Panthera tigris tigris) left in Nepal, that are generally confined to five protected areas across the Terai Arc Landscape, genetic studies are needed to provide crucial information on diversity and connectivity for devising an effective country-wide tiger conservation strategy. As part of the Nepal Tiger Genome Project, we studied landscape change, genetic variation, population structure, and gene flow of tigers across the Terai Arc Landscape by conducting Nepal’s first comprehensive and systematic scat-based, non-invasive genetic survey. Of the 770 scat samples collected opportunistically from five protected areas and six presumed corridors, 412 were tiger (57%). Out of ten microsatellite loci, we retain eight markers that were used in identifying 78 individual tigers. We used this dataset to examine population structure, genetic variation, contemporary gene flow, and potential population bottlenecks of tigers in Nepal. We detected three genetic clusters consistent with three demographic sub-populations and found moderate levels of genetic variation  and genetic differentiation (FST = 0.14) across the landscape. We detected 3–7 migrants, confirming the potential for dispersal-mediated gene flow across the landscape. We found evidence of a bottleneck signature likely caused by large-scale land-use change documented in the last two centuries in the Terai forest. Securing tiger habitat including functional forest corridors is essential to enhance gene flow across the landscape and ensure long-term tiger survival. This requires cooperation among multiple stakeholders and careful conservation planning to prevent detrimental effects of anthropogenic activities on tigers.With fewer than 200 tigers ( Reduction in prime habitat and loss of genetic diversity are influential factors leading to the extirpation of wildlife populations  and extiPanthera tigris) is a species of global conservation concern as its range has declined more than 90% in past two decades, and the species now occupies only 7% of its historic range [2) of the Eastern Himalayan eco-region [Panthera tigris tigris) are the most diverse globally and represent half of the extant genetic diversity in the species [The tiger .2) that stretches along the southern lowland areas of the country (Shorea robusta) forests. This global priority landscape [This study was conducted across the TAL-Nepal landscape  and surveyed 54 grid cells  samples were collected from protected areas and connecting wildlife corridors across the TAL-Nepal. We divided the study area into 108 grid cells each measuring 15 X 15 km  following the manufacturer’s instructions. Species identification polymerase chain reaction (PCR) was performed using tiger-specific primers (TIF/TIR)  41] tha tha41] tSixteen microsatellite markers used by previous studies , 43–45 wPCR amplification was carried out in a 7 μL reaction volume containing 3.5 μL of Qiagen master-mix . The PCR conditions for microsatellites amplification included an initial denaturation (95°C for 15 min) with a touchdown PCR step for 10 cycles . This was followed by 25 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 90 s and extension at 72°C for 60 s, and a final extension at 72°C for 10 mi. PCR product (0.7 μL) were sized against LIZ-500 size standard in ABI 310 genetic analyzer . Three to five PCR replicates were run per sample. Microsatellite alleles were scored in GENEMAPPER, version 4.1 . To finalize consensus genotypes, at least three identical homozygote PCR results were assessed to determine homozygote, and each allele was observed in two independent PCR for a heterozygous genotype .(ID)) and siblings (P(ID)sib) were estimated using Gimlet, version 1.3.3[(ID)sib [PCR amplification success rates and genotype accuracy (GA) were based on results from the last two PCR runs . PCR ampion 1.3.3. A minim[(ID)sib , 49 for [(ID)sib  was usedo) and expected heterozygosity (He) using GenAlEx, version 6.41[HP-RARE, version 1.0 [ST, inbreeding coefficients (FIS) and to test the statistical significance of the pair-wise FST values between the populations in the TAL, using 10,000 permutations [EST was used as an alternative metric for genetic distances between the populations, as it outperforms FST as an accurate and unbiased metric of the level of differentiation between populations when the sample size is high and the number of loci is low [EST across the loci for each population was calculated using the web-based program SMOGD [Genetic diversity was quantified by estimating observed heterozygosity , was allowed to vary between 1 and 5; we performed 10 independent runs for each value of k. This analysis was run with admixture models with correlated frequencies using a burn-in of 500,000 Markov chain Monte Carlo (MCMC) steps followed by an additional 1,000,000 iterations without prior information on the sampling sites. The optimal value of k was inferred by examining the likelihood curve, q value bar plots, and using the Evanno method [To visualize genetic similarities among regions and individuals, we performed a multivariate principal coordinate analysis (PCoA) in GenAlEx. Furthermore, an individual-based Bayesian clustering approach was implemented using STRUCTURE , version 2.3.4  and TESSo method  implemeno method . The indo method  with a gk = 2 to 10 with 100 replications per k. The average of the 10% of the lowest Deviance Information Criteria (DIC) values was used for each kmax. DIC values were taken for estimating the number of optimal kmax (genetic sub-populations) [kmax and most likely value of kmax was selected by visually assessing the point at which DIC first reached a plateau and the number of clusters to which individuals were proportionally assigned.The admixture models  were run using TESS  with 100lations) . DIC valr = 0) by generating 95% confidence intervals (CI) for each distance class via permutation  and bootstrapping (999 repeats). We correlated genetic distances and spatial distance matrices and generated auto-correlation matrices for each spatial distance class ranging from 0–250 km based on the distribution of tigers in TAL. Results were visualized as correlograms and the location of first x-intercept represents the extent of non-random