Fis values and heterozygote deficiency were observed in populations from the marginal  and discontinuous zones . However, populations from the continuous zone were in HW equilibrium . There were no significant latitudinal effects on gene diversity (Hs), allelic richness (AR), or population differentiation (Fst). Bayesian and NJT (neighbor-joining tree) analyses demonstrated the presence of a population structure that was partly consistent with the geographic origins of the populations. The impact of population fragmentation on the genetic structure of EWC is to create a positive inbreeding coefficient, which was two to three times higher on average than that of a population from the continuous zone. This result indicated a higher occurrence of selfing within fragmented EWC populations coupled with a higher degree of gene exchange among near-neighbor relatives, thereby leading to significant inbreeding. Increased population isolation was apparently not correlated with a detectable effect on genetic diversity. Overall, the fragmented populations of EWC appear well-buffered against effects of inbreeding on genetic erosion.We tested the hypothesis that marginal fragmented populations of eastern white cedar (EWC) are genetically isolated due to reduced pollen and gene flow. In accordance with the central-marginal model, we predicted a decrease in population genetic diversity and an increase in differentiation along the latitudinal gradient from the boreal mixed-wood to northern coniferous forest. A total of 24 eastern white cedar populations were sampled along the north-south latitudinal gradient for microsatellite genotyping analysis. Positive  Climate is among the most important ecological processes that strongly shape the range and genetic diversity of a species Hewitt . The welAbies balsamea [L.] Miller), and the northern coniferous forest, which is dominated by black spruce (Picea mariana [Miller] BSP). The present-day transition between these two boreal zones is controlled by both climate and fire  and involved all of the tree species that are presently found within the area Richard . Since 7Thuja occidentalis L.)   is ill-aFst = 0.073) within six EWC populations. In contrast, Perry and others showed that six northern populations were not differentiated (Fst = 0.016) and, indeed, were in Hardy–Weinberg equilibrium  and yellow birch  bioclimatic domain and represents an area where eastern white cedar is common. The discontinuous zone is in the balsam fir and white birch (Betula papyrifera Marsh.) bioclimatic domain and marks the northern edge of the continuous distribution, where eastern white cedar becomes less common in the forest matrix. The marginal zone is in the black spruce (Picea mariana [Mill.] B.S.P.) and feather moss bioclimatic domain, where only a few isolated populations are found. The site occupation rates by EWC along the gradient were estimated to 55%, 9%, and 3% in the continuous, discontinuous, and marginal zones, respectively , seven in the discontinuous zone , and nine in the marginal zone  . Populat2, 1 pmol each of forward and reverse primers, 0.2 μL of 10 mmol/L dNTP Mix, 1 μL 10X NovaTag Hot Start Buffer, and 0.25 U NovaTag Hot Start DNA Polymerase . The best results were obtained by performing a touchdown PCR that decreased the annealing temperature by 0.2°C every other cycle. At the end of each cycle, we added a final 72°C extension step. Loci developed by O'Connell and Ritland ). Amplification was performed by a gradient polymerase chain reaction (PCR) in a total volume of 10 μL using a 96-well GeneAmp PCR System 9700 . Each reaction mixture contained 2.5 μL of DNA extract, 2.5 mmol/L MgCl Ritland  for Thujo et al.  for Chammorphism . Prior tFst] with and without ignoring the null alleles at each locus . The genetic estimates that we compared included AR, Ho Nei , gene dii s] Nei , Fis . The neighbor-joining tree was bootstrapped 1000 times.To reveal genetic structure, and test if the samples could be clustered according to their respective distribution zones, we used STRUCTURE v. 2.3.2 software  , ancestral population size at the time population started to decline or expand (N1), and time (in generations) since population started to decline or expand (T). The change in population size was determined by the ratio r (r = N0/N1) where r < 1 indicates decline, r = 1 indicates stability, and r > 1 indicates expansion (Beaumont http://cran.r-project.org/).We tested for a recent population genetic bottleneck using the program BOTTLENECK v. 1.2.02  were obtained when either excluding or not excluding the null alleles  to 17 (Locus TP12) . Our rescus TP10 . MICRO-C alleles .a ranged from 5.3 (MZ6) to 10.0 (MZ8), with an average of 7.8. The mean Ne was 4.9, with lowest value being 3.5 (CZ3) and the highest being 6.4 . Ho had a mean value of 0.7 and was lowest in population MZ3 (0.463) and highest in population CZ6 (0.883). The mean He was 0.77, ranging from 0.712 (CZ2) to 0.826   to 6.9 . NZ3, CZ7) .When populations were pooled, AR was quite similar among the three distribution zones , as was Na. Ho showed an increase from the marginal zone (0.657) to the discontinuous zone (0.697), further, to the continuous zone (0.831) . The popFis = 0.145; PHW = 0.0125). Positive Fis values and heterozygote deficiency were also observed in populations from the marginal  and discontinuous  distribution zones. However, populations from the continuous zone were in HW equilibrium   showed a significant deficiency of heterozygotes and a departure from Hardy–Weinberg equilibrium (data not shown). None of the populations from the continuous distribution zone exhibited significant departure from HW equilibrium (data not shown). When populations were pooled, the global HW test revealed a significant departure from equilibrium and a slight heterozygote deficiency ( 0.3625) .o among the populations from the three zones was highly significant (P = 0.003), as were differences for Fis (P = 0.002) and Relc (P = 0.005). We did not find any significant differences for AR, Hs, Fst, and Rel among the populations from the three zones  and between the marginal and continuous zones (P = 0.001) (data not shown). Similarly, the differences between populations for Fis and Relc were only significant between the discontinuous and continuous zones  and between the marginal and continuous zones  (data not shown).Further comparisons revealed that the difference in HBayesian analysis demonstrated the presence of population structure. The three clusters detected by STRUCTURE  are dispThe results of the NJT that were based on Nei's Nei  standardAMOVA revealed a significant level of differentiation among the EWC populations, with 7.7% of the variation found among populations and 92.3% within populations . When thr = 0.645, P = 0.001). However, this correlation became non-significant when the populations from Chibougamau  were excluded from the analysis   . Moreover = 0.87). Several populations  had r-ratios slightly below 1, which indicated a slight decline in population size (r > 1)  under both TPM and SMM models. However, population MZ4 had a normal L-shaped allelic distribution, indicating that the bottleneck was not recent or that the population is not completely isolated. Bayesian MSVAR detected a population decline in marginal population MZ8 (ion size . The rem (r > 1) .Fis, possibly because the fragmentation was too recent. In many published studies, the sampled adults were established before fragmentation occurred . It was also higher than those values that were reported in EWC populations by Matthes-Sears et al.  as et al.  (1.6%). s et al. . The evis et al. . GeneticThe majority of studies that have examined geographic variation in genetic diversity have used a ‘categorical approach' in which only groups of peripheral and central populations were sampled  from Chibougamau and one population (DZ6) from Abitibi were assigned to one cluster, even though more than 400 km separated DZ6 from the Chibougamau marginal populations. One explanation may be that these populations followed the same post-glacial migration route. Apparently, the four populations from Témiscamingue  belonged to the same cluster, indicating that gene flow  was high among them. Some sub-branches of the NJT were significant , such as the sub-branch clustering of DZ1 and DZ5 or that of DZ2 and DZ3. These populations that clustered together are genetically closer and may have followed similar post-glacial migration routes. Fourteen populations  were assigned into a single (yellow) cluster.Our results converged to demonstrate that spatial isolation of marginal EWC populations is not associated with low genetic diversity. Therefore, increased inbreeding does not lead to a loss of genetic variation in northern EWC populations, and therefore they have the potential to respond and adapt to environmental changes. The actual distribution and expansion of white cedar at the northern edge of its range has been limited by climate in association with fires Paul . This li


Timema cristinae stick insects. We quantify whether loci that are highly divergent between geographically separated  populations exhibit unusual patterns of introgression in admixed populations. To the extent this is true, highly divergent loci between allopatric populations contribute to reproductive isolation in admixed populations.Genetic divergence during speciation with gene flow is heterogeneous across the genome, with some regions exhibiting stronger differentiation than others. Exceptionally differentiated regions are often assumed to experience reduced introgression, i.e., reduced flow of alleles from one population into another because such regions are affected by divergent selection or cause reproductive isolation. In contrast, the remainder of the genome can be homogenized by high introgression. Although many studies have documented variation across the genome in genetic differentiation, there are few tests of this hypothesis that explicitly quantify introgression. Here, we provide such a test using 38,304 SNPs in populations of As predicted, we find a substantial association between locus-specific divergence between allopatric populations and locus-specific introgression in admixed populations. However, many loci depart from this relationship, sometimes strongly so. We also report evidence for selection against foreign alleles due to local adaptation.Loci that are strongly differentiated between allopatric populations sometimes contribute to reproductive isolation in admixed populations. However, geographic variation in selection and local adaptation, in aspects of genetic architecture , and in stochastic evolutionary processes such as drift can cause strong differentiation of loci that do not always contribute to reproductive isolation. The results have implications for the theory of ‘genomic islands of speciation’. Levels of genetic differentiation are highly variable across the genome-5. In paBut many processes other than selection and reproductive isolation can promote genetic differentiation. If overall gene flow levels are very low, for example due to geographic isolation, then