coalesced about 0.26 million years ago . Lineage A contains individuals mainly from central and southern Wuzhi Mountain on Hainan Island, whereas lineage B mainly comprises individuals from other sites on the island plus the adjacent mainland. The estimated coalescence times within lineages A (0.05 million years ago) and B (0.13 million years ago) fall within a period of cyclical land-bridge formation and disappearance in the Pleistocene. A spatial analysis of molecular variance identified two distinct population groupings: I, primarily containing lineage A, and II, mainly consisting of lineage B. However, haplotypes from lineages A and B occur sympatrically, suggesting that gene flow is ongoing. Neither Wuzhi Mountain nor Qiongzhou Strait and Gulf of Tonkin act as barriers to gene flow among C. versicolor populations. Analyses of the data using mismatch distributions and extended Bayesian skyline plots provide evidence of a relatively stable population size through time for Group I, and moderate population expansions and contractions during the end of the Pleistocene for Group II. We conclude that the phylogeographical patterns of C. versicolor are the combined product of Pleistocene sea-level oscillations and nonphysical barriers to gene flow.The Oriental garden lizard ( The geographical distributions and genetic structure of organisms in the present have been shaped by factors such as climatic oscillations and geological events during the Pleistocene Significant geological events, such as the formation of mountain ranges, are often treated as a source of long-term biogeographical barriers when inferring present or past patterns of gene flow Island systems can provide compelling environments for evolutionary studies of species Hainan Island is characterized by two zoogeographical regions, the mountain provinces and plain provinces, which are defined by altitude, climate, vegetation, and distribution of terrestrial vertebrates Calotes versicolor, an insectivorous agamid that is distributed across Asia. It ranges from Oman, across southern and South-East Asia to Indo-China, the Maldives, Réunion, Mauritius, and Seychelles, and was recently introduced to Florida in the USA C. versicolor one of the most widespread non-gekkonid lizards in the world et al.There are 28 lizard species belonging to eight families on Hainan Island. Among these is the Oriental garden lizard, Calotes versicolor is common on Hainan Island and the adjacent mainland . This provides an opportunity for assessing the phylogeographical effects of past land bridges and contemporary geographical separation. In the present study, we sampled individuals across most of the geographical range of C. versicolor on Hainan Island, and conducted a range of phylogenetic and population-genetic analyses of their mitochondrial DNA. The objectives of our study are: (1) to estimate the impact of Pleistocene climatic variations on the genetic structure of the species; (2) to test whether Qiongzhou Strait and Wuzhi Mountain impede gene flow between populations; and (3) to investigate past population dynamics of C. versicolor, especially on Hainan Island.Calotes versicolor is not endangered or protected by any law. All samples obtained in these locations in field were not required any specific permissions because all sampled locations are not privately owned or protected. Collections of tissue samples were carried out in strict accordance with “Regulation for the Collection of Genetic Resources (HJ 628–2011)”, and all practical efforts were made to ameliorate specimens suffering throughout this study. This study was approved by the Animal Ethics Committee of Chengdu Institute of Biology, Chinese Academy of Sciences, and animal experiments were carried out in line with the institutional guidelines.According to “Law of People's Republic of China on the Protection of Wildlife” and “Regulations for the Implementation of the People's Republic of China on the Protection of terrestrial Wildlife” (State Council Decree [1992] No. 13), this study did not require any ethical or institutional approvals since C. versicolor from 39 sites between 2007 and 2009. These included 191 individuals from 35 sites across Hainan Island, covering the entire range of the species on the island to allow us to characterize its lineage differentiation, biogeographical patterns, and historical demography. In order to infer the expected impact of sea-level fluctuations at lower latitudes on the species, we also sampled 21 individuals from 4 sites on the Chinese mainland  and Vietnam  and H5162 (5′-GGT TGA RAG TAR TCA TCG AGT TAA GAA CGAC-3′), as well as L5037 (5′-GAG TAG ACC CAG GAA CCR AAG TTC-3′) in combination with H6448 (5′-GTA TAC CGG CTA ATC CAA GCA TGT G-3′). Standard polymerase chain reactions (PCR) were performed in 50 µL reactions, including approximately 100 ng of template DNA (1 ng/µL), 2 µL of each primer (each 1 µmol/µL), 5 µL of 10×Ex-Taq buffer (Mg2+ Plus), 4 µL dNTPs (each 2.5 mmol/L), 0.3 µL of Ex-Taq DNA polymerase (5 U/µL), and sterile distilled water. PCR was conducted with the following conditions: an initial denaturing step at 95°C for 4 min; 35 cycles of denaturing at 94°C for 35 s, annealing at 65°C for 45 s, and extending at 72°C for 90 s; and a final extending step of 72°C for 8 min. PCR products were electrophoresed in 0.8% agarose gels, visualized with ethidium bromide. These products were purified and sequenced directly by Invitrogen Trading (Shanghai) Co., Ltd, using the corresponding PCR primers. Where products could not be sequenced by the primers above, we used L3705 in combination with L4375 (5′-CAC CTA CAT CAY CCT AAC-3′). All individuals were sequenced in both directions. All new sequences were deposited in GenBank with accession numbers KC875609 to KC875820.Total genomic DNA was extracted from tissue samples following a universal protocol of DNA extraction ND2 and COI regions, suggesting that the sequences were not nuclear copies of mitochondrial sequences. We used DnaSP v5.0 h) and to estimate nucleotide diversities (π) Nucleotide sequences were checked and aligned by eye. No premature stop codons were observed in the a priori assumptions about the age of one or more clades to calibrate the substitution rate. This is most commonly achieved by using fossil calibrations. In the absence of fossil calibrations for C. versicolor, we expanded the data set to include various agamid taxa from GenBank , between populations within regions (SCF), and between all populations (STF). We examined several hypotheses, including: (i) no overall regional structure; (ii) structure between Hainan Island and its adjacent mainland (the “Qiongzhou Strait isolation” hypothesis); (iii) structure between the mountain and plain provinces on Hainan Island ; and (iv) structure caused by the Changhua-Wanquan River  with 10,000 permutations were assessed in Arlequin v3.1 an River  presentiC. versicolor across the sampled region, pairwise values of STF/(1−STF) were plotted against the logarithm of geographical distance  STF values were calculated with Arlequin 3.1 using the TrN model of evolution selected by the hierarchical likelihood-ratio test. Reduced major axis regression was used to estimate the slope and intercept of the isolation-by-distance association. To include geographical structuring among populations, a third matrix (indicator matrix) was included so that populations were grouped by regions based on the results of AMOVA and SAMOVA tests and the “Qiongzhou Strait