spatial genetic structure. Individuals below this threshold share a higher proportion of genes than spatially distant individuals. Within a given correlogram, significant spatial auto-correlation was confirmed only when a positive r-value fell outside the 95% CI (derived from the permutation test), and when the 95% CI about r (derived from bootstrapping) did not intercept the axis of r = 0[We also examined isolation-by-distance (IBD) and spatial auto-correlation patterns to characterize spatial genetic structure of tigers in TAL using GenAlEx. First, we tested whether a significant correlation existed between pair-wise co-dominant genotypic distance and geographical distance by applying simple Mantel tests with 9,999 permutations. Secondl of r = 0.home/ Lmax) was applied to identify migrants. This method uses the Bayesian criteria of Rannala & Mountain (1997) [home/ Lmax beyond which a sample is treated as a migrant. We also carried out assignment tests in program STRUCTURE incorporating the geographical sampling sites as prior information (LOCPRIOR) without changing the other parameter settings as described above. Since we did not have prior knowledge about migration of individuals (MIGPRIOR) between potential sub-populations, we used the default setting.To examine whether the individuals were born in the location from which they were sampled, assignment/exclusion tests were performed in GENECLASS, version 2.0 . The Bayn (1997)  along wi7 MCMC iterations and a 107 burn-in with different seed numbers and delta values confirmed the final parameters and ensured their convergence. Both immigration and emigration rates between populations were considered as contemporary migrations.Rates of recent immigration over the last several generations among the sub-populations were estimated using program BayesAss+, version 3.0 . This Baeq). Simulations were performed under three mutation models: infinite allele model (IAM), single stepwise model (SSM), and two-phase model (TPM). The simulation values were then compared to real data values to obtain the distribution of Heq. For the TPM, we used the generic values of 0.95 and 0.12 for frequency of single-step mutations and variance, respectively [We used two approaches to test for the genetic signature of a severe demographic contraction (i.e. bottleneck) in the tiger population across the TAL. First, we used test M-ratio  implemenectively . A Wilcoth and 20th centuries. There has been a 97% increase in agriculture and settlement areas over the past 200 years in the TAL , fecal samples were collected from grid cells (n = 36) within four protected areas . No fresh scats were found in Banke National Park. In the second phase (2012–2013), putative tiger samples were only found and collected in grids (n = 5) of the two known corridors (Khata and Basanta). No fresh tiger scats were found in corridors: Kamdi, Laljhadi, Barandabhar, and Bramahadev.(ID) and P(ID)sib were estimated to be 1.5E-06 and 3.2E-03 (<0.01) respectively, and 3.1 E-02 for our 6 least powerful loci. We obtained a consensus genotype at 6–8 loci for 212 samples (51% genotyping success) and identified 78 individual tigers . Only four scat samples collected outside of protected areas were of tiger  and two corridors (Khata and Basanta) within the TAL, a total of 412 were confirmed to be tiger scat  Table 1Table 1. P < 0.05 (P≤ 5.95E-04) was detected among three pairs of loci with no apparent pattern . Overall average genetic diversity of the TAL tiger population was as follows: observed heterozygosity of 0.54, expected heterozygosity of 0.61, mean number of alleles per locus of 6.0, and mean allelic richness of 3.51. Levels of genetic variability varied among the protected areas, with the highest genetic diversity found in CNP and the lowest in SuNP (IS = 0.16) in the smaller SuNP population relative to other sites in the TAL  at P < 0.05 . No locu in SuNP . The ave the TAL , which iST value (level of differentiation) for the TAL was found to be moderate (FST = 0.14 ± 0.07). Results of AMOVA showed that genetic variation among the sites was 13.7%, while the residual variation among individuals within the sites was 86.3%  were found to be low to moderate and statistically significant (P < 0.05). Comparatively low FST and DEST were found between the BNP and SuNP populations. In contrast, levels of genetic differentiation were high (FST = 0.21) for SuNP and CNP populations , and k = 3 based on delta k method. Four clusters showed a high standard deviation relative to k = 3  . Resultsto k = 3  and sub-to k = 3 . Hence, analysis . At k = st cluster ; 2ndcluster ; and third cluster  , with variable forest connectivity between them  with the central cluster (BNP) showing the highest degree of admixture: 11% SuNP) . Within een them , Fig 7.k = 3, and the standard deviation increased with an increasing value of kmax , BNP (2), and SuNP (3) as three distinct sub-populations with some evidence for sub-structure within CNP.Results from the non-spatially explicit clustering analysis in STRUCTURE were corroborated with the spatially explicit analyses in TESS. Based on the DIC model selection criteria, graphs tended to plateau at  of kmax . The harr = 0.48, P = 0.0001) between geographical distance and genetic distance . An x-intercept of r at ~ between 75–100 km . The BNP sub-population appeared to be receiving the migrants from both CNP and SuNP, with a total migration rate of 0.17 , which is below the threshold value of 0.68. This suggests that the tiger population suffered a bottleneck event that caused a severe decline in the population size in the recent past. These results were supported among all sub-populations. The Bottleneck software detected heterozygote excess using the sign test, with 5 to 6 loci, depending upon the mutation models. However, results of the two-tailed Wilcoxon signed-rank test showed that the signature of a bottleneck event was significant only for the CNP population under the Infinite Allele Mutational (IAM) model  along TAL. We screened the duplication of samples at two stages: at field, we only collected the fresh scat samples at each encounter and avoided collection of the same scats samples by putting in the red marker on