pronounced genetic differentiation might arise due to the stochastic effects of genetic drift-31. ThesThere are several ways to test if highly differentiated regions reside in regions of reduced introgression. First, mapping studies could test whether phenotypic traits involved in divergent adaptation or causing reproductive isolation map to regions of strong divergence between natural populations,39. SecoTimema cristinae stick insects. The genotyping-by-sequencing approach which generated these data used restriction enzymes to cut up the genome into DNA fragments that are distributed across the genome, sequenced tens of millions of these fragments on the Illumina next-generation sequencing platform, and aligned the fragments to discover genetic variation . This approach was thus aimed at surveying genome wide patterns of differentiation, rather than focusing in on specific genes that causally affect adaptation and speciation. Previous work used this dataset to test which ecological and geographic factors affect variability in genetic differentiation [Here, we adopt this third approach following using a  in FST). The res in FST). PreviouST) between two allopatric  populations of T. cristinae. This step included, but was not restricted to, the identification of high-FST outlier loci [Our approach involves three steps Figure. First, lection). Second,lection). HoweverTimema are wingless, herbivorous insects inhabiting Southwestern North America[T. cristinae, which uses two host species (Adenostoma fasciculatum: Rosaceae and Ceanothus spinosus: Rhamnaceae)[ America. The curmnaceae). Populatmnaceae).T. cristinae are sometimes separated from patches of the alternative host, usually via regions containing unsuitable hosts. All populations found in such geographically separated patches are termed ‘allopatric’. We study here two allopatric populations, LA and PRC, which are known from past work to be strongly genetically differentiated, undergo little or no between-host gene flow, and are thus ‘non-admixed’[ST) here thus all stem from LA and PRC [1,6 = 13.76, p = 0.021). Our estimates of introgression here thus all stem from the admixed populations HV and MR.In contrast to allopatric scenarios, the two host species are sometimes distributed in adjacent patches that are in direct geographic contact with one another. Previous work has shown that such adjacent populations undergo extensive between-host gene flow and are ‘admixed’,47-50. Wanothus). NonetheThe analytical approach we use is that of ‘genomic clines’. GenomicT. cristinae. We were not concerned with the geography of introgression, but rather with the movement of genetic material from one genomic background into another within a geographic region i.e., genomic introgression[j with hybrid index H = h inherited a gene copy at locus I = i from LA . The base probability of LA ancestry for a locus is equal to an individual’s hybrid index. The genomic cline center parameter, α, specifies an increase  or decrease  in the probability of LA ancestry for a locus relative to the base expectation. The genomic cline rate parameter, β, specifies an increase  or decrease  in the rate of transition from low to high probability of LA ancestry as a function of hybrid index and measures the mean ancestry-based pairwise linkage disequilibrium between a locus and all other loci. More formally,We thus measured genomic introgression of LA and PRC genetic regions in admixed HV and MR gression,20,52-54where Φ_ij is given by a simple transformation of Φ_ij to ensure 0≤Φ≤1 and that Φ is a monotonically increasing function of hybrid index. Simulations have demonstrated that selection against specific hybrid genotypes , whether arising from single locus (underdominance) or multilocus (Dobzhansky-Muller) incompatibilities, affects α and β, but the effect of selection on α is often more pronounced, particularly if dispersal from parental populations is limited,52. UndeT. cristinae system demonstrated that multiple geographic and ecological factors each leave clear patterns in the genome[Related work in the e genome. We stress that this analysis relies strictly on the departure of individual loci from the genome-wide hybrid index , rather than on average hybrid index (h) itself.Here, we extend this previous work in two major ways. First, we provide novel analyses testing whether local adaptation results in selection against foreign alleles, a process that can reduce introgression and promote the genetic differentiation that drives speciation. The admSecond, we test for correspondence between locus-specific differentiation between allopatric populations and locus-specific introgression in admixed populations. Here we focus on absolute  levels of α because although loci with highly negative versus highly positive α values differ in their probability of LA versus PRC ancestry, both loci with highly negative and highly positive α values exhibit exceptional patterns of introgression in admixed populations. A wide range of outcomes is possible because reproductive isolation can evolve via selection or drift and might evolve via similar or different processes in allopatric versus admixed populations. We focus here on some likely possibilities.ST are geographic variation in the genomic architecture of local adaptation or in local selection pressures. Both these types of variability among populations can cause high FST and α to occur in some geographic regions, but not in others. Such factors could also explain loci with high α but low FST. We enumerate the frequency of these different outcomes and discuss the results with respect to understanding speciation.First, some regions might exhibit strong divergence between the allopatric populations and high (absolute) α values in the admixed populations. These likely represent regions that truly differentiated between LA and PRC via divergent selection, and which contribute to reproductive isolation in admixed populations . Second, some regions will exhibit low divergence and low (absolute) α values. These likely represent neutrally evolving regions that do not readily differentiate and which do not contribute to reproductive isolation. Third, some regions might exhibit strong divergence in allopatry but low (absolute) α in the admixed populations. Such regions might not harbor genes under divergent selection or those reducing introgression, but have nonetheless differentiated strongly in allopatry due to genetic drift. Alternatively such regions could harbor genes experiencing divergent selection in allopatry that are unrelated to reproductive isolation in admixed populations. Possible mechanisms causing such discordance between α and FST and genomic cline parameter estimates[ST estimation and methods for delimitation of ‘outlier loci’ with exceptionally high FST values are described in full detail in[ST, because it fully accounts for uncertainty in genotypes, allele frequencies, and FST caused by missing data and low sequence coverage. Low sequence coverage will simply cause greater uncertainty in FST  and will mean that FST for the locus will be more similar to the prior expectation . In other words, for a locus to be an outlier or have a high FST there must be sufficient data for that locus for the likelihood (based on the data for that locus) to overcome the prior (based on the genome-average FST).The data we analyzed come from six of the eight populations examined by than expected by chance in admixed populations. The genomic cline parameter α represents a deviation from the ancestry probability predicted solely from hybrid index and is constrained to sum to zero. Thus, we used the expectation that 50% of outlier loci should have α > 0 as a null hypothesis. We tested for a significant deviation from this expectation based on a binomial probability distribution with pLA = 0.5. Additionally, we obtained Bayesian posterior estimates of the probability that FST outlier loci had estimates of α > 0 by specifying a binomial likelihood for the number of FST outlier loci with α > 0 and an uninformative beta prior on pLA . We also conducted these analyses with all 38,304 loci for comparison.The zones of admixture we studied were geographically closer to LA than to PRC. If local adaptation varies relatively continuously along environmental gradients  and thus selects against foreign PRC alleles in the admixture zones, we predict an excess of loci with elevated LA ancestry , relative to those with elevated PRC ancestry . We thus tested whether high-FST and locus specific absolute values of α. We used the empirical quantiles of the estimated FST and absolute α values to delimit different combinations of high and low values for these two parameters . For FST, we used the 95th quantile to determine loci with ‘low FST’ . For α, we are interested in extreme negative and positive values. Therefore we used the 2.5th and 97.5th empirical quantiles .We used correlational analyses to test for relationships between locus-specific FWe observed substantial variation in genomic introgression Figures. We deteST outlier loci from the allopatric population pair there was a consistent and significant excess of highly positive α values relative to highly negative α values in the admixed populations  α values  α despite high FST (3.7%).Here we focus on absolute levels of α because although loci with highly negative versus highly positive α values differ in their probability of LA versus PRC ancestry, both loci with highly negative and highly positive α values exhibit exceptional patterns of introgression in admixed populations. We detected a highly significant positive correlation between FWhen populations diverge in the face of gene flow, exceptionally differentiated genomic regions are often assumed to harbor genes undergoing reduced introgression whereas, in contrast, the remainder of the genome is homogenized by high introgression,5,8. HowHere we provided such a test and found some correspondence between locus-specific divergence and locus-specific introgression. Specifically, we detected accentuated ancestry from either allopatric population for highly differentiated loci, consistent with such highly divergent regions contributing to reproductive isolation in the admixed populations. There are reasons to expect such correspondence in the system examined; population sizes are large, introgression in admixed populations is known, admixed populations are relatively old, and gene flow from allopatric populations into admixed populations is low. These aThe observed correspondence between divergence and introgression is consistent with previous work in other systems documenting genomic islands of divergence,23-26,56ST outlier loci with positive α values in admixed populations; that is, with accentuated LA ancestry. This result is suggestive of local adaptation to the environmental conditions around LA, which might be expected given that LA is more proximate to the admixture zones than is PRC. Most previous work on this system focused on the effects of divergent host adaptation[Timema system more generally. In particular, it would be interesting to estimate genomic