isolation” hypothesis. Partial Mantel tests took these groups into account while determining the significance of genetic and geographical distance relationships.To test for associations between genetic and geographical distance among populations or within lineages, isolation-by-distance analyses C. versicolor, mismatch distributions were calculated for each group in Arlequin 3.1. To compare the observed data with the expected data under the sudden- and spatial-expansion models, we conducted goodness-of-fit tests based on the sum of squared deviations and Harpending's raggedness index DFsFs and Tajima's D suggest a recent population expansion, whereas multimodal mismatch distributions suggest that populations were recently influenced by migration, are subdivided, and/or have undergone historical contraction t = τ/2uτ is measured in mutations per sequence, and u is the mutation rate per sequence per generation. The u value was calculated by formula u = 2μk, where μ is the mutation rate per nucleotide per generation and k is the number of nucleotides . Finally, the approximate expansion time measured in years was estimated by multiplying t by the generation time of C. versicolor (one generation per year).To investigate past changes in the population size and range of 7 steps. The initial 10% of steps was discarded as burn-in. We obtained consistent demographic inferences across three replicates of the analysis.In order to examine the shape of population growth over time, we analysed the data using the extended Bayesian skyline plot http://www.worldclim.org/) at 30 s resolution DID measures the similarity of two habitat niches, comparable to a percentage overlap I measures the probability distributions of two ecological niche models We firstly used niche models to test the ecological divergence between the different groups using Maxent version 3.3.1 C. versicolor sampled in our study, we sequenced 2663 bp of mitochondrial DNA, spanning TrptRNA, ND2, and COI. Among the 118 haplotypes identified, only one  was shared by individuals from three sites . Two haplotypes (11.9%) were shared by individuals from two localities, whereas the remaining haplotypes (87.3%) were restricted to single sampling localities. The sequence alignment contained 239 variable nucleotide sites, of which 160 were parsimony-informative.For each of the 212 individuals of π) and haplotype diversity (h) within populations are summarized in π and h varied considerably among populations. The number of haplotypes (N) within each population ranged from 1 to 11, with the greatest number of haplotypes in the Lingshui population. Values of π ranged from 0 to 0.0012, whereas h ranged from 0 to 1.0000. Among all samples (excluding Gaoligong and Xishuangbanna populations), mean values of h and π were 0.991 and 0.0108, respectively, indicating a high haplotype diversity but relatively low nucleotide diversity  of 1.0  of 1.0 . The sam) of 1.0 . None of) of 1.0  are foun) of 1.0 .The coalescence time of all of the sampled individuals was estimated at 0.26 million years ago , during a period of marked glacial cycling in the Pleistocene. The estimated coalescence times of lineages A and B are 0.05 Ma (95% CI: 0.01–0.13 Ma) and 0.13 Ma (95% CI: 0.02–0.31 Ma), respectively.CTF values did not have the highest differentiation among groups when K = 2; however, one or more groups contained a single population when K≥3. Therefore, we retained the configuration of K = 2. For the AMOVA analysis, based on several hypotheses (see the Methods), the maximal CTF was achieved with two groups . While controlling for indicator matrix based on the two most parsimonious geographical groups I and II inferred from both AMOVA and SAMOVA tests, the partial Mantel test was significant . To test the “Qiongzhou Strait isolation” hypothesis, partial correlation of genetic distance and geographical distance was r = 0.28 (one-sided P<0.0001) while controlling for indicator matrix. In contrast, when geographical distance was controlled, partial correlation was r = −0.05 (one-sided P = 0.76).We found a significant relationship between genetic and geographical distances for all populations. The reduced major axis regression had a slope of 3.48 (SE = 0.12) and an intercept of −6.78 (SE = 0.25), with an explanatory variation of 0.10. The Mantel test for matrix correlation of log-genetic and log-geographical distances was significant  from the null distributions obtained from the pseudoreplicated data sets.The predicted distribution of ecological niche models of the two genetic groups was shown in P<0.001). Moreover, there are no significant differences among groups along the first three PC axes .The principal-components analysis revealed three components that cumulatively explain more than the 89% of the variation, with each principal component (PC) responsible for 42%, 33%, and 14% of the variation, respectively. Precipitation variables dominated PC1  and PC2 . PC3 loaded mainly on minimum temperature of coldest month as well as mean temperature of driest quarter. The MANOVA analyses indicated that climatic conditions differed significantly between the two groups  , corresponding to a period of pronounced glacial cycling during the Middle Pleistocene C. versicolor colonized Hainan Island recently (long after its formation), probably within the Middle Pleistocene when land bridges formed. An alternative possibility is that they reached the islands much earlier but were isolated recently, as a consequence of Pleistocene climatic oscillations and sea-level fluctuations, as appears to be the case for other species in this region Our phylogenetic analyses revealed two mitochondrial lineages, A and B, across the sampled populations of C. versicolor within our study area share a relatively recent evolutionary history, there has been sufficient time for them to acquire unique haplotypes. However, given that there has been limited gene flow occurring between Groups I and II, despite the absence of any obvious physical barrier, we propose that non-physical barriers have led to geographical subdivision. There are several plausible scenarios for this, as explained below.Our analyses of molecular variance identified two distinct groups, I and II, with restricted geographical distributions. Levels of sequence divergence and frequencies of private haplotypes are high for intraspecific data across such a small geographical area. This pattern suggests that although the populations of C. versicolor are consistent with the isolation-by-distance model C. versicolor, but it is believed that lizards have limited dispersal abilities One possibility is that species that have small dispersal distances are likely to show distinct genealogical lineages even without geographical barriers to gene flow C. versicolor. Further studies involving additional molecular markers and data on behavior and landscape processes will improve our understanding of the evolutionary history of this species.The second possibility is that non-physical barriers were stronger in the past. Our ecological niche modeling and niche-identity tests indicate significant niche differentiation between Groups I and II with respect to the selected bioclimatic variables . This reIn the ‘North’ clade, a member of lineage B see , three pLeiolepis reevesii), populations of C. versicolor on both sides of Wuzhi Mountain did not become reciprocally monophyletic and were embedded within lineage A. This implies that Wuzhi Mountain does not constitute an important