the scat (dried) itself and/or placed a twig into soil at the site of fresh samples. At the analysis phase, each of the duplicate samples (if scored) were taken as recapture.e = 0.61) across the landscape and similar levels across sub-populations (He ≈ 0.57), perhaps due to moderate population size and/or gene flow resulting from potential connectivity among tiger-bearing protected areas across the TAL in Nepal (n = 5) and India (n = 10).Our estimates of genetic variation were lower in comparison to genetic diversity estimates for Bengal tigers overall (He = 0.72) [e = 0.70) [e = 0.53, Sumatran, Indochinese, Malayan and Siberian) [e = 0.58 in the southeastern Ghats [e = 0.67 in the northeast landscape [e = 0.76 in the Western Ghats [e = 0.81 in the central Indian landscape [Tigers in the TAL displayed moderate levels of genetic variation (H = 0.72)  or the I = 0.70)  using miiberian) . Landscarn Ghats , He = 0.andscape , He = 0.rn Ghats , and as andscape . HoweverP = 0.42). At the sub-population level, SuNP showed a weak sign of local inbreeding (FIS = 0.16), which approached statistical significance (P = 0.06). This suggests the importance of connectivity in averting inbreeding depression in the small SuNP sub-population[The average inbreeding coefficient across the sub-populations in the TAL-Nepal suggests weak inbreeding that was statistically non-significant  results where populations (sites) observed moderate differentiation showing three genetic clusters confirming that total population in Terai Arc may not be total panmixia. Our Bayesian clustering results support three distinct genetic clusters (sub-populations) within the TAL-Nepal, representing the three tiger bearing protected areas  confirming our a priori hypothesis. Our field data suggested demographic contiguity in and around these protected areas [ST/DEST showed higher connectivity between CNP and BNP, which are farther apart, than between SuNP and BNP, which are closer together.Both population- and individual-based tests for assessing the level of genetic sub-division revealed moderate levels of differentiation across the landscape. This is in concordant with AMOVA and fixation index  moving between the sub-populations. Tigers are known to disperse long distances (~200 km) based on long-term camera-trap data . FurtherResults of the BayesAss+ analysis showed high estimates of recent migration of tigers among the sub-populations. High net immigration rates were detected in BNP. In the medium and large sub-populations (BNP and CNP), we found evidence of one to two male tigers dispersing between the populations. There was also evidence of one male and two females moving from SuNP to BNP. If these tigers breed, this will likely avert the detrimental effects of inbreeding depression, thus flattening the slopes of extinctions curves accordingly . Results2 of the Pilibhit Forest Division as a tiger reserve by the Indian government is an important step towards increasing connectivity between the western TAL in India and SuNP in Nepal .The SuNP population, while small, holds the highest density of tigers, likely due to high prey density . This coWhile both analysis techniques revealed population bottlenecks in the TAL-Nepal, there was a disagreement in the resulting number of detected population bottlenecks. The M-ratio test revealed a population bottleneck in all populations, in contrast to the heterozygous excess test showing a bottleneck only in the CNP population. Both tests have been found to be effective at detecting bottlenecks, but each works under different assumptions. Peery et al.  suggesteWe provide evidence of three genetically admixed sub-populations across the TAL-Nepal based on spatial (TESS) and non-spatial (STRUCTURE) Bayesian clustering techniques, suggesting that tigers have been able to move between the populations and breed, at least in the recent past. Contemporary gene flow measures of tigers were estimated in the TAL based on both likelihood and Bayesian approaches. Improved connectivity between the protected areas, facilitated by male and female tiger dispersal, appears to be able to avert the negative consequences of inbreeding depression following bottleneck events. Thus, there is a need to maintain connectivity throughout the TAL-Nepal landscape and beyond. We did not find migrants from CNP into SuNP or vice versa in any of the migrant detection tests. However, the dispersal among SuNP and CNP is more likely to be a stepping-stone process .In recent times, connectivity in the Nepali landscape has been improved by the protection of large forest blocks after the nationalization of forests in the 1960s , improveS1 Tablebp: base pair; F: forward; R: reverse.(DOC)Click here for additional data file.S2 TablePCR: polymerase chain reaction; min: minute; sec: second; “x” indicates times; F: forward; R: reverse.(DOC)Click here for additional data file.S3 TableA, number of alleles; Ho, observed heterozygosity.N(DOC)Click here for additional data file.S4 TableP value (α = 0.05).df = degree of freedom, (DOC)Click here for additional data file.S5 TableNet migration rates (immigration-emigration) were estimated as 0.02 for CNP, -0.08 for SWR, and +0.10 for BNP; “+” indicate migrant receiving from other population; “-” indicate contributing migrant to other population.(DOC)Click here for additional data file.S6 TableP<0.05 signifies significant heterozygous excess, suggesting that a bottleneck event occurred in CNP only.IAM: Infinite Allele Model; TPM: Two-Phase Mutation Model; SMM: Stepwise-Mutation Model. Assuming any mutation model, a Wilcoxon test results with (DOC)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigBoth models selected three genetic clusters across the landscape. Error bars represent standard deviations.(TIF)Click here for additional data file.S5 Fign = 78) shows the genetic correlation coefficient (r) as a function of geographic distance across defined spatial distance classes. Dashed red lines represent upper (U) and lower (L) bounds of the null hypothesis based on 9,999 random permutations. Error bars represent 95% confidence intervals about r based on 999 bootstraps.The spatial correlogram for tigers ((TIF)Click here for additional data file.