introgression in admixed populations that are near PRC (which do exist in nature). The prediction would be an excess of loci with PRC ancestry, and this should occur for loci that had excess LA ancestry in the current study .We documented an excess of high-Faptation. Howeveraptation. Thus, fFinally, we note that we did observe some loci that were strongly divergent between allopatric populations and had highly negative (rather than positive) α values. Although many selective pressures in the admixed populations studied here might mirror the conditions in LA, some might mirror those in PRC, particularly if selection is highly multifarious. Thus, these loci with highly negative α values could represent loci responding to conditions that mirror those in PRC . Again, further studies of genomic introgression in admixed populations near PRC could test this prediction.ST and α. However, many loci depart from this relationship, leading to the different outcomes discussed previously. If FST and α were completely independent, we would expect the frequency of the four following outcomes to be as follows (at the 95th quantile level): high α, high FST = 0.05 * 0.05 = 0.25%; low α, low FST = 0.95 * 0.95 = 90.25%; high α; low FST = 0.05 * 0.95 = 4.75%; low α; high FST = 0.95 * 0.05 = 4.75%, etc. Instead, we observed an excess of loci falling into the first two categories, and a slight deficiency of loci falling into the latter two. This is exactly what is expected given the correlation between FST and α .Our results show a general association between FST and α is expected in T. cristinae. For example, allopatric populations undergo little or no gene flow[T. cristinae exist in a mosaic patchwork and there is evidence that divergence in host use arose multiple times[ST and α. In summary, our results are consistent with some regions of strong differentiation harboring genes subject to divergent selection, but also often representing different outcomes such as divergence by drift or driven by geographic variation in genetic architecture or local selective regimes.Some discordance between Fene flow,47,50. Tle times,59. ThusThe findings shed insight into the theory of ‘genomic islands of speciation’-26. In tST and absolute values of α, even in the absence of selection[ST and absolute values of α, but only for loci with low FST values[ST values , the relationship between FST and α in simulated datasets was erratic and not consistently positive.There are three main caveats that should be taken into account when interpreting our results. First, loci that are more differentiated among populations carry more information about ancestry, and this could affect estimates of α. Thus, variation in the ancestry-information content among loci can generate a relationship between Felection. PreviouT values. For locST and α in our data for loci with varying minimum FST values . This was done for each admixed population separately. In the data, correlations between FST and α decrease within increasing FST, particularly for loci with FST > 0.08  than the allopatric populations are from each other , as estimated from the size of the host-plant patch each population occupies (which is highly correlated with insect population size in this species) and as eT. cristinae. These experimental results can then be coupled with the genomic clines analyses presented here and functional genomic studies to gain a more complete picture of the associations among genetic divergence, selection, reproductive isolation, and speciation.The approach and results presented here provide a test of the idea that genomic regions of strong divergence can harbor genes that are important for speciation. However, a number of further lines of evidence could be used to strengthen this conclusion, and to refine the results. For example, it will be of interest to learn the genomic distribution of the loci examined and to test which regions are linked to phenotypic traits involved in divergent adaptation. Such mapping studies are currently underway. Additionally, several field experiments designed to measure selection at the genomic level have now been implemented in Timema spanning a wider range of divergence  in an attempt to reconstruct how the relationship between genetic divergence and reproductive isolation might change as the continuous process of speciation unfolds.Finally, the current study examined populations that are at a particular point in the speciation process, and an early one where reproductive isolation is far from complete. The resThe authors declare no competing interests.PN, TP, JF, and ZG conceived the project. PN, TP, and ZG conducted the analyses. PN, TP, JF, and ZG wrote the manuscript. All authors read and approved the final manuscript.Figure S1. TMap of the eight study populations examined by Nosil et al.[l et al.. The curClick here for fileFigure S2. The relationship between the number of outlier loci  and the geographic distance between populations , for geographically separated and geographically adjacent population pairs . Both the effects of geographic distance itself, and of geographic arrangement (separated versus adjacent) were statistically significant. The thick arrow labels the point at which zero gene flow between population pairs was achieved, where gene flow was estimated from genomic data using Approximate Bayesian Computation. Also shown is a picture of the study organism. Modified from Nosil et al.[l et al..Click here for fileFigure S3. The distribution of FST values under different degrees of geographic separation. The top panel shows the distribution of FST values across individual loci. The bottom panel presents a barplot of the distribution of point estimates for logit(FST) across the genome. The dashed black line is the genome-wide distribution of logit(FST) ). The vertical red line in each pane denotes the 95th quantile of the genome-wide distribution, which was used to delimit high FST outliers. In all instances, some outlier loci were detected but the FST distribution tended to be the most ‘L-shaped’ for geographically-adjacent pairs and became less ‘L-shaped’ with increasing geographic separation of populations. Modified from Nosil et al.[l et al..Click here for fileFigure S4. Results of genomic clines analyses for the population pair MR. a) the 95% credible intervals for genomic cline parameters α. Loci are sorted by the point estimate of α and 95% CI's that do not include zero are shown in black . There are over 30,000 lines, thus individual 95% CI’s are difficult to see. b) Genomic clines for 1000 representative loci. Black lines denote clines with α values whose CI do not include zero. Grey lines denote loci whose α values had CI that did include zero. c) The correlation between FST and α. See text and TablesClick here for fileFigure S5. The correlation coefficient for the relationship between locus-specific FSTand locus-specific absolute α values (y-axis) for loci with FST values above certain thresholds (x-axis). Open circles are HV. Closed circles are MR. Numbers of loci that had FST values above each threshold are as follows .Click here for file


Locustella luscinioides), mitochondrial DNA sequences (ND2) and microsatellites were analysed in c.330 individuals of 17 breeding and two wintering populations. Phylogenetic, population genetics and coalescent methods were used to describe the genetic structure, determine the timing of the major splits and model the demography of populations. Savi’s Warblers split from its sister species c.8 million years ago and have two major haplotype groups that diverged in the early/middle Pleistocene. One of these clades originated in the Balkans and is currently widespread, showing strong evidence for population expansion; whereas the other is restricted to Iberia and remained stable. Microsatellites agreed with a genetic break around the Pyrenees, but showed considerable introgression and a weaker genetic structure. Both genetic markers showed an isolation-by-distance pattern associated with the population expansion of the eastern clade. Breeding populations seem to be segregated at the wintering sites, but results on migratory connectivity are preliminary. Savi’s Warbler is the only known migratory bird species in which Iberian birds did not expand beyond the Pyrenees after the last glaciation. Despite the long period of independent evolution of western and eastern populations, complete introgression occurred when these groups met in Iberia. Mitochondrial sequences indicated the existence of refugia-within-refugia in the Iberian Peninsula during the last glacial period, which is surprising given the high dispersal capacity of this species. Plumage differences of eastern subspecies seemed to have evolved recently through natural selection, in agreement with the glacial expansion hypothesis. This study supports the great importance of the Iberian Peninsula and its role for the conservation of genetic variation.In order to describe the influence of Pleistocene glaciations on the genetic structure and demography of a highly mobile, but specialized, passerine, the Savi’s Warbler ( Historic events, such as changes in climate and topography, have been shown to leave genetic signatures that subsist to the present time However, species-specific patterns are found in the timing of divergence between major genetic lineages, in the number and location of refugia as well as on post-glacial colonization routes, even if only temperate species are considered Locustella luscinioides), a small, insectivorous passerine that breeds exclusively in medium to large reed beds across the Palaearctic, and winters along a narrow belt in sub-Saharan Africa L. l. fusca occurs. A third subspecies (L. l. sarmatica) is sometimes recognized, occurring from the Sea of Azov to the Black Sea (i.e. on the southeast part of the western range). These subspecies were described on the basis of subtle plumage colour differences, with eastern birds having paler underparts, olive rather than brown upperparts, and more obvious white tips to under-tail coverts In this study, we describe for the first time the phylogeography of the Savi’s Warbler  and between its major genetic lineages, thereby assessing the importance of the Pleistocene glaciations to a specialized, naturally-fragmented species; and (3) identify the number and location of refugia within Europe, as well as the major post-glacial expansion routes, comparing with other species.We analysed the phylogeography and population genetics of birds belonging to 17 breeding populations, plus a few birds from two wintering sites. Our purpose was to: (1) evaluate the mitochondrial and nuclear genetic structure, identifying evolutionary units that could potentially be of conservation value; (2) determine the timing of divergence of the Savi’s Warbler from its sister species  was amplified and sequenced from both ends using the primers L5215 In addition, eight microsatellite loci were used for population genetic analyses: Ase64 and Ase18 eneious 5.1 D and Fu’s Fs neutrality tests were calculated in Arlequin 3.5.1.2 naSP software rlequin and compared with models of