barrier to gene flow between C. versicolor populations. This was not surprising, considering that the specific habitat requirements of L. reevesii are associated with littoral sand and not with the montane conditions in Wuzhi Mountain and high altitudes (>150 m) C. versicolor which inhabits this mountain ranges L. reevesiiC. versicolor. Our results suggest that it is necessary to study several species within the same region to see if they share a common phylogeographical pattern, in which case those patterns are likely to have specific geographical causes.Unlike those of Reeves's butterfly lizard (Diadophis punctatus) Desmognathus wright) Ambystoma maculatum) Our analyses show that Group I has experienced long-term population stability through time, whereas Group II has undergone a moderate population expansion and contraction. The population size and range of Group I was not negatively affected by major Pleistocene climate oscillations, despite changes in sea level affecting the size of Hainan Island. To allow such population stability across the complex environmental gradient throughout alternating periods of glacial and interglacial episodes, intrinsic and extrinsic factors might have limited species dispersal Pipistrellus abramus) Rhinolophus affinis hainanus) L. reevesii) Bambusicola thoracica thoracica) The historical demography of Group II seems to be more complex than that of Group I. The skyline plot indicated that the expansion of Group II preceded the last glacial maximum, corroborating studies of other taxa at low latitudes, such as Japanese pipistrelle bat  on Hainan Island and the adjacent mainland. We identified two mitochondrial lineages occupying specific habitats separated by putative nonphysical or ecological barriers. Although our study is based on a single locus , we have been able to gain some useful insights into the demographic history of C. versicolor. We proposed that Pleistocene sea-level fluctuations had profound effects on lineage diversification and regional genetic structure. However, the demographic history of the species was influenced by habitat requirements and competitive interactions between lineages, rather than changes in climate during the last glacial maximum. Our study highlights the importance of nonphysical barriers, such as ecological processes and geographical distance, in shaping the present-day distribution of a species at lower latitudes in tropical China. We suggest that the addition of ecological niche modeling to phylogeographical analyses will offer further insights into the mechanisms that determine the distributions of other organisms in this region.We have presented the first comprehensive phylogeographical analysis of the Oriental garden lizard Click here for additional data file.Figure S2Observed and expected mismatch distributions showing the frequencies of pairwise differences of Group I and Group II based on models of sudden expansion  and spatial expansion .(TIF)Click here for additional data file.Figure S3Principal-components analysis of climatic niche differences between Groups I and II . (a) X-axis indicates PC1, and Y-axis indicates PC2. (b) X-axis indicates PC2, and Y-axis indicates PC4.(TIF)Click here for additional data file.Table S1Accession numbers for the outgroup sequences retrieved from GenBank and used for phylogenetic analyses of Fiure S1.(DOC)Click here for additional data file.Table S2Results of the principal components analysis of 19 bioclimatic variables.(DOC)Click here for additional data file.


Iberolacerta monticola, an endemism considered to be facing a high risk of extinction in several populations.The study of the factors that influence population connectivity and spatial distribution of genetic variation is crucial for understanding speciation and for predicting the effects of landscape modification and habitat fragmentation, which are considered severe threats to global biodiversity. This dual perspective is obtained from analyses of subalpine mountain species, whose present distribution may have been shaped both by cyclical climate changes over ice ages and anthropogenic perturbations of their habitats. Here, we examine the phylogeography, population structure and genetic diversity of the lacertid lizard Northwestern quadrant of the Iberian Peninsula.b (598 bp) loci, as well as at 10 nuclear microsatellite loci from 17 populations throughout the distribution range of the species.We analyzed the mtDNA variation at the control region (454 bp) and the cytochrome According to nuclear markers, most sampling sites are defined as distinct, genetically differentiated populations, and many of them show traces of recent bottlenecks. Mitochondrial data identify a relatively old, geographically restricted lineage, and four to six younger geographically vicariant sister clades, whose origin may be traced back to the mid-Pleistocene revolution, with several subclades possibly associated to the mid-Bruhnes transition. Geographic range fragmentation of one of these clades, which includes lowland sites, is very recent, and most likely due to the accelerated loss of Atlantic forests by human intervention.Iberolacerta. However, the changes operated during the Holocene severely compromise the long-term survival of those genetic lineages more exposed to the anthropogenic perturbations of their habitats.Altogether, the data fit a “refugia within refugia” model, some lack of pattern uniformity notwithstanding, and suggest that these mountains might be the cradles of new species of  The mean height of these mountains falls off considerably after the so-called Picos de Europa massif, and Sierras de Peña Prieta, Peña Sagra and Híjar, on entering Cantabria, which turns out to be a determinant feature of the eastern limit of the I. m. cantabrica distribution  must have been unsuitable for the species during the last ice age, a situation that most likely took place repeatedly during the cyclical climate changes of the Pleistocene ribution . WesternI. monticola, which may have been shaped both by Pleistocene glacial cycles and Holocene habitat fragmentation. But then we would like to address several issues concerned with the conservation of this species, by assessing the effects of population decline on the levels of deleterious mutation load and evolutionary potential to respond to an ever-changing environment.Molecular data openly question the subspecific rank of the population from Serra da Estrela and, conversely, point out an unexpectedly high degree of differentiation between the populations of Galicia and the Cantabrian Range, but fail to produce conclusive evidence on the location of putative glacial refugia, or the eventual dispersal routes from them I. monticola was designed to include a full representation of the known distribution of this species. Ethics approval of all procedures involving vertebrate animals is legally required under the Spanish legislation , which is a transposition of the European Directive 86/609/EEC. In agreement with article 18 and annexes VII and XI of the said Royal Decree, all animal procedures performed as part of the experimental work described in this paper have received prior and explicit approval from the competent authorities, defined in article 3e of the Law, and substantiated in the corresponding regulations of the Spanish autonomous communities. Thus, permissions for fieldwork and the concomitant experimental procedures were issued by the Xunta de Galicia, Junta de Castilla-León, and Principado de Asturias, in Spain, and, in application of an analogous transposition of the European Directive, by the Instituto da Conservação da Natureza in Portugal. Altogether, 316 lizards were genotyped, from 17 populations and 24 sites covering most of the species range , corresponding to position intervals 14,193–14,763 and 16,311–16,773, respectively, of the Lacerta viridis mitochondrial genome (GenBank acc. no. AM176577); for amplification details, see I. monticola populations, and then to 15 representative localities (http://dx.doi.org/s2479). This is made explicit in this section, since two of the sampling sites happened to harbor not I. monticola, but a different, so far undescribed, new Iberolacerta taxon (see Results). DNA extractions from tissue samples and genotyping were carried out as described elsewhere odonCode Alignerv. 