Here, we used the mitochondrial cytochrome H. leucisculus populations diverged between 1.61–2.38 Ma. Bayesian species delimitation analysis using 12 nuclear DNA loci indicated the three lineages probably represented three valid taxa. Isolation-with-migration (IM) analysis found substantial gene flow has occurred among the three lineages. Demographic analyses showed that lineages B and C have experienced rapid demographic expansion at 0.03 Ma and 0.08 Ma, respectively.Our mitochondrial phylogenetic analysis identified three major lineages . Lineages B and C showed a relatively narrower geographic distribution, whereas lineage A was widely distributed in numerous drainages. Divergence dates suggested that Hemiculter leucisculus populations in drainages in southern China comprise three mtDNA lineages, and each of which may represent a separate species. Intense uplift of the Qinghai–Tibetan Plateau, evolution of Asian monsoons, changes in paleo-drainages, and poor dispersal ability may have driven the divergence of the three putative species. However, gene flow occurs among the three lineages. Climatic fluctuations have a prominent impact on the populations from the lineages B and C, but exerted little influence on the lineage A.The online version of this article (10.1186/s12862-017-1058-0) contains supplementary material, which is available to authorized users. Haloptilus longicornis [Microtus agrestis [In recent years, numerous phylogeographical studies have shown that species with wide distribution ranges are frequently subdivided into genetically distinct groups –4. The mgicornis  and Micragrestis ), these Recent improvements in molecular and analytical tools have aided in investigations of cryptic speciation. Multilocus sequence data involving newly developed theoretical models can be particularly helpful to generate species trees of closely related taxa . SeveralGeologic changes and climatic instabilities are important factors that influence the evolutionary history of many taxa. Geologic changes have frequently shape the genetic structure of many species in East Asia –14, inclClimatic instabilities during the Quaternary Period have resulted in periodic expansions and contractions of the population sizes and distribution ranges of species , 24. ForCyprinids represent one of the most diverse freshwater fish groups and are a major component of the primary freshwater ichthyofauna of Eurasia . The widRhynchocypris oxycephalus [Opsariichthys bidens [Zacco platypus [Hemiculter leucisculus (Cyprinidae: Cultrinae), a small cyprinid fish, reaches a size up to 23.0 cm long, and is native to fresh and brackish water habitats with a pH of 7.0, a hardness of 15 dH, and a temperature of 18 to 22 °C [. This species has a wide distribution in the drainage basins of China, North Korea, South Korea, Japan, Mongolia and Russia  and multiple nuclear DNA (nDNA) markers to investigate the phylogeographical structure and evolutionary history of the http://iacuc.usc.edu/).All experimental protocols were approved by the Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences. The policies were enacted according to Chinese Association for Laboratory Animal Sciences, and coordinated with the Institutional Animal Care and Use Committee (IACUC) protocols  was amplified for all individuals using the universal primers L14724 and H15915 [The total genomic DNA was extracted from muscle tissue or fin tissue using standard salt extraction . The pard H15915 . We sequd H15915 . Nine pud H15915 . The ampThe nucleotide sequences were initially edited using the DNASTAR multiple package , aligned using MUSCLE  and optiH. leucisculus of the Cytb data were reconstructed using Bayesian inference (BI) and maximum-likelihood (ML) methods. Hemiculter bleekeri (GenBank no: KF029693) and Culter alburnus (GenBank no: KM044500) downloaded from GenBank, were used as the outgroup. Nucleotide substitution models (GTR + I + G) were selected using the Akaike information criterion in MRMODELTEST version 2.3 [Phylogenetic analyses of sion 2.3 . For BI sion 2.3 , startinsion 2.3  to ensursion 2.3  using a sion 2.3  and nodesion 2.3  to constIn addition, BI and ML analyses were employed for the nDNA loci using the identical parameter settings as well. The optimal nucleotide substitution model of each nDNA locus  could have affected the posterior probabilities of the models [0 were implemented in BPP:  τ = ; ɵ =  τ = ; and ɵ =  τ = . We ran the rjMCMC analyses for 1,000,000 generations, sampled every five generations, and discarded samples from the first 50,000 generations as burn-in. Each analysis was repeated at least twice to confirm consistency between runs. Topology based on Cytb was used as a guide tree.To examine whether the three lineages inferred from Cytb trees represent different species, we used Bayesian Phylogenetics and Phylogeography (BPP) v3.0 , 62 withe models , three dH. leucisculus were incorporated in the program BEAST 1.6.1 [Divergence times among the major lineages of ST 1.6.1 . For molST 1.6.1 . LackingST 1.6.1 –68. ThusPotential gene flow among major lineages was estimated using the isolation-with-migration (IM) model in the IMa2 program . We usedh) and nucleotide diversity (π) of each population and lineage were calculated using DnaSP v5 [The number of haplotypes (n), private haplotypes (ph), haplotype diversity (DnaSP v5 . In addiDnaSP v5  as impleST) was calculated using Jukes and Cantor distances between populations having more than five samples (17 populations fulfilled this condition) in ARLEQUIN 3.5. A Mantel test was used to investigate isolation by distance using genetic differentiation (ɸST/(1-ɸST)) and the geographical distance (in kilometers) measured as the straight-line distance, in ARLEQUIN 3.5. Geographic distances were estimated using GOOGLE EARTH