sudden population expansion (estimated from 100 bootstrap replicates) using the sum of squared deviations test.The ND2 sequences were aligned and edited in GyML jModelTesteneious produced similar results and are not shown.A haplotype network was calculated in TCS east 1.6.1 east was also used to determine the age of the major nodes in two models: (1) a constant population model that includes all Savi’s Warbler haplotypes plus the River Warbler sequences; and (2) a population expansion model, in which all sequences of Savi’s Warblers (including repeated haplotypes) were analysed. For both models, lognormal and strict molecular clocks were implemented with a uniform prior for substitution rates varying from 0.0095 to 0.0115 b gene . The models were run multiple times in Beast and evaluated in the program Tracer 1.5, in order to assess the influence of different priors and chain length on the effective sample size and convergence. Final models were run for 20 million generations and sampled every 2000 generations, which resulted in 10000 trees that were summarized and visualized with TreeAnnotator 1.6.1 and FigTree 1.3.1 (discarding the first 10% of trees). Beast was also used to model the demographic changes of the two major clades using the Bayesian skyline analysis racer.In addition, we used the coalescent analysis implemented in Bicro-checkerenAlEx 6.1 DestenAlEx for better visualization of the genetic relationships among populations.The microsatellites were first evaluated with Mtructure 2.3.3 was also used to detect population structure 4 and 106 Markov Chain Monte-Carlo interactions. Multiple runs varying the priors and number of interactions resulted in identical conclusions. The number of clusters was determined using the ad hoc statistics (?K) following Evanno et al. tructure Harvester 0.3 The individual population assignment method implemented in SThe collection of all samples was conducted under the licenses required by the corresponding national authorities. Permits were given by the following institutions: Bulgarian Ministry of Environment and Waters (N76/06.07.2006), Conselleria de Medi Ambient, Aigua, Urbanisme i Habitatge, Generalitat Valenciana (440066), Consejería de Medio Ambiente de la Junta de Andalucía (6305), National Environmental, Conservation and Water Inspectorate (14/2436-3/2006 and 14/140/3/2007), Krajsky urad Jihomoravskeho kraje (JMK 31771/2006), Instituto de Conservação da Natureza (73/2005), Centre de Recherches par le Baguage des Populations d’Oiseaux (381805EH44805), State Committee for Hunting and Fishing of Tyva Republic.We analysed the complete (1041 bp) ND2 gene of 330 individuals sampled from 17 breeding and two wintering sites, and also included one sequence available in GenBank from Krasnodar, Russia The haplotype network  and the The two Savi’s Warbler clades have different structures and diversity, as well as geographical distribution . The mosFs =  –0.009, P = 0.5; Tajima’s D = 0.036, P = 0.5), and similar results are obtained for the mistmatch and Bayesian skyline analyses.Clade B, which occurred exclusively in the Iberian Peninsula, has a lower diversity and is more structured than clade A. Indeed, three of the haplotypes  that differ from the most frequent one (h46) by 3–5 substitutions are clustered together in the minimum spanning network as well as in the ML tree  and 2. Teast produced a maximum clade credibility tree similar to the ML tree, and full posterior support for the nodes splitting the clades A-B, and B1–B2 , and the split between clades A and B , and between clades B1 and B2  were slightly older than in the population growth model , the divergence between Savi’s and River Warblers occurred 8.34 MYBP, whereas between clades A and B and B1/B2 was 1.07 and 0.23 MYBP, respectively. The expansion models with this molecular rate yielded divergence dates for clades A/B and B1/B2 of 0.79 and 0.13 MYBP, respectively.Neutral tests and mismatch analysis of the sampled populations show strong evidence for population expansion in the Czech Republic, Hungary and Shabla, Bulgaria, but not for the populations in the Iberian Peninsula, France or Russia .ΦST was highly significant  and varied between zero and 0.63 (Doñana vs. France). It was particularly high between the Iberian sites and the remaining populations, and Russia also stands out being significantly different from the remaining populations (ΦST/(1-ΦST)+0.519; R2 = 0.325; P = 0.06), but the remaining sites still showed a very significant pattern of IBD +0.216; R2 = 0.345; P<0.008).The overall ulations . The AMOulations . When thulations , in agreulations . Within Only 14 out of the 112 tests for HWE were significant. In one population  three out of eight loci were not in HWE, and one locus (Pdoμ5) was not in HWE in six out of 14 populations. However, the fixation index was very low for most loci except Gf05 , probablGST , varying between zero and 0.117  than in the GST analysis  three out of eight loci were not in HWE, and one locus (Pdoμ5) was not in HWE in six out of 14 populations. However, the fixation index was very low for most loci except Gf05 , probablGST , varying between zero and 0.117  than in the GST analysis .We also carried out an AMOVA comparing microsatellite diversity between individuals from the Iberian Peninsula that carried mtDNA haplotypes of clade A versus those with clade B. The percentage of variation between these two “populations” was very low (1%), but significant  ΦST/(1-ΦST)+0.066; R2 = 0.009; P = 0.407) but, as with mtDNA, the remaining sites showed a highly significant pattern of IBD +0.052; R2 = 0.366; P<0.001).The individual-based analysis implemented in SThe two wintering populations show evidence for little mixing: the birds from Senegal belong to the Iberian populations, both according to the mtDNA  and the Phylogeographic analysis has revealed common patterns in the genetic structure of numerous species, suggesting that at least some historical events were extremely influential at the community level Our study on a migratory, habitat-specialist bird revealed considerable genetic structure, especially within the populations belonging to the nominate subspecies. It was shown that Savi’s Warblers have two major mitochondrial lineages that originated in the early or middle Pleistocene and evolved separately for a period of time spanning several glacial and interglacial periods. The distribution of these lineages and their genealogy is consistent with the existence of two refugia: one in Iberia and the other in the Balkan region; and the latter was the source of individuals that colonized most of the Palaearctic. Our results indicate that the two clades met only very recently, rather than during previous interglacial periods, as mismatch and Bayesian skyline analyses are consistent with population expansion only after the last glacial maxima. Our results also support the observation of eastern populations expanding earlier and more extensively than western populations, either because there are fewer barriers to dispersal, larger populations, larger patches of suitable habitat and/or the climate ameliorated comparatively earlier in the east than in western Mediterranean In Savi’s Warblers, the post-glacial colonization proceeded towards Europe and Asia from the Balkans and resulted in an IBD pattern ΦST takes into account the distance between haplotypes, whereas GST only considers differences in allele frequencies among populations. These differences in the measurement of genetic distance (as well as homoplasy and greater effective population size of microsatellites) might be the reason for some Iberian sites appearing much closer to central European sites in nuclear than in mitochondrial DNA , the Green Woodpecker (Picus viridis) and the Red-legged Partridge (Alectoris rufa), all of which are sedentary species Phylloscopus ibericus) also meet with European Chiffchaffs (P. collybita) in the Pyrenees, but their divergence is much older than the species considered earlier, as the mtDNA divergence between these species is 4.6% Acrocephalus arundinaceus), Iberian birds did colonize Europe after the last glaciation . But the pattern is still not clear for other species like the Reed Bunting (Emberiza schoeniclus), for which there are two near-endemic Iberian subspecies Acrocephalus scirpaceus), in which Iberian and African birds seem to be genetically distinct from the rest of the Palaearctic  Overall, our results agree well with other phylogeography studies from Europe Phylloscopus trochilus), whose expansion around the Baltic led to the evolution of distinct migratory directions and a hybrid zone in mid-Scandinavia GST between birds with mtDNA belonging to the two clades within Iberia, which could perhaps reflect the asymmetrical introgression from the local birds to the expanding population expected from theory Second, despite the long period of isolation between the two refugial populations, post-glacial expansion resulted in complete introgression in the Iberian Peninsula, rather than in a suture or hybrid zone. Hybrid zones can occur among populations that are much more closely related than Savi’s Warbler’s populations, as in the case of Willow Warblers (Picus vaillantii) are distinct from Iberian birds Ficedula speculigera) Chersophilus duponti), which seems to have had two isolated refugia in these regions during the last glaciation Third, genetic structure within the Iberian clade is consistent with the existence of two refugia within the Iberian Peninsula during the last glaciation. Refugia within refugia have been found in amphibians, reptiles and small mammals in Iberia, in which mountain ranges and, sometimes, major rivers seemed to constitute insurmountable barriers for dispersal This study shows that Savi’s Warbler’s populations of the Iberian Peninsula deserve a special conservation effort, as they hold a high genetic variation that includes all the western haplotypes, and should be considered a distinct management unit Finally, genetic variation within the Savi’s Warbler seems to be large enough to determine broad patterns of migratory connectivity Table S1Haplotype frequencies per population. Colours indicate the haplotypes belonging to clades A (blue), B1 (red) and B2 (brown). Population names are as described in (EPS)Click here for additional data file.Table S2Microsatellite statistics. Mean and standard error (SE) of microsatellite statistics across populations (above) and across loci (below): number of individuals (n), number of alleles (Na), effective number of alleles (Ne) observed heterozygosity (Ho), expected heterozygosity (He), unbiased He (UHe) and fixation index (F). Population names are as described in (EPS)Click here for additional data file.Figure S1Maximum clade credibility tree. Result of a coalescent analysis implemented in BEAST with strict clock (uniform distribution 0.0095–0.025 substitutions per million years) and constant population size as priors. Horizontal blue bars represent the 95% HDP of the age of major nodes; branch length and posterior probability of the nodes are also indicated.(EPS)Click here for additional data file.