3.5.7 (CodonCode Corporation). Newly reported sequences have been deposited in GenBank . A sequence from I. cyreni (constructed from EF121834 and AY267232) was used as outgroup to root the phylogenies.Sampling of es range . In evericrosatelliteToolKithobs) and expected under Hardy-Weinberg equilibrium (hHW), and numbers of alleles for each locus and population (an), together with the corresponding averages across loci for each population  statv. 2.9.3 ISf , and possible non-random allelic associations between pairs of polymorphic loci (2100 permutations). The significance level of all these tests was adjusted by the sequential Bonferroni procedure enepopv. 4.0.The Mottleneckv. 1.2.02 M-ratio software ottleneck, a two-phase mutation model with 5% of multi-step changes was assumed, using 10,000 iterations, and Wilcoxon signed rank tests of significance. For the M-ratio test, the bottleneck hypothesis was tested under three different population scenarios, corresponding to different values of the mutation parameter θ (4Neµ), as in θ = value estimated from the data, θ = 10, and θ = 0.5. See footnote at We checked the analyzed populations for evidence of recent bottlenecks using both Bi.e., with no a priori assumptions about populations) were taken to visualize the similarities among multilocus genotypes. First, by constructing a UPGMA tree of individuals microsatelliteanalyser (MSA) v. 4.05 eighbor program in PHYLIP v. 3.6 ega 4 enetixv. 4.05.2 Two geographically “blind” approaches  within I. monticola, we used both the method suggested in the original Structure paper, based on scoring mean log likelihoods penalized by one-half of their variance ), and the approach developed by Evanno et al. k values (Δk). We used an admixture model of genetic clustering with correlated allele frequencies, run for 50,000 generations after a burn-in of 100,000 generations, assuming that there were up to 14 possible partitions of the data (k = 2 to k = 16), and ran 10 parallel chains to estimate what number of genetic clusters had the highest probability. Membership coefficients of individuals to each of the clusters were plotted with Distructv. 1.1 At the population level, we first constructed a neighbor-joining tree of the populations, based on Cavalli-Sforza & Edwards chord distance, v. 4.0 ega 5 p-distances between sequences). MP trees were obtained using the Close-Neighbor-Interchange algorithm, with search level 3, and 100 random addition sequence replicates. The NJ tree was based on distances obtained by the Tamura-Nei model, allowing for a heterogeneous pattern among lineages and gamma-distributed rates among sites. The value of the gamma shape parameter (0.20) was obtained using a combination of ancestral sequence inference and maximum likelihood estimation amma , based on 10,000 bootstrap replicates. For ML, we first determined the model of sequence evolution that best fitted each of the four partitions of the data, namely (i) control region, (ii) 1st, (iii) 2nd, and (iv) 3rd codon positions of the cytb, by the Bayesian Information Criterion (BIC), using the metaPIGA v.2.1.3 development (http://www.metapiga.org) jModelTestmetaPIGA, choosing the best-fit model selected by BIC for each partition . We used loose Neighbor-Joining (20% range) to generate the starting trees, based on a HKY85 distance matrix, with rate heterogeneity across sites (shape parameter of the Γ distribution = 0.20). A ML test of the molecular clock hypothesis for the consensus topology thus obtained was carried out with Mega 5. As for the Bayesian phylogenetic inference, we used MrBayesv. 3.2 rBayes default priors until the standard deviation of split frequencies dropped below 0.01, and the potential scale reduction factor for all parameters lied close to 1.0. Two simultaneous, completely independent analyses starting from different random trees were run. For the Markov chain Monte Carlo (MCMC) sampling of the target distribution, three heated chains and one cold chain were used. The first 25% samples from the cold chain were discarded as burn-in. We used Bayes factor comparisons to test several topological hypotheses. Marginal model likelihoods were estimated by the stepping-stone method; strength of the evidence in favor of the better model was then assessed by the magnitude of the log-difference, following Kass and Raftery cyreni, and from 1.9 to 2.9 My on the monticola-galani-martinezricai split. These “hard” bounds are based on Carranza et al. (2004) Iberolacerta) fall well within the 95% confidence intervals produced by nearly all the methods and models tested by Hipsley et al. (2009) igTreev. 1.3.1 The topological congruence between the phylogenies obtained with the two markers was assessed by the incongruence length difference (ILD) test tcsv. 1.21 Since tree-building methods tend to resolve intraspecific gene genealogies poorly when the different mitochondrial types are separated by few mutations, and ancestral haplotypes are still present in the populations lignment Transformation EnviRonment (ALTER) The Aa priori continuous or fragmented distributions of the species  or DC) against geographic distance (linear or log-transformed) Π), obtained with Megav. 4, considered as a proxy of historical population connectivity r2. Geographical distance matrices were generated with GDMG v. 1.2.3 Genetic samples were separated into two groups, corresponding to cies see . Pairwisrlequinv. 3.5 FST over all loci) were used to compute the distance matrix. Significance tests were based on 1,000 permutations.Using the same two groups of populations, an analysis of molecular variance (AMOVA) in Acytb) produced essentially similar phylogenetic signals , so that they were concatenated to produce a two-gene data set . The null hypothesis of equal evolutionary rate throughout the ML tree was not rejected at a 5% significance level (P<0.878), and Bayes factor comparisons rendered very strong evidence in favor of a strict molecular clock (log-difference of 10 units), which was accordingly assumed for all the phylogenetic reconstructions. Two of the samples, a priori from I. monticola, showed an exceptionally large number of nucleotide differences as compared with the others . They correspond to individuals from the Sierra de Gistredo, at the SW of the Cantabrian Mountain Range, which is part of those largely independent lesser mountain groups found in the province of León, to the south of the Minho-Sil river basin, and east from the Ancares-Courel groups  and I. monticola (to the northwest). As shown at ca. 1.6 Million years ago , thus probably meriting the consideration of its full species or subspecies status (see discussion).The sequences of the