v.4.3. All calculations implemented in ARLEQUIN 3.5 used 1000 permutations to assess significance.To investigate population structure, an analysis of molecular variance (AMOVA)  was perfFs statistics [Sample size allowing observed lineages was screened for historical demographic events in each deme. Selective neutrality test of Fu’s atistics  based onatistics , 77. We H. leucisculus including 98 novel sequences, 283 sequences from the study by Fan & He (2014) [A 1040-bp segment of Cytb was obtained from 390 individuals of e 2014)  and 9 se014  and The ML and BI trees based on Cytb produced similar gene tree topologies and congruently revealed three well-supported monophyletic lineages , although the support is not very strong in some nodes Fig. . The thr0 did showed consistent results.The nuclear data strongly supported that the three lineages defined by the Cytb data represent three different species with perfect statistical support  = 2.92–4.65 Ma). Lineage A diverged at 2.38 Ma , and lineages B and C split at 1.61 Ma .The null hypothesis of clock-like evolution was not rejected at the 5% level. Therefore, we estimated the divergence times for the monophyletic Cytb lineages identified in the phylogenetic analyses  and unidirectional migration events among the three lineages , with the highest within-lineage h occurring in lineage C (h = 0.9573 ± 0.0082), and was similar in lineages A (h = 0.868 ± 0.0400) and B (h = 0.891 ± 0.0320)  (Table P < 0.001) occurred among groups . A significant correlation between the pairwise genetic differentiations (ɸST/(1-ɸST)) and the geographical distance was found over the entire dataset , indicative of isolation by distance (IBD).Results from the two-level hierarchical AMOVA suggested that FSstatistics  based on Cytb sequences. Lineages A and B display a highly geographical structure. Members of lineage A originate from the middle and lower Pearl River drainage and Hainan Island, and most representatives of lineage B are limited to a small area  in the Upper Yangtze River. Regarding lineage A, the populations from middle and lower Pearl River drainages and Hainan Island were unexpectedly found to show a close relationship, though they are separated by the Qiongzhou Strait. This may be a result of the fact that the Gulf of Tonkin and the Qiongzhou Strait were once part of the coastal plain of the Asian continent during the Pleistocene glaciations [In this study, we clarify the phylogeographical structure and hidden diversity of ciations , 79. PopH. leucisculus populations between the Yangtze River basin and the four coastal rivers in southeastern China  may due to their spatial proximity. The spatial proximity among these rivers enables H. leucisculus populations from the Yangtze River basin to expand to nearby rivers easily due to floods in summer. The hypothesis of active river capture in southwestern China [In contrast, lineage C is poorly correlated with its geographic distribution and is extensively distributed in many isolated drainage basins, including the Lancangjiang River , the Upper Pearl River , the Yangtze River, the Minjiang River, the Jiulongjiang River, the Lingjiang River and the Qiangtangjiang River  supported a deep divergence among platypus , 83, Opss bidens , Rhyncho9-6.87% sientalis .H. leucisculus originated in the drainages of southern China at 3.80 Ma , suggesting that this species originated near the beginning of the Late Pliocene [We speculate that Pliocene , 85. ThiPliocene , 87. ThePliocene , 89, whiPliocene , 90, 91.Pliocene  and othePliocene , 31, 94.Lineages B and C, which comprised most individuals from the Yangtze River basin, separated at ~1.61 Ma , which corresponds to the hypothetical formation of the current Yangtze River , 96 and H. leucisculus populations. Firstly, the lower dispersal potential inferred from Mantel analyses indicates that geographic distance acts as a barrier for gene exchange between populations and may lead to genetic divergence. Furthermore, Cytb analyses suggest that H. leucisculus is geographically restricted to local areas, which may be due to the poor dispersal potential. For example, approximately 78.4% of Cytb haplotypes were only detected in single locality, which mainly distrubuted in Localities 3–12 and 17–26  and EBSPs, consistently indicated that rapid population expansion has occurred in the two lineages. The absence of sublineages probably further reflects an expansion event for the two lineages. Lineage B mainly occurred in narrow regions  and exhibited low genetic diversity, which was easily influenced by fluctuant climate. In contrast, the expansion time of lineage C (~0.08 Ma) was much earlier than that of lineage B, occurring in the Late Pleistocene with the Riss-Würm interglaciation (0.073–0.125 Ma), when the climate in China was warm and wet [H. leucisculus survival and dispersal. Furthermore, the floods frequently occurred in summer may be a potential factor that can promote the population expansion of this species. During annual rainfall periods, the floods are common, allowing fluvial connectivity restoration and providing a contemporary condition for H. leucisculus population expansion. Certainly, the decreasing of competition and predation may also contribute to the population expansion of these two lineages.In our study, lineages B and C of  and wet , 102. DuH. leucisculus [H. leucisculus inhabiting these regions might have remained in stable niches and did not experience drastic demographic fluctuations. The high level of genetic diversity reflects this scenario. Similar examples were reported in other taxa in southern China [However, we found no evidence of long-distance dispersal and population expansion in lineage A, which is principally distributed in southernmost China. In these temperate regions and the