To genotype and evaluate a panel of single-nucleotide polymorphisms for individual identification (IISNPs) in three Chinese populations: Chinese Han, Uyghur, and Tibetan.forID 52-plex markers, were pooled and analyzed. Four SNPs were included in both panels. In total, 132 SNPs were typed on Sequenom MassARRAY® platform in 330 individuals from Han Chinese, Uyghur, and Tibetan populations. Population genetic indices and forensic parameters were determined for all studied markers.Two previously identified panels of IISNPs, 86 unlinked IISNPs and SNPe) ranged from 0.144 to 0.500 in Han Chinese, from 0.197 to 0.500 in Uyghur, and from 0.018 to 0.500 in Tibetan population. Wright's Fst values ranged from 0.0001 to 0.1613. Pairwise linkage disequilibrium (LD) calculations for all 132 SNPs showed no significant LD across the populations (r2<0.147). A subset of 58 unlinked IISNPs (r2<0.094) with He>0.450 and Fst values from 0.0002 to 0.0536 gave match probabilities of 10−25 and a cumulative probability of exclusion of 0.999992.No significant deviation from Hardy-Weinberg equilibrium was observed for any of the SNPs in 3 populations. Expected heterozygosity (HThe 58 unlinked IISNPs with high heterozygosity have low allele frequency variation among 3 Chinese populations, which makes them excellent candidates for the development of multiplex assays for individual identification and paternity testing. Single-nucleotide polymorphisms (SNPs) are often used as a supplementary tool to short tandem repeats (STRs) analysis  since thst values <0.06 for 44 major populations across the world (forID consortium (www.snpforid.org) developed a 52-plex SNPs assay for individual identification  who had provided written informed consent. Ethical approval was received from the Review Board, Institute of Forensic Sciences, Ministry of Public Security.® DNA blood midi kit . DNA quantification was performed with adding 1.5 μL DNA samples in solution on NanoDrop® ND-1000 spectrophotometer .DNA was isolated and purified with a QIAamp® platform with the iPLEX GOLD chemistry  following the manufacturer’s protocols. Polymerase chain reaction (PCR) primers and locus-specific extension primers were designed using MassARRAY® Assay Design software package (v. 3.1). DNA template of 50 ng was used in each multiplexed PCR well. PCR products were treated with shrimp alkaline phosphatase  before the iPLEX GOLD primer extension reaction. The single base extension products were desalted with SpectroCLEAN® resin (Sequenom), and then an aliquot of 10 nL of the desalted product was spotted onto a 384-format SpectroCHIP® with the MassARRAY® Nanodispenser. Mass determination was done with the MALDI-TOF mass spectrometer. The MassARRAY® Typer 4.0 software was employed for data acquisition.SNPs genotyping was performed using the Sequenom MassARRAYe), Hardy-Weinberg equilibrium (HWE), and Wright's Fst value, were calculated for each SNP marker with Genepop v4.2. Fst and δ values were used to measure variance in allele frequencies among populations. Random match probability (RMP), probability of exclusion (PE), and other forensic parameters were calculated with PowerStats v. 12. HaploView v. 4.2 genetics software was implemented to estimate linkage disequilibrium (LD) values (r2) of each pair of SNPs. HapMap genotype data were used for population comparison analyses. All individual and marker data were filtered with more than 20% of missing alleles.Population indices including allele and genotype frequency, expected heterozygosity . There were no significant LD values among the SNPs (pair-wise r2<0.147). He ranged from 0.144 to 0.500 in Han, from 0.197 to 0.500 in Uyghur, and from 0.018 to 0.500 in Tibetan population. Wright's Fst values were from 0.0001 to 0.1613. Allele frequencies ranged from 0.114 to 0.922 in Han, from 0.118 to 0.889 in Uyghur, and from 0.055 to 0.991 in Tibetan population. Pair-wise δ values ranged from 0 to 0.324 (Supplementary Table 2)We typed 132 SNPs in 330 individuals from Han, Uyghur, and Tibetan subpopulations (Supplementary Table 1)2<0.094) separated on 22 autosomal chromosomes. This subset consisted of 41 markers from the Pakstis' panel (20 recommended ones), 14 markers from the SNPforID panel, and 3 markers from both panels. He values were >0.450 and Fst values ranged from 0.0002 to 0.0536. Match probabilities of Han, Uyghur, and Tibetan populations were 5.91 × 10−25, 7.79 × 10−25, and 6.61 × 10−25, respectively. The cumulative probability of exclusion for these three populations was 0.999992.Among the 132 IISNPs, we identified a set of 58 unlinked markers (re>0.450) and very similar frequencies (Fst<0.060) across the three Chinese population samples. One hundred and thirty-two autosomal IISNPs were screened in three Chinese populations. These markers have previously been tested in many populations . The panel meeting this criterion should have high power of discrimination and allow for universal uses of individual identification. Thus, we considered the subset of 58 unlinked SNPs, which had previously been tested for a large number of world-wide populations, an excellent panel for practical use in Chinese populations. It had match probabilities of 10−25, which is comparable to the CODIS STR panel and other existing SNP typing systems (Each of the 58 unlinked markers from two IISNP panels had high heterozygosity (Hulations ,18,19. I systems ,8,19. As systems ,21 chrom systems ,23 shoulIndividual identification requires an easy-to-use multiplex SNP genotyping system, which can be read with capillary electrophoresis. Furthermore, a stable and reliable detection system should include more population samples. In conclusion, the 58 unlinked IISNPs identified here would be an ideal panel for further multiplex system optimization and validation among Chinese populations.


Macaca mulatta is the most widely distributed non-human primate. It may be the most suitable model to test hypotheses regarding the genetic consequences of orogenesis on an endotherm.Pliocene uplifting of the Qinghai-Tibetan Plateau (QTP) and Quaternary glaciation may have impacted the Asian biota more than any other events. Little is documented with respect to how the geological and climatological events influenced speciation as well as spatial and genetic structuring, especially in vertebrate endotherms. M. mulatta formed around 2.31 Ma , and divergence of the two major matrilines was estimated at 1.15 Ma . The western super-haplogroup exhibits significant geographic structure. In contrast, the eastern super-haplogroup has far greater haplotypic variability with little structure based on analyses of six variable microsatellite loci using Structure and Geneland. Analysis using Migrate detected greater gene flow from WEST to EAST than vice versa. We did not detect signals of bottlenecking in most populations.Using a large dataset of maternally inherited mitochondrial DNA gene sequences and nuclear microsatellite DNA data, we discovered two maternal super-haplogroups exist, one in western China and the other in eastern China. M. mulatta. The difference likely reflects inheritance mechanisms of the maternally inherited mtDNA genome versus nuclear biparentally inherited STRs and male-mediated gene flow. Dramatic environmental changes may be responsible for shaping the matrilineal history of macaques. The timing of events, the formation of M. mulatta, and the divergence of the super-haplogroups, corresponds to both the uplifting of the QTP and Quaternary climatic oscillations. Orogenesis likely drove divergence of western populations in China, and Pleistocene glaciations are likely responsible for genetic structuring in the eastern super-haplogroup via geographic isolation and secondary contact.Analyses of the nuclear and mitochondrial datasets obtained large differences in genetic patterns for  Three phases–A, B, and C, and occurring about 3.6 million years ago (Ma), 2.6 Ma, and 1.7 Ma, respectively–characterize the last orogenic push of the QTP The Pliocene uplifting of the Qinghai-Tibetan Plateau (QTP) and Quaternary glaciation may be the primary drivers of genetic divergence and patterning of the Asian biota. With a mean elevation of about 4000 m above sea level and an area of approximately 2.5 million kmMacaca mulatta, the rhesus macaque, may be the most suitable model to investigate the relationship between the non-human primates and the genetic consequences of orogenesis. Its geographic range extends from Afghanistan in the west to the coast of the East China Sea in the east. The species occurs southwards from the Himalayas and central China to central India and central Indochina. This range, which exceeds that of all other primates except for humans Geological and climatological events influenced speciation and spatial and genetic structuring, especially among ectotherms. However, much remains to be documented with respect to endothermic vertebrates. Macaca appears to have originated in North Africa approximately 5.5 Ma M. fascicularis complex are well documented Macaca fascicularis forms the sister group of the other three species of Macaca, M. cyclopis, M. mulatta, and M. fuscatafascicularis species group, which also contains rhesus and cynomolgus macaques, shared a common ancestor about 3.2 Ma Genus M. mulatta. For example, a large degree of genetic divergence occurs between Indian and Chinese populations. Smith and McDonough Diversity in the mitochondrial genome of rhesus macaques is reported from restriction fragment analyses Macaca mulatta, the maternal genetic structure, especially in the form of maternal genealogy, is likely to better reflect the history of troop dispersal and colonization than that of paternal history.Captive populations form the basis of most genetic assessments. Relatively few wild samples are assessed. Often these samples are missing specific locality data other than their provinces of origin We assess the extent of genetic diversity and the evolutionary history of the most widely distributed nonhuman primate, the rhesus macaque, by using a large dataset of maternally inherited mitochondrial DNA gene sequences and nuclear DNA microsatellite data. We test a suite of hypotheses related to the geographic structure of