two mitochondrial markers (CR and I. monticola sensu stricto (i.e. excluding Gistredo) is concerned, the mitochondrial results indicate that the most recent common ancestor (MRCA) of their different haplotypes dates back to ca. 0.9 Mya (0.67–1.25). The samples from the Cantabrian Mountain Range (CAMOR) give rise to two basal haplotype clusters, with strong statistical support, namely Xistral-Courel-Ancares in Galicia, on the one hand . The high diversity of the system formed by these mountains stands in sharp contrast with the unexpectedly low diversity and close similarity of the haplotypes observed in the main (eastern) part of the Cantabrian Range (clade II), in spite of the high census size and the large extension occupied by the corresponding populations, without apparent geographic discontinuity among them (ca. 0.4 Mya (0.19–0.62). The single haplotypes found at Pindo and Queixa-1, the two remaining northwestern populations, also began to diverge approximately 0.4 Mya (0.10–0.75). They are joined in a separate clade (VI) both by the MP and the Bayesian reconstruction, but its statistical support is relatively low , which is most closely related to clade V, although the statistical support for this topology (favored by a very strong log-difference of 23 units in Bayes factors comparisons) is not conclusive , which might be due to segregating null alleles, namely A5 at Queixa-L6A, C113 at Courel-L9A, and PB20 at Courel-L9B and Leitariegos. Applying a conservative criterion, these data were not taken into account for estimating genetic distances among populations, nor for evaluating the evidence of past bottlenecks (see later). However, it should be mentioned that the number of positive fIS estimates for each locus and sample nearly doubles the number of negative estimates, and that only two negative global FIS scores were obtained for the different samples . On the other hand, individuals from each locality generally group together in a UPGMA tree based on allele-sharing distances (tructure unveils a clear hierarchy in the partition of population samples among clusters (L(k) or Δk as optimization criterion , CAMOR+E and NWP groups become apparent , all the populations except Eume, Xistral, Ancares, Estrela and Leitariegos, show traces of recent bottlenecks.As regards the demographic history of these populations, the results of BST-based distances. This finding, together with the clear geographic subdivision of the genetic diversity of I. monticola (and the ensuing reduction of sample sizes), also hindered any attempt to infer the demographic history of its different lineages by mismatch-distribution analyses of pairwise mtDNA differences.In principle, all CAMOR populations should be connected by gene flow, since no apparent geographical or ecological barrier has been found within their range see , whereasP<0.000001). Lower values are related to differences among populations within groups , and still lower to differences among groups . Results from several one-way AMOVAs, together with different measures of genetic diversity in continuous and fragmented populations are shown in FST score (0.215 vs. 0.107), whereas the mean number of alleles per locus and expected heterozygosis at HWE are considerably lower in them . Actually, OCCIDENTAL_R shows lower average numbers of alleles in 10 out of the 11 loci analyzed, and lower heterozygosis in nine of them . In addition, on average, large populations show an excess of homozygotes over HW expectations in a significant proportion of loci ; this observation is consistent with mean FIS scores for these categories, which can be easily obtained from θ estimates derived from mean expected heterozygosis point out a reduction of 48% in the scores obtained from reduced populations (1.62), as compared with large populations (3.10), which, assuming equal mutation rates in both classes, amounts to an equivalent descent in their corresponding mean effective population sizes.Results from a two-way AMOVA indicate that most of the observed microsatellite variation is explained by differences within populations , karyology and osteology, will be necessary to establish the definitive taxonomic status of this population, together with microsatellite data to investigate its genetic structure (in preparation).Comparative phylogeographic analyses have shown strong genetic subdivisions, indicative of extended periods of population isolation, for many species and species complexes in the Iberian Peninsula, and especially for those with limited effective dispersal that live in mountains. This is most likely due to their survival throughout the Pleistocene ice ages in multiple refugia or sanctuaries, which tend to occur in mountain ranges icai see  and I. gions see . But theI. monticola into I. m. monticola at Serra da Estrela, and I. m. cantabrica elsewhere, falls too short to describe the actual pattern of diversification within this species. With the conspicuous exception of Serra de Ancares, which shows extensive mitochondrial admixture, most other populations are characterized by parapatrically distributed clades and subclades. According to their estimated coalescence times, this pattern could be the outcome of the relatively recent (re)colonization of these areas from different refugial populations, which, as in other well documented cases vs. Xistral (clade III) in the CAMOR group, or the low-altitude Artabrian Gulf vs. the moderate-high altitude populations of Serra da Queixa (clade V) in the OCCIDENTAL group, whose dating (mean node ages of 0.4–0.5 Mya) agrees quite well with the second distinct climate change of the Quaternary, the mid-Bruhnes event, about 0.43 Mya The mitochondrial results are consistent with the simultaneous vicariance of not less than four, and a maximum of six populations after the mid-Pleistocene revolution (about 0.9 Mya), one of the two major climate transitions of the Quaternary period, with drastic effects on the geographical distributions of many species I. monticola populations, might also be explained by a recent selective sweep, as suggested in other cases Lacerta schreiberi), will be necessary for a more thorough understanding of the evolution of these populations, which undoubtedly have experienced multiple episodes of admixture.The primarily east-west orientation of the Cantabrian Range may have facilitated the survival of populations simply by moving up or down mountains, as the general climate worsens or ameliorates Iberolacerta bonnali, which inhabits rocky habitats at 1,600–3,300 m asl, and whose current distribution is most likely a consequence of post-glacial range fragmentation after the end of the Last Glacial Maximum, would perfectly fit into the first category Iberolacerta monticola should be expected to lie somewhere in-between the two extremes. Its mitochondrial lineages seem to have Pleistocene origins, with diversification times estimated to be younger than 1.5 Mya, in contrast to other Iberian species characterized by the persistence of generally much older lineages through glacial cycles Iberolacerta do in fact exist, but they are nowadays associated to different, closely related species. In terms of the phylogeographic models of “S” (sanctuary) and “R” (refugia) species I. monticola  is actually similar to the genetic signature of “R” species .On general grounds, perhaps the main factor determining the level of genetic and regional subdivision of species inhabiting European mountains is the duration of the cycles of range contraction