tropics, the relatively mild Pleistocene climate in southern China , 29, 103cisculus . The suirn China , 31, 104H. leucisculus under the phylogenetic species concept [H. leucisculus may have undergone cryptic speciation. However, the formal taxonomies of the DNA-based cryptic lineages are problematic [H. leucisculus detected by unlinked molecular markers merely conform to the primary species hypotheses, and more morphological analysis and phylogenetic evidence are needed to clarify formal taxonomic assignments of each lineage under the criteria of secondary species hypotheses [Many hidden species (cryptic species) with the same morphological characteristics have been recognized using molecular tools and integrative taxonomy, which enrich species diversity but challenge traditional morphological taxonomy . In our  concept . Thus, g concept , H. leucblematic . The crypotheses .IM analysis provides unambiguous evidence of gene flow from lineages B to A, from C to A, and from C to B Fig. . In the H. leucisculus in the drainages of southern China. Three mtDNA lineages were observed, and each lineage represents a valid species. Intense uplift of the Qinghai–Tibetan Plateau, evolution of Asian monsoons, changes in paleo-drainages, local adaptation and poor dispersal ability may have driven the divergence of the three putative species. However, gene flow occurs among the three lineages. Demographic analyses suggest that lineages B and C have experienced rapid demographic expansion, whereas lineage A remained stable long term during the cyclical Pleistocene glacial periods. Our results reveal the phylogeographical structure, cryptic diversity and evolutionary history of H. leucisculus populations and reinforce the notion that the number of widespread Chinese cyprinid species has been underestimated.Our study documents the phylogeographic structure and cryptic speciation of Additional file 1: Table S1.Hemiculter leucisculus and outgroups. Locality numbers correspond to Fig. Summary of sample localities for Additional file 2: Table S2.Table S3 Summary statistics for the 13 loci used in this study. bp, length of locus in bases; n, number of sequences; ps, polymorphic sites; pis, parsimony informative sites; LNR, length of the longest non-recombining regions, h, number of haplotypes. Table S4 Nucleotide substitution models used in tree reconstruction. Pinvar, proportion of invariable sites; Gamma, gamma shape parameter. Table S5 Nucleotide substitution models for Cytb and three nuDNA loci used in extend Bayesian skyline plots. Table S6 Genetic diversity statistics for each population based on Cytb. n, individual numbers; Nh, haplotype numbers; ph, private haplotype within each population; Hap, Cytb haplotype; h, haplotype diversity; π, nucleotide diversity. Table S7 Genetic distance based on Cytb among the three lineages estimated by K2P distance (%). Figure S1 The entire distribution of Hemiculter leucisculus in global scale. The map derived from http://www.discoverlife.org. (DOCX 267 kb)Information of primer pairs used in our study.


SNPs) for inferring mountain pine beetle population structure across its geographic range. Both adaptive and neutral SNPs, and their combination, allowed range‐wide structure to be distinguished and delimited a population that has recently undergone range expansion across northern British Columbia and Alberta. Using an equal number of both adaptive and neutral SNPs revealed that adaptive SNPs resulted in a stronger correlation between sampled populations and inferred clustering. Our results suggest that adaptive SNPs should not be excluded prior to analysis from neutral SNPs as a combination of both marker sets resulted in better resolution of genetic differentiation between populations than either marker set alone. These results demonstrate the utility of adaptive loci for resolving population genetic structure in a nonmodel organism.Assessments of population genetic structure and demographic history have traditionally been based on neutral markers while explicitly excluding adaptive markers. In this study, we compared the utility of putatively adaptive and neutral single‐nucleotide polymorphisms ( Neutral genetic markers have been considered essential to research on population structure , occurs throughout much of western North America as a native pest of mature pine forest , western white pine (Pinus monticola Douglas ex D. Don), and lodgepole pine , a novel host , D. monticolae  sequences and amplified fragment length polymorphisms (AFLPs) showed isolation‐by‐distance (IBD) patterns around the US Great Basin , with emphasis on Canadian populations. Genomic DNA was extracted from late instar larvae and adults using the DNeasy 96 Blood and Tissue Kit , following the manufacturer's instructions, or phenol–chloroform extraction as outlined in Samarasekera et al. . GenomicSequenom  at the GSequenom . SelectiSequenom .Based on their identification as outliers by Janes et al.  using BaFST measures for all 62 populations were calculated to evaluate genetic differentiation among MPB populations and tested with 10,000 permutations using Arlequin 3.5.2 (Excoffier and Lischer K) of genetically related individuals. The number of retained principal components (PC) was determined using the optim.a.score function for each data set. We retained seven PCs for the neutral SNPs, 24 for the adaptive SNPs, and 14 for the combined panel of SNPs. The Bayesian information criterion was used to select the optimal number of K clusters.Population genetic structure was determined using three data sets: (1) all available SNPs, as “combined”; (2) putatively adaptive SNPs; and (3) putatively neutral SNPs. Pairwise K 1–10 using the following parameters: admixture ancestry model and correlated allele frequencies, 10,000 burn‐in, 100,000 MCMC