genetic diversity in wild Chinese rhesus macaques based on mtDNA and microsatellite markers. Further, we test hypotheses on the relative roles played by dramatic environmental changes, in particular orogenesis of the Qinghai-Tibetan Plateau (QTP), and Quaternary climatic oscillations in shaping genetic structure.The Anhui experimental rhesus monkey Center, Chengdu pingan animal Breeding and Research Center and Primate experimental animal center of KIZ have provide the provided the blood samples. Blood samples were salvaged from routine health checks for newly captured wild macaques at these breeding centers. We have obtained permission from the centers to analyze the blood samples for this study. These captures are usually as the founder populations of the breeding populations. And These captures of rhesus monkey were authorized by the specific government agency, for example, the Sichuan Department of Forestry, Yunnan Department of Forestry, or An Department of Forestry which is in charge of a certain sampling region. We collected the fecal samples from the Henan Taihang Macaque Nature Reserve, and the Zhangjiajie National Forest Park. We have obtained permission from the above administrative departments to observe the animal behavior of rhesus monkey and collect the stool samples. We also confirm that the behavioral observations did not impact the animals in any way.M. mulatta across its entire range in China  gene (882 bp) and part of rRNA 16S (861 bp). Partial fragments of the hypervariable D-loop were initially sequenced for all 337 specimens. Subsequently, 16S and Cytb fragments were sequenced for a subset of samples (66 specimens) that represented the major haplogroups identified from the genealogical reconstruction of the D-loop data . Final extension at 72°C was conducted for 10 min. We used the same procedure for Cytb and 16S but with annealing at 55°C and 59°C, respectively. Primers sequences were summarized in To reconstruct the matrilineal genealogy, three fragments from the mitochondrial genome were selected for sequencing. A partial fragment of the D-loop included 510 base pairs (bp) in length. Another two mitochondrial fragments included part of the cytochrome b . For the second dataset, phylogenetic relationships were also inferred using Bayesian inference as implemented in MrBayes v.3.1.2 Cytb: GTR+G, D-loop: HKY+I+G) was selected based on the Akaike Information Criterion as implemented in Modeltest v.3.7 For the first dataset, associations among the 214 D-loop haplotypes were also visualized by a full median-joining network rlequin v.3.11 STF, against the corresponding log10 transformed geographical distance.Using the first dataset, we tested the null hypothesis of a correlation between genetic and geographical distances, i.e. isolation-by-distance (IBD), for the matrilines using both all ten sampling localities and the six western sites that had specific locality data . Approxirlequin v.3.11. This analysis compared the observed frequencies of pairwise differences of haplotypes with those expected under a single sudden expansion model DFs statistics rlequin v.3.11, were also used to seek evidence of demographic expansions within individual haplogroups and subhaplogroups. Statistical significance was tested with 1000 permutations. The null hypothesis was that of a stable population. Additionally, we calculated the statistics of genetic diversity, neutrality tests, and mismatch distributions for the main populations of Macaca mulatta in China.Historical population dynamics of the major haplogroups assigned in the median-joining network were examined with mismatch distribution analysis beast v.1.5.3 odeltest v.3.7 Divergence times were estimated using a Bayesian MCMC method implemented in M. sylvanus and the other Asian macaques. Instead of using hardbounded calibration points, we relied on published dates and specified a normal distribution prior, with a mean of 5.5 Ma and a standard deviation of 0.5 Ma. Two replicates were run for 100 million generations while sampling trees and parameters every 1,000 generations. The adequacy of using a 50% burn-in to generate MCMC trees and for convergence of all parameters was assessed visually using Tracer v.1.4.1 ogCombiner v.1.5.2 reeAnnotator v.1.5.2 ig Tree v.1.2 As a calibration point, we used the divergence time of 5.5 Ma  convertrlequin v.3.11 to check if the microsatellite data departed from Hardy-Weinberg expectations. Tests for linkage disequilibrium were checked by using the GenePop v.4.0 AN), allelic richness (R), gene diversity (GD) and allele frequencies were calculated in Fstat v.2.9.3.2 All loci were collected as an EXCEL file, and then converted into the computable files using ottleneck v.1.2.02 ottleneckThe presence of genetic bottlenecks was tested by using the heterozygosity excess method FST values calculated in Arlequin v.3.11 and the Bayesian clustering approach implemented in Structure v.2.3.3 tructure, the admixture model with correlated allele frequencies was chosen and the number of genetic groups K was set from 1 to 8. Each run used five replicates, which consisted of a burn-in period of 1 000 000 MCMC steps followed by 1 000 000 MCMC steps. Plots of the mean value of the log posterior probability of the data [mean of lnP(D)] were examined to check the maximum lnP(D) value eneland v.4.0.3 First, we assessed population structure by using pairwise migrate v.3.3.2 We also used the maximum likelihood and Bayesian inference The D-loop-only dataset included 168 variable (polymorphic) sites of which 26 were autapomorphic and 142 potentially parsimony informative. Inclusion of 16 indels resulted in 214 haplotypes, 92 of which we generated and 122 downloaded from GenBank. The haplotypic composition of each sampled population was summarized in Analysis of the D-loop data did not yield a well-resolved MP tree  whereas Haplogroup EAST mainly corresponded to haplogroup ChiE and Ind1, defined by Smith and McDonough M. mulatta. The WEST haplogroup was further divided into two subhaplogroups. Subhaplogroup WEST B was represented by 21 haplotypes from Yunnan and Sichuan  and four others were from Sichuan (SCJY). Subhaplogroup WEST A, from Sichuan and Yunnan, had two haplotypes from SCDB, two from SCJY, and two from Yunnan. Unfortunately, the two samples from Yunnan in WEST A and all the samples from SCJY were missing specific locality data. EAST was comprised 35 haplotypes, which included all sampling locations other than Yunnan and Sichuan. One haplotype from Yunnan (YN) (Hap_89YNSM) clustered in EAST.For the concatenated data, the Bayesian inference and maximum parsimony genealogies had identical topologies and similar support values . The hypSTF estimates based on the D-loop sequence data from the ten populations of Macaca mulatta that had specific sampling locality information was 0.769. The two populations from Sichuan had a far larger STF value (0.984) compared with the four populations from Yunnan (ST = F0.0784). IBD was observed for both all the ten populations and the six western populations that had sampling data (structure (below).Although we had relatively few populations with specific sampling information, a positive correlation between geographical distance and genetic differentiation was obtained. The average pairwise ing data . The corM. mulatta . For the main populations of Chinese M. mulatta, there was considerable variation in haplotype diversity (h) and it ranged from 0 to 1.0000±0.063 (π) ranged from 0 to 0.06052±0.00521. These values were highest in ZJ, FJ, GX, and Viet Nam, a reflection of the sympatric occurrences of multiple haplotypes. In addition to the haplotypes from above, four provinces occupied central locations in EAST suggesting that these haplotypes had a greater heterogeneity and an earlier history of divergence than other groups in EAST. The very low haplotype and nucleotide diversity in HA and AH corresponded to their peripheral locations in the median-joining network , and the separation of the western and eastern haplogroups was dated to 1.15 Ma (0.78–1.55). The age of the most recent common ancestor (MRCA) of the western haplogroup was estimated at 0.92 Ma (0.6–1.27) while that of the eastern haplogroup was ca. 0.81 Ma (0.5–1.11).The main nodes  were datM. mulatta were found by e-PCR in the UniSTS database of NCBI. All loci were unlinked and showed no departures from Hardy-Weinberg equilibrium. The total number of alleles (AN), number of private alleles (PAN), allelic richness (R), and gene diversity (GD) were summarized in AN/locus was found in population YNJG (AN/locus = 9.5), and the highest allelic richness and gene diversity were found in population SCJY . In contrast, populations HI and SCDB had the lowest values . Using the Wilcoxon signed-rank test, we detected an excess of heterozygotes in three populations . Except for four populations , the allelic frequencies of nearly all the populations fit the normal L-shaped distribution in the mode-shift test. These results indicated that most populations had not experienced bottlenecking. Pairwise STF values (STF = 0.033). Structure resolved one unique genetic group for M. mulatta  and the other in eastern China . The lineages have high levels of genetic divergence between them, as previously reported in molecular and morphological studies. For the first time and in contrast to haplogroups, our analyses of the microsatellite data do not detect significant population differences . The haplotype network  as reported by Li et al. The matrilineal pattern in EAST differs from that of WEST. A meaningful analysis of IBD is not possible because of only four specific sampling sites. Notwithstanding, the 35 haplotypes have a different pattern and this rejects the null hypothesis of all populations being equal. Samples from Zhejiang, Fujian, and Guangxi (China) and Viet Nam contain a large mix of haplotypes . Nearby structure analysis of microsatellite data does not detect significant population structure . This date is similar to prior estimates of 2.6 Ma M. mulatta dates to 1.61 Ma , which is congruent with the 1.2 Ma in a previous study M. mulatta are not equivalent for three reasons: they occur for different events; one is based on a single