and expansion triggered by climate changes. For species dwelling at upper elevation  habitats, vicariance events are most likely associated to warm, relatively short interglacials, thus giving rise to shallow phylogenies. On the contrary, according to the displacement refugia model Iberolacerta, except I. monticola, are confined to mountains in the centre and NE of the Iberian Peninsula, probably because of the low thermal quality and scarcity of refuges of lower elevation environments at those regions I. monticolaI. monticola populations, which can be traced through the results of all our analyses of microsatellite variation, but particularly of FCA and Mantel tests. The significant association of Cavalli-Sforza chord distance (DC), but not FST-based indices, both with connectivity and with log-distance between populations, probably reflects the better performance of DC in recovering the topology of microsatellite trees I. monticola populations have low statistical support. This still on-going process of fragmentation has produced a patchy distribution of I. monticola in the NW of the Iberian Peninsula, which includes large, moderate and small sized populations, and both montane and lowland habitats (OCCIDENTAL group).In addition to the large effects of glaciations on the diversification of this species, the combined evidence from microsatellites and mitochondrial markers clearly shows that its current fragmented distribution at most nearby lowland populations has a very recent origin. All the species of I. monticola is so far not conclusive, and mostly based on differences in home-range sizes of the closely related species I. cyreniI. monticola, so that it is probably a contributing factor to the phylogenetic pattern observed at Estrela. But there are other, not mutually excluding possibilities as well. One plausible scenario is that after the few hundred thousand years that followed the vicariance episodes, during which the different mitochondrial lineages of I. monticola evolved in isolation, there would have been a colonization of Serra da Estrela from northeastern populations (Courel-Ancares). A mitochondrial “occidental” allele may have drifted by chance to high frequency at the population of Estrela, even though the nuclear genomes bear the trace of that secondary contact. If the above scenario is correct then there may be alleles from the Cantabrian lineage III still present elsewhere in the Serra da Estrela.Although mtDNA data suggest that the Estrela population is most closely related to other occidental populations (at Serra da Queixa and the Artabrian Gulf), thus implying that it has been separated from the CAMOR populations for nearly 1 million years, the microsatellite data do not support this conclusion, and indicate that it has been last connected with the populations of Courel and Ancares, at the western part of the Cantabrian Range, instead. Sex-biased gene flow is the most common explanation for such discordant patterns of biparental nuclear and maternal mitochondrial markers I. monticola show footprints of genetic erosion, such as a reduction of standing levels of genetic variation and, insofar as FST is accepted as a valid index of drift load FST values exceeding 0.2 I. monticola is threatened by extinction in these isolated sites seems justified, particularly so if considering that there has been scarcely time for these populations to reach mutation-drift equilibrium, so that the observed STF value of 0.215  is most likely to be an underestimate. However, there are other considerations that must be taken into account on this respect.Small isolated populations of I. monticola at pre-fragmentation stages may have been much lower than for an undivided population. It all depends on whether the species is expected to largely fit a metapopulation model or not. In species displaying low natal and breeding dispersal, such as many lizards FST scores, such as those observed among “continuous” populations of I. monticola, the expected reduction in inbreeding depression and mutation load of the metapopulation as compared with an undivided population can be quite important First, the frequency of segregating deleterious mutations and the mutation load of I. monticola show no evidence of overall excess of homozygotes, not even Lambre, a very small population that may have gone extinct by now Second, small isolates of endless formsThe tempo and mode of the building-up of genetic differences among populations that ultimately become new species may assume The Holocene may represent an inflexion point for the long-term survival of those genetic lineages more exposed to the anthropogenic perturbations of their habitats. Thus, successful heirs of many thousand years of evolution, with unique genetic characteristics forged in the engine of the cyclic climate changes of the Pleistocene, might not get through the next glacial stage, but become extinct relatively soon.Figure S1Bayesian phylogenetic tree of I. monticola populations based on mitochondrial sequences. The tree is rooted using I. cyreni as outgroup, and it includes representative sequences from I. martinezricai and I. galani. See legend of (DOC)Click here for additional data file.Figure S2Different estimates of genetic variation at microsatellite loci, for the different geographical samples of I. monticola.(A) Expected heterozygosity in HW equilibrium (He); bars correspond to standard deviations. (B) Average allelic richness (blue bars) and private allelic richness (red bars), corrected for sample size.(DOC)Click here for additional data file.Figure S3tructure.Results of the analysis for genetic clustering using S Colors correspond to the different clusters inferred by the analysis. The log likelihood of each assumed number of clusters (lnL) is shown, together with the % of independent runs that correspond to the represented partition of populations among clusters.(DOC)Click here for additional data file.Figure S4Identification of the most likely number of I. monticolatructurev. 2.1 populations by the analysis of microsatellite data with S. (A) Estimated log probability of data for the different number of inferred clusters (K); bars correspond to standard deviation, after 10 independent runs. (B) Rate of change in the log probability of data between successive k values (Δk). Both figures were obtained with the aid of Structure Harvesterv. 0.56.4, http://taylor0.biology.ucla.edu/struct_harvest/.(DOC)Click here for additional data file.Table S1Information about the populations and the genetic samples of I. monticola used in this work.(DOC)Click here for additional data file.Table S2Information summary of the DNA markers used in this work.(DOC)Click here for additional data file.Table S3Genetic variation at microsatellite loci in populations of I. monticola .(DOC)Click here for additional data file.