repetitions, and 10 iterations of each K. The Delta K method of Evanno et al. . Using this data, ObStruct generates a multiple R2 statistic that is used to explain how much of the variability is explained by the sampling locations. This correlation, presented as a single R2 statistic, is interpreted such that a low R2 indicates no significant structure between populations and a high R2 indicates strong population structure.Comparisons of STRUCTURE results from each of the data sets  were conducted with ObStruct . FST values between northern Canadian populations and southern Canada averaged 0.106, whereas average pairwise distances within these two regions were 0.019 and 0.026, respectively. South Dakota and Arizona had the largest differences from other regional groupings, ranging from 0.267 for US Rocky Mountain populations  to an average of 0.376 in comparisons with northern Canadian populations.Pairwise K = 2 as optimal, separating northern Canadian populations from the southern Canadian and US populations. All randomized data sets reflected this optimal K = 2. STRUCTURE analysis at K = 3 for each of the data sets also distinguished the populations from South Dakota, Arizona, and part of Colorado . For adaptive SNPs, population groupings are less distinct in each plot, and although some genetic differentiation of west coast US populations can be seen, adaptive SNPs did not identify the Manning Park and Whistler populations as a separate cluster. Further analysis (K = 5) did not differentiate US Rocky Mountain populations  from the remaining US populations.In each data set , STRUCTURE analysis showed K = 2 for each data set. Unlike the STRUCTURE results, the optimum K value for DAPC was K = 8, resulting in several broadly overlapping clusters that were difficult to interpret . However, the bar graph plots for DAPC for K = 3 to K = 5 showed substantial similarity to the corresponding STRUCTURE plots for each of the data sets , indicating that the variability of the data was best explained by sampling locations, than each of the three randomized subsets of neutral SNPs (R2 = 0.76–0.77). Assignments based on the full set of neutral SNPs resulted in an increase in the R2 statistic (R2 = 0.83), as did the combined set containing both neutral and adaptive SNPs (R2 = 0.85).Ancestry profiles from STRUCTURE for Using the combined data set, BayeScan was used to identify loci that might show differing degrees of selection within the different regional groupings of MPB populations. The clustering assignments from STRUCTURE and DAPC were used to partition the different populations for outlier detection. No outliers were detected within the US clusters , or within northern Canadian populations.Examination of southern Canadian/northern US populations identified 12 outlier loci, two of which were noncoding. The SNPs in coding regions with a putative protein function included the following: a gene encoding a ribonuclease CAF1 (locus 0103), ribosomal protein L9 (locus 0147), vacuolar protein sorting‐associated protein 51 (Vps51) (locus 0369), proliferation‐associated protein 2G4 (locus 0821), ABC transporter G family member (locus 0950), major facilitator superfamily (MFS) protein (locus 0981), family with sequence similarity 60 member A (FAM60A) protein (locus 1045), Beige and Chediak‐Higashi (BEACH) domain‐containing protein (locus 1069), and a cold shock‐containing protein (locus 1529).This study documents the first explicit use of adaptive SNPs to assess the range‐wide population structure of an invasive insect pest. Adaptive SNPs detected in MPB populations may be important for identifying local variation and selection processes in regions where ecological adaptation to different conditions is leading to increased fitness  that is an RNA‐binding protein with a potential role in cell‐cycle control. The second is a FAM60A protein, a subunit of the SIN3A‐HDAC complex that acts as a regulator of gene expression by deacetylating histones  and adaptive loci (R2 = 0.81) showed that sampling locations for each individual had a stronger correlation with their inferred population cluster using the adaptive markers. According to Gayevskiy et al.  in addition to combining both adaptive and neutral markers (R2 = 0.85), indicating that increasing the number of markers for STRUCTURE analysis leads to more differentiation between inferred populations. Taken together, these results show that the removal of adaptive loci prior to analyses is not always advisable as these loci may be helpful in identifying differentiation between populations when combined with neutral markers, and can potentially identify fine‐scale structure to define management units.Adaptive and neutral markers showed largely concordant results for the overall population structure of MPB Fig. . Howevery et al. , the lowThis study shows that outlier loci can be highly informative for determining the population structure of MPB across its entire range. This challenges the traditional population genetic paradigm of using only neutral markers. Moreover, with the increasing availability of SNP data, this demonstrates the value of including adaptive loci, whether alone or in conjunction with traditionally neutral loci, for population genetic studies.None declared.http://dx.doi.org/10.5061/dryad.540bt).All data used in this article will be available in Dryad Digital Repository ; southern Canada and Idaho, Montana, and Washington (green); Oregon, California, and Nevada (orange); Utah and Wyoming Colorado, Arizona, and South Dakota (blue).Figure S2. DAPC clustering of each marker set for K = 8.Click here for additional data file.Table S2. Pairwise values of FST calculated between mountain pine beetle populations.Click here for additional data file.Data S1. SNP list.Click here for additional data file.