locus and the other on multiple loci; and our estimate is for the historical split of the matrilines only and we note that our study demonstrates different sex-biased patterns. Perhaps most important, the SNP data estimate the cessation of biparental gene flow between the two groups as mediated by male dispersal. Thus, it is likely the difference reflects inheritance mechanisms of the two genomes, i.e. the maternally inherited mtDNA genome versus nuclear biparentally inherited SNPs and male-mediated gene flow.We estimate the matrilineal split between M. mulatta. Macaca mulatta likely diverged from its ancestor shared with fascicularis about 2.31 Ma in or near northern Indochina. About 1.61 Ma , M. mulatta appears to have become widely distributed via a northward expansion using two different dispersal routes: Myanmar–India and Viet Nam–China , a time of great change in Chinese and Southeast Asian environments. The last orogenic push of the QTP is associated with the formation of Asian monsoons, the beginning of Chinese loess (aeolian sedimentary deposits), and the appearance of the Yellow River M. mulatta seems to have dispersed southwards and repeatedly supplanted local matrilines. This scenario best explains the great extent of mitochondrial DNA sequence differentiation between WEST and EAST. During the Pleistocene, two refugia might have existed: the western montane areas of southwestern China, and montane eastern China During the Quaternary Period, when climatic deterioration compressed suitable habitats southwards, Glaciation appears to have more severely affected EAST than WEST. The environment of East China experienced dramatic changes during the Quaternary We detect multiple mtDNA haplogroups in Zhejiang, Fujian, and Guangxi, China and in Viet Nam. The absence of overall geographic structuring and the apparent absence of IBD may have resulted from either the southward retreat of multiple haplogroups to a refuge during glacial periods, or a population expansion during interglacial periods M. mulatta come from human habitat destruction and illegal poaching. These activities are responsible for the protection of the species as listed in Category II of the Chinese Wildlife Protection Act (1989) and in Appendix II of the Convention on the International Trade in Endangered Species of Fauna and Flora (CITES) M. mulatta corresponds to the traditional subspecific taxonomy or not, and this initiative is underway.The most serious threats to The western and eastern haplogroups indicate an important level of genetic structuring. Thus, WEST and EAST might be regarded as separate evolutionarily significant units (ESUs). These groups are experiencing shrinking distributions, increasing genetic isolation, and decreasing haplotype diversity. Attention should be paid to conserving populations in Henan and Hainan because both have very low genetic diversity, small residual areas of habitat, and significant morphological differences Figure S1A maximum parsimony tree for wild Chinese rhesus macaques, Macaca mulatta, derived from 214 D-loop haplotypes. Four outgroup haplotypes were used to root the tree. Bootstrap support from maximum parsimony (1000 replicates) (≥50% retained) were shown at the nodes.(TIF)Click here for additional data file.Table S1Macaca mulatta. The first 92 haplotypes were sequenced in this study and the last 122 haplotypes were downloaded from GenBank. The 67 haplotypes colored in red were selected for concatenation with other mtDNA genes.The distribution of 214 D-loop haplotypes in each sampling location of wild Chinese rhesus macaques, (XLS)Click here for additional data file.Table S2Macaca mulatta in China. SSD = Sum of Squares Deviations.Statistics of genetic diversity, neutrality test and the mismatch distribution analysis. These analyses were performed for main populations of (DOCX)Click here for additional data file.Table S3Macaca mulatta.The STR data for the wild Chinese rhesus macaque, (XLSX)Click here for additional data file.


Geographic surveys of allozymes, microsatellites, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) have detected several genetic subdivisions among European anchovy populations. However, these studies have been limited in their power to detect some aspects of population structure by the use of a single or a few molecular markers, or by limited geographic sampling. We use a multi-marker approach, 47 nDNA and 15 mtDNA single nucleotide polymorphisms (SNPs), to analyze 626 European anchovies from the whole range of the species to resolve shallow and deep levels of population structure. Nuclear SNPs define 10 genetic entities within two larger genetically distinctive groups associated with oceanic variables and different life-history traits. MtDNA SNPs define two deep phylogroups that reflect ancient dispersals and colonizations. These markers define two ecological groups. One major group of Iberian-Atlantic populations is associated with upwelling areas on narrow continental shelves and includes populations spawning and overwintering in coastal areas. A second major group includes northern populations in the North East (NE) Atlantic (including the Bay of Biscay) and the Mediterranean and is associated with wide continental shelves with local larval retention currents. This group tends to spawn and overwinter in oceanic areas. These two groups encompass ten populations that differ from previously defined management stocks in the Alboran Sea, Iberian-Atlantic and Bay of Biscay regions. In addition, a new North Sea-English Channel stock is defined. SNPs indicate that some populations in the Bay of Biscay are genetically closer to North Western (NW) Mediterranean populations than to other populations in the NE Atlantic, likely due to colonizations of the Bay of Biscay and NW Mediterranean by migrants from a common ancestral population. Northern NE Atlantic populations were subsequently established by migrants from the Bay of Biscay. Populations along the Iberian-Atlantic coast appear to have been founded by secondary waves of migrants from a southern refuge. Engraulis encrasicolus, are widely distributed in near-shore pelagic waters in the Eastern Atlantic from the North Sea and into the Mediterranean and Black Sea and as far south as southern Africa. Populations in the North East (NE) Atlantic and Mediterranean are partitioned into several spawning groups that are isolated from one another by complex shorelines and oceanic regimes European anchovies, While migration, genetic drift and adaptive selection may explain much of the genetic structure of anchovy populations, deeper genetic partitions with coalescences dating to tens of thousands of years more likely reflect ancient climate events during the Pleistocene ice-ages. During each ice age, continental glaciers expanded across North America and Eurasia, leading to lower ocean temperatures and sea levels Previous studies of European anchovies have been limited in two ways. First, most studies of European anchovies have been regional, where samples have been restricted to one of the many sub-basins in the Mediterranean Sea. Populations in the North Sea and Bay of Biscay and along the Iberian-Atlantic coast have received less attention. The analysis of these populations will provide a deeper understanding of population structure and history. Second, the power of some molecular markers to detect some aspects of population structure is weak. Most studies have used neutral molecular markers, which are interpreted in terms of gene flow and genetic drift (population size) The goal of this study was to examine variation at several SNP loci to better resolve the genetic structure of anchovy populations in the NE Atlantic. A multi-locus approach is more likely to yield a fuller snapshot of population structure than the analysis of a single marker Anchovies were provided either by commercial vessels or by oceanographic institutes that collected the samples during scientific acoustic surveys . All surveys followed local regulations and guidelines for such research. Anchovies were collected following fishing without unnecessary suffering of the animals and following usual procedures. No experimentation with animals was performed. No other ethical issues applied to the present research project.N = 30) were collected from the entire geographic range of this species and its spawning areas (N = 188) were sampled in the Mediterranean Sea along an east-west axis. Several locations were sampled in the Eastern Atlantic (N = 443), including 127 fish from the North Sea and English Channel (6 locations), 106 fish from the Bay of Biscay (3 locations) and 210 fish from Galicia to South Africa (7 locations). Most samples were collected from 2007 to 2009. Genomic DNA was extracted from muscle tissue using the DNeasy 96 Tissue Kit (Qiagen), following manufacturer’s protocol. A specific protocol was used  for tissues preserved in RNAlater® (Applied Biosystems), as was the case for North Sea and English Channel samples.A total of 626 anchovies . Hence, it was discarded from the final SNP panel of 47 nDNA SNPs, representing 35 independent markers including 26 individual SNPs and 9 haplotype blocks. We used FSTAT 2.9.3 FIS for each sample.The 90 novel SNPs, including 75 nuclear (n) DNA and 15 mtDNA SNPs, described by Zarraonaindia et al. included in Molecular Ecology Resources Primer Development Consortium et al. TaqMan® OpenArray™ genotyping system. DNA concentrations and reactions for amplification and detection of the SNPs followed the TaqMan® OpenArray™ Genotyping System User Guide. Genotypes were scored using Autocaller 1.1 (Applied Biosystems).The 626 anchovies from 20 localities were screened for the 47 nDNA SNPs panel and 15 mtDNA SNPs with K groups. Ten independent runs were conducted for each value of K (K = 1–10) using 500,000 Markov Chain Monte Carlo iterations, after a burn-in of 50,000 iterations. We assumed a mixed ancestry model and correlated allele frequencies locprior model. We used CLUMPP 1.1.2 K) that best explained the pattern of genetic variability was evaluated using the approach of Evanno et al. Nuclear SNPs were examined in three ways. First, population structure was inferred from individual assignments