Arabidopsis lyrata subsp. petraea populations that differ in demographic histories since the Pleistocene. As expected, northern European populations have genetic signatures of a strong population bottleneck likely due to glaciation during the Pleistocene. Levels of diversity in the northern populations are about half of that in central European populations. Bayesian estimates of historical population size changes indicate that central European populations also have signatures of population size change since the last glacial maxima, suggesting that these populations are not as stable as previously thought. Time since divergence amongst northern European populations is higher than amongst central European populations, suggesting that the northern European populations were established before the Pleistocene and survived glaciation in small separated refugia. Estimates of demography based on expressed genes are complementary to estimates based on microsatellites and transposable elements, elucidating temporal shifts in population dynamics and confirming the importance of marker selection for tests of demography.Demography impacts the observed standing level of genetic diversity present in populations. Distinguishing the relative impacts of demography from selection requires a baseline of expressed gene variation in naturally occurring populations. Six nuclear genes were sequenced to estimate the patterns and levels of genetic diversity in natural  Demographic factors shape baseline genetic variability within and between populations and can obscure detection of molecular evolution signatures and identification of adaptive alleles Understanding the role of demography in genetic variation is important for the implementation of population genetic theory to tease apart selection and population structure and size fluctuations. Inferring the best fit model explaining the source and patterning of genetic variation in a system is hinged on an understanding of the demographic history which shapes the baseline variation of the genome. However, baseline genome variation falls in a distribution of values. Demography impacts the distribution of baseline variation across the genome, shifting all loci including outliers Dissecting the genetic impacts of demography is further confounded by the temporal fluctuations of population size and population connectivity. Within several generations, the genome-wide impacts of bottlenecks may no longer be statistically detectable  Arabidopsis thaliana  has long been the model plant used in genetic and developmental studies due to its small genome size, ease and quickness of growth and propagation in laboratory settings, and plasticity to genetic modifications. This model system status has resulted in a large knowledge base of gene function and a sequenced, well characterized genome. Genus Arabidopsis is also a model for speciation and evolutionary ecology due to the interesting life history differences present in populations of closely related species Determining the extent that demography can impact genetic variation in naturally-distributed populations requires closely related populations with differing demographic histories as well as an excellent evolutionary and ecological background on both the population and the species levels. A. thaliana is A. lyrata subsp. petraea (A. l. petraea)A. l. petraea have therefore been found to be stable with isolation by distance detected in microsatellite loci A. thalianaThe closest relative of A. l. petraea have differing demographic histories A. l. petraea across the entire range. This finding is in contrast to the results cited using microsatellite markers, which indicate stability in central European populations A. l. petraea a great system to study the impacts of demography on genetic diversity of naturally-distributed populations.European populations of A. l. petraea. Coding regions of the genome are better suited to divergence population genetics than are microsatellites, transposable elements or chloroplast markers, due to the availability of more consistent and predictive mutation accumulation models, especially for coalescent-based analyses. We estimated the baseline impact of alternative demographic histories on coding gene diversity as an independent distribution for use by the ecology and evolutionary genetics community to dissect the impact of selection in relevant functionally-important candidate genes in genus Arabidopsis. This analysis can also serve as a reference point for future comparisons across different systems to test broader comparative genomics hypotheses.We developed and utilized expressed haplotypes of single copy nuclear loci (SCNL) to determine the signatures of demographic histories between populations of A. l. petraea in northern and central Europe for a total of 148 sampled plants  to do the following: sort genes by length of longest exon, remove genes with sequences consistent with transposable element histories, include only genes that are expressed (have ESTs), and remove genes with too much nucleotide sequence similarity precluding distinction between gene copies versus alleles for PCR amplification simplicity. Only regions with long exons (greater than 500 basepairs) were considered. Genes that contained transposable elements were removed from the list to avoid confounded mutation models and evolutionary histories in coalescent models. Next, genes without representatives in the EST library were removed. Finally, to verify single copy status, only genes that had little similarity to the closest BLAST hit were retained using the reference point similarity score of paralog and ortholog detectability in gene families in other systems with which we have published 2) concentrations and annealing temperatures were adjusted as necessary amplification. Single bands were verified by gel electrophoresis. Excess primers and dNTPs were removed by Exonuclease and Antarctic Phosphatase treatment. Direct sequencing reactions were carried out with both forward and reverse primers on PCR products using ABI Big Dye protocols by the Max Plank Institute for Chemical Ecology and the Clemson University Genomics Institute (CUGI).Single copy nuclear loci (SCNL) were developed as markers using searches of the http://en.bio-soft.net/dna/BioLign.html). Alignments were trimmed to contain only high quality base calls  and to insure the same length for all sequences. Heterozygous sites were called by eye using the presence of a double peak from sequence reads in both directions. Sequences were doubled for each individual and pseudo-haplotypes were created by random assignment of heterozygous base calls. PHASE version 2.1 Sequences were aligned and checked for quality using CodonCode Phred/Phrap and BioLign version 4.0.6.2  using Akaike Information Criteria (AIC) as in MODELTEST Phr2 was TrN The best fit mutational models were identified for each gene using FINDMODEL , pairwise ФST between each population, exact tests for population differentiation, and Hardy-Weinberg equilibrium. We used 10,000 permutations to assess significance of AMOVAs and pairwise ФST, while 100,000 steps were used to assess significance of exact tests and HWE. Bonferroni corrections for multiple tests were used to determine appropriate P-value cutoffs for statistical significance.Arlequin version 3.1 CRecombination break points in each gene were determined using the 4 gamete test Best fit population structure was inferred using STRUCTURE version 2.2 ef) over time. Each run used the HKY mutation model and a strict molecular clock with a 1.5×10−8 substitutions/site/generation. The mutation rate is based on that estimated for the Brassicaceae family BEAST version 1.4.8 ef), migration between populations (Nefm), ancestral population size (NefA) and time since divergence (T). All populations were analyzed on a pairwise basis. Longest non-recombining blocks (LNRB) were determined from the 4 gamete test in SITEs, and used for all analyses in IMa. Each population comparison was run in M-mode with wide value cutoffs for all parameters. After the initial run, three runs with different random number seeds and smaller cutoff values that were based on the distribution of parameter values from the first run. All runs had 50,000,000 MCMC steps after a burn-in of 100,000 steps. Each run had 10 chains with a mixing rate of 5 chain swaps per step. All three M-mode outputs are checked for convergence and nested models were tested in L-mode. Using nested models, which tests for likelihood of data fitting the model with and without migration, we found that in all cases the models that included migration fit the data better than the no migration models. Values were scaled based on a substitution rate of 1.5×10−8−9 has been calculated in A. thaliana−8 mutation rate. All IMa runs were computed on the Condor cluster at Clemson University using primarily an extensive web-enabled system to simultaneously manage and monitor performances of each set of input priors.IMa D, Fu’s FS or Rozas R2 were significantly different from neutral-equilibrium model expectations  and Watterson’s θ are much lower in northern European populations compared to central European populations . The SjoA. l. petraea can be inferred by the closest connections to the A. thaliana ecotypes. These connections are most often to central populations, but the Cori1 and Fdh networks have these connections with northern populations. This could be reflective of older divergence times between northern and central populations. The most common haplotype (largest circle) for each gene is made up of a high percentage of central European individuals.Haplotype networks  indicateST values for all 6 loci  are insufficient to detect significant differences using this test.Analyses of molecular covariance (AMOVAs) revealed highly significant Фl 6 loci  with higulations . However−34 and 10−28 respectively. The two Icelandic populations group with the Spiterstulen population and all other populations group separately. The grouping of the three northern populations is due to a small number of shared haplotypes. One individual from the Schaerftal population is more similar to individuals in the Plech population. This individual was checked for placement in the haplotype networks to validate that this specific individual was not mis-assigned to the Schaerftal sampling. All alleles for this individual grouped very closely with other alleles in individuals from Schaerftal and so it was kept in the dataset.The best fit number of populations is six (K = 6), as estimated in STRUCTURE , with thef) for all 6 loci. In contrast, the Neutras and Plech populations increased in Nef over at all 6 loci since the LGM. In all cases, expansion does not appear to be exponential, which could explain the non-significant values of Fu’s FS and Roza’s R2 test statistics.The Bayesian skyline plots indicate expansion since the last glacial maximum  in at least one locus for all populations . The Hasef) in the northern populations  also indicate much smaller effective population sizes  and Bayesian skyline plots (BSP) of population size changes over time also indicate much lower Nef values for the northern populations. These estimates coincided with what we expect based on studies using other markers ef estimates between IMa and BSPs. IMa uses LNRB, which have a reduced amount of polymorphism, while BSPs use the full sequence. High recombination rates are likely not restricted to the central European populations, but may not be detected in northern populations due to lower levels of nucleotide diversity. Therefore, northern populations may have expanded at a greater rate since the LGM, but detection is difficult at slow evolving loci with low levels of starting variation due to a strong bottleneck.Here we present evidence of differing demographic histories amongst European populations of A. thaliana ecotypes, differences in the distribution of ancestral alleles among populations can be inferred. The distribution of ancestral alleles supports the theory that northern populations survived the Pleistocene as separate, possibly transitory refugia  support pre-glacial divergence between northern and central populations. However, the best fit model suggests post-glacial divergence between the central European populations. These estimates contrast with those from a study that used the Plech population as a reference population in demographic modeling A. l. petraea is in Austria rather than Germany It has been suggested that the center of origin for ST estimates) indicate strong population structure between populations and low levels of gene flow for all except the three northern populations. However, the best fit isolation-migration models estimate higher rates of gene flow and more recent divergence times amongst the central populations. This discrepancy amongst algorithms is likely due to high within-locus recombination rates in the central populations, resulting in an increase of private alleles in the central populations that are not detected when using the longest non-recombining block of sequence for IMa analyses. The underlying models for these algorithms are also very different in that IMa uses a coalescent approach for allele divergence while STRUCTURE uses current haplotype frequencies to determine relatedness of populations. The grouping of northern populations in STRUCTURE is likely due to a small number of shared haplotypes between these populations, however most alleles in each population are diverged. When a mutation model is incorporated into IMa, the number of mutations between the diverged haplotypes gives a more accurate estimate of divergence. Therefore, our data supports pre-glacial divergence between northern populations. This finding is consistent with high divergence rates between Scandinavian and Icelandic populations of A. l. petraeaPopulation connectivity revealed in STRUCTURE and the molecular covariance estimate of variance due to among population diversity  intervals for NefA and T, but with little impact on point estimates for these two parameters. Only very high values of gene flow from a third population (Nefm >0.5) have a strong impact on estimates efm. The overall impact of gene flow from a third population is difficult to estimate and this violation is most likely the reason for high HPD-90 intervals for NefA and divergence time (T) in all runs.It is important to note that we cannot rule out possible gene flow from a third population in the pairwise population analyses. Isolation-migration model fitting in IMa assumes no gene flow from a third population. A simulation study A. l. petraea reported at microsatellite loci Gene sequences of nuclear loci complement patterns of demographic histories for European populations of A. l. lyrata and A. halleri since initial divergence has impacted the level of diversity on a genome-wide scale A. l. petraea and other closely related species. Of special interest is A. arenosa, which maintains a stable hybrid zone with A. l. petraea in Austria . It would be informative to know if there are regions of the genome undergoing different levels of gene flow and if those regions coincide with regions that are differentially shared between A. l. lyrata and A. halleri. These regions might harbor ‘speciation genes’ or genes that maintain reproductive isolating barriers. This approach would be a nice complement of candidate gene approaches for understanding mechanisms of speciation in plants  information.(XLSX)Click here for additional data file.Table S2Diversity information for full length and LNRB for each of the 6 loci.(XLSX)Click here for additional data file.