based on the analysis of the nuclear SNPs using the Bayesian model-based clustering algorithm implemented in STRUCTURE 2.3.2 Path Distance implemented in ArcGis 9.2. Matrix comparisons by Mantel’s method were carried out with the program ZT Second, we estimated genetic distances between samples and used these distances in additional analyses. Reynolds genetic distance FST) FST were determined by jackknifing, and statistical significance was determined with 15,000 permutations. We used the Bonferroni correction probabilities for multiple tests FST between samples and by significant values of FST with other populations Lastly, genetic differentiation among samples was estimated with the unbiased fixation index (nh), haplotype diversity (h), and Tajima’s DFSD and FS were tested with 1000 coalescent simulations. Mutational relationships among haplotypes were represented by a median-joining (MJ) network http://www.fluxus-technology.com). The SNPs Cyt-b-318, Dloop-323 and Dloop-336 were not used to construct the network, because their high levels of polymorphism would reduce the resolution among haplotypes.The number of SNP mtDNA haplotypes . IBD among the NE Atlantic samples from the Bay of Biscay to the Canary Islands (including the Alboran Sea) was highly significant . The addition of the distantly located sample from South Africa lowered the correlation between geographical distance and genetic distance .A similar pattern of population differentiation appeared in genetic distances between populations. The NJ tree of Reynolds genetic distances also showed two major groups supported by a 90% bootstrap value . The firFST ranged from 0.04, between two samples from the Bay of Biscay, to 0.318 between samples from Denmark and South Africa, and averaged 0.078±0.012  and nearby Tarragona (2009) in the NW Mediterranean and between samples from the North Sea (2006–2009) were not significant. Based on these pairwise FST values, the original 20 samples were pooled into 10 groups, which showed within-group homogeneity but between-group heterogeneity. The largest amount of divergence between these 10 groups was FST = 0.270±0.053 between the South African and Aegean Sea samples (FST = 0.013±0.007) was detected between samples from nearby locations in the Bay of Biscay , which were placed into two different pooled groups. Both pooled groups (BISC-1 and BISC-2) showed a larger amount of divergence from the populations in the North Sea and English Channel pooled group (NSEC),  than from NW Mediterranean samples (NWMD) .Pairwise 78±0.012 . No sign samples . ImportaCyt-b-318, D-loop-323 and D-loop-336. The MJ Network showed two phylogroups, A and B, separated by 3 mutational steps, including a transition, a transversion and an indel  was similar to haplotype diversity in phylogroup B . Phylogroup A displayed a star-like haplotype network, and both Tajima’s D and Fu’s FS were negative, but not significant . In contrast, the phylogroup B network was more reticulated, with two predominant haplotypes. Neither D  nor FS  was significant. The mismatch distribution for both phylogroups was unimodal (not shown). In contrast, the two phylogroups together showed a bimodal mismatch distribution, with a positive and significant Tajima D value . Both phylogroups appeared in each sample, but at different frequencies (h = 0.606) and South Africa (h = 0.669), whereas the largest diversities appeared in the Bay of Biscay  and English Channel (h = 0.913).The 15 mtDNA SNPs defined 55 haplotypes, but only 23 haplotypes after excluding the highly polymorphic SNPs, an indel . Haplotyquencies . PhylogrThe present study combines the use of mitochondrial and nuclear SNP markers, together with a large coverage of the distributional range of the European anchovy to provide a deeper understanding of the factors shaping the genetic population structure of the species and to disentangle historical from ecological factors reflected in contemporary populations. On the one hand, the analysis of maternally inherited, non-recombining mtDNA complements nuclear SNPs and provides insights into ancient events that have produced deep genetic/genomic partitions in European anchovies. On the other hand, nuclear SNPs enhance the power to detect population divergences resulting from genetic drift.FST values that were 7–8 times larger between samples than FST values estimated with 7 microsatellite loci FST between the Bay of Biscay, Gulf of Cadiz and Mediterranean anchovy samples was 0.029±0.013 FST was 0.224±0.057 (present study). Furthermore, microsatellites were not powerful enough to find genetic differences among one Bay of Biscay sample and western Mediterranean samples When comparing the resolving power of nDNA SNPs with other types of markers, such as microsatellites, the use of 4–12 nuclear SNPs is expected to have the same resolving power as a microsatellite locus Regarding the sample design, the strategy followed was successful to reveal both, small and large spatial scale population structure. First, samples were taken from oceanic and near-shore populations to test for possible genetic differences between anchovies showing different life-history patterns. Second, we concentrated our sampling effort in the NE Atlantic, an area that had previously not been well studied. Populations of anchovies in northern areas of the NE Atlantic were of particular interest, because these areas have been open to colonization in only the past few thousand years On large geographical scales, two genetic groups of populations were identified , 3 that On geographical scales of hundreds of kilometers, genetic data resolved at least ten homogeneous population groups  that likGenetic homogeneity among anchovy samples in the North Sea-English Channel area and genetic divergence between this northern-most population group NSEC,  and thosRegarding southern European populations, the genetic division found in the east Iberian Atlantic area appears to correspond to morphological differences. Caneco et al. In the Mediterranean, our sampling does not allow us to define the geographical boundaries between the four genetically distinct groups , but ourSmall, but significant, amounts of divergence appeared between anchovy populations in the Bay of Biscay  that areGenetic relationships among populations within regions are here interpreted to reflect ancestral relationships that have been overlain with patterns of contemporary gene flow, but not to the extent that historical signals of dispersal have been erased. The NE Atlantic experienced strong ocean-climate shifts during the Pleistocene. Periodic drops in sea surface temperatures and lower sea levels led to the elimination of anchovy habitats in northern areas during the last glacial maximum about 18,000 years ago. Even after continental glaciers largely receded, a land bridge existed between the British Isles and continental Europe until about 7500 years ago FST between northern North Sea-English Channel and some populations in the Bay of Biscay indicate a close genetic relationship between these groups that may reflect historical biogeographic relationships. Present-day northern populations and those in the Bay of Biscay have increased in abundance since the 1990s Other regional relationships also appear to reflect ancient dispersals and founder events. For example, small values of Our genetic results also confirm dispersals of anchovies from European waters across the equator to southern Africa. The major European mtDNA lineages also occur in southern African anchovies FST than did microsatellite markers and, hence, provided a greater amount of power to detect population structure. The results of our study and of several other studies demonstrate hierarchical genetic differences between populations on different temporal and spatial scales. Some divergences between populations in the Mediterranean and in the Atlantic can be explained by random drift in populations isolated by current gyres, by frontal systems, or by distance. However, some small-scale differences between populations in the Bay of Biscay and in other areas may be due to adaptive divergences in life-history traits. These life-history contrasts are ideal starting points for genomic studies to understand the genetic basis of adaptation.The analyses of multiple nDNA and mtDNA SNP markers confirm and expand previous population genetic and phylogeographic hypothesis for European anchovy populations. The nuclear SNPs yielded larger values of Our multi-locus approach also revealed two major groups of European anchovies that are associated with different oceanic systems. One group inhabits productive wide-shelf ecosystems with physical mechanisms that enhance nutrient enrichment, an abundance of larval food and egg and larval retention, the ‘ocean triads’ The analysis of the mtDNA resolves two deeply separated phylogroups in European anchovies. The frequencies of these lineages vary among populations and give insights into patterns of dispersal and colonization during the Pleistocene climate cycles. One conundrum is the genetic similarity between some anchovy populations in the Bay of Biscay and those in the NW Mediterranean. This genetic similarity can only be explained by post-glacial dispersals into these two areas by anchovies migrating from a common ancestral population. Since these are ‘wide-shelf’ populations, the ancestral refuge population may have been in the Mediterranean, or along Atlantic Africa in an area with ‘ocean triad’ characteristics. The colonization of the Bay of Biscay was followed by stepping-stone dispersals into northern coastal areas, including the English Channel and the North and Baltic seas. The genetic discontinuity at Galicia is likely due to secondary contact between the established populations in the Bay of Biscay and later secondary colonizers from southern refugia located in a narrow-shelf ocean ecosystem.Table S1Summary of nuclear DNA SNP statistics in 626 samples of Engraulis encrasicolus by locus. Number of alleles or haplotypes for each locus , Allelic Richness (A) for a minimum sample size of 15 individuals, mean heterozygosity (he) and FIS values with their Standard Error (SE).(DOCX)Click here for additional data file.