California red‐legged frog (Rana draytonii) has undergone extensive declines in heavily urbanized southern California, where the range edge has rapidly contracted northward while shifting its cardinal orientation to an east‐west trending axis. We studied the genetic structure and diversity of these frontline populations, tested for signatures of contemporary disturbance, specifically fire, and attempted to disentangle these signals from demographic events extending deeper into the past. Consistent with the genetic expectations of the ‘abundant‐center’ model, we found that diversity, admixture, and opportunity for random mating increases in populations sampled successively further away from the range boundary. Demographic simulations indicate that bottlenecks in peripheral isolates are associated with processes extending tens to a few hundred generations in the past, despite the demographic collapse of some due to recent fire‐flood events. While the effects of recent disturbance have left little genetic imprint on these populations, they likely contribute to an extinction debt that will lead to continued range contraction unless management intervenes to stall or reverse the process.Populations forming the edge of a species range are often imperiled by isolation and low genetic diversity, with proximity to human population centers being a major determinant of edge stability in modern landscapes. Since the 1960s, the  A variety of evolutionary and ecological phenomena create greater instability in populations at the edge of a species' distribution compared with those located more toward the interior. These can include maladaptation to environments at or beyond the current boundary, increased competition with other species, reduced immigration due to greater isolation, low genetic diversity, smaller effective population sizes, or interactions between these and other variables Holt . When anBecause these phenomena have the potential to reduce the performance of fitness‐related traits and adaptive potential, peripheral populations are controversial targets for conservation (Hunter and Hutchinson Rana draytonii). Since the 1960s, a gap between extralimital populations in the San Pedro Martir Mountains of Baja California and southern California has been widening (~500 km), with the southern range limit in California contracting northward along the coast , East Las Virgenes by the Topanga Fire of 2004 (98 km2), and Aliso by the Crown Complex Fire of 2004 (73 km2) and the Station Fire of 2009 . Sampling at San Francisquito consisted of a temporal series, one representing a preburn group (collected in 2002) and two representing postburn groups (2005/06 and 2009). We combined the 2005–06 cohorts due to small sample sizes and because both were sampled after anomalous El Niño storm events that heavily impacted aquatic habitat in the canyon. East Las Virgenes and Aliso were each represented by a single time series; the former was a postburn sample collected in the wake of the Topanga Fire, and the latter was collected after the Crown Fire but during the Station Fire.We collected toe clips from all known HincII, followed by ligation of an SNX linker to these fragments. Biotinylated probes that included di‐, tri‐, and tetranucleotide repeats were then used to isolate and separate microsatellite loci from the rest of the DNA extraction. These fragments were PCR amplified and sequenced on a 454‐automated pyrosequencer (Roche) by the Evolutionary Genetics Core Facility (EGCF) at Cornell University.We isolated microsatellite loci from genomic DNA using a modification of the Hamilton et al.  protocol®. All genotyping runs were performed on an ABI 3730 in the CSUPERB Microchemical Core Facility at San Diego State University. We edited and scored the raw data in gene‐marker v1.90 (SoftGenetics) and used microchecker . We divided these loci into four groups and performed multiplex PCRs using a Qiagen Multiplex PCR Kitstructure v2.3  to which individuals could be assigned. For each K, we performed 10 separate runs  using both the correlated and uncorrelated allele frequency models with admixture. To approximate the number of clusters, we used structure harvester  score for each of the 10 runs against K, where the number of clusters was based on the asymptote of the lnP(D|K) curve. Based on these results, we performed a second round of assignment tests on clusters from the initial analysis that included multiple sampling locations  for two individuals in each population and analyzed the phylogeography of haplotypes across the same geographic area sampled for microsatellites. We used the primers MVZ15‐L , expected heterozygosity (HE), Queller and Goodnight's relatedness index (R), and the inbreeding coefficient FIS in genalex v6.0  using approximate Bayesian computation (ABC) in OneSAMP ), we specified a noninformative, flat prior on Ne (5–1000) based on Ne estimates across a range of amphibian taxa, including several North American and European ranids ; Waples Ne estimate for the San Francisquito population, given that we had three samples separated by multiple generations.We estimated effective population sizes  are below the observed M‐ratio. We also considered low allelic richness, low heterozygosity, and increased numbers of monomorphic loci to be an indicator of a bottlenecked population, as excess heterozygosity diminishes over time if the population size remains small.We used two approaches to infer population bottlenecks. First, we tested for heterozygote excess using a two‐phase mutation model and a Wilcoxon signed‐rank test in Ne for each population. Our approach consisted of two steps: (1) simulating data sets based on scenarios in which the historical effective size Ne1 exceeded the current effective size Ne0 at time T in the past (measured in number of generations); (2) estimating a posterior for T through an additional set of simulations by setting a prior based on the most likely scenario(s) from step (1). We inferred the ‘best‐fit’ scenarios from step 1 using the closest 10 000 simulated data sets and the logistic and direct approaches described in Cornuet et al.  and Ne1 (51–10 000). Prior bounds for Ne0 were based on the upper limits of the 95% CIs inferred for Ne(ABC) and Ne(LD), whereas the bounds for Ne1 were restricted to values above 50 but were otherwise noninformative.Having detected bottlenecks in the three fire‐affected edge populations , we used coalescent‐based simulations under different demographic scenarios to approximate the timing of major reductions in t et al. ; see alsTn and Ne as free parameters with uniform prior distributions. For San Francisquito, we implemented the same priors for Ne as described above and bounded the Tn prior (3–200) according to the number of generations separating our temporal groups . This forced the bottleneck to predate our earliest sample, which is reasonable given that the 2002 sample showed a significant bottleneck based on multiple techniques. For Aliso and East Las Virgenes, we expanded the lower prior bound for Tn to 1–200 because these sites were limited to single point samples. We estimated Tn using the closest 10 000 simulated data sets to the observed data based on the same summary statistics and regression approach as described above. All simulations were conducted in DIY‐ABC v1.0.4.46 . We lumped the 2005–06 samples because of small sample sizes for those 2 years  and because both were obtained in the aftermath of debris flows that were directly caused by the Copper Fire. We also performed an analysis of molecular variance (amova) in arlequin v3.5  curve and the ΔK statistic indicated six clusters as an estimate for KMAX, three of which were exclusive to individuals belonging to each range‐edge population .We found no qualitative differences in the cluster assignments based on models using correlated versus uncorrelated allele frequencies; thus, we discuss only the results from the uncorrelated analyses. Visual inspection of the lnP formed an exclusive group, a pattern also recovered in the nested structure analyses. Nested cluster analyses revealed further evidence of population substructuring, with most sampling areas separating into distinctive clusters. Some exceptions occurred for groups of sites in adjacent drainages on the south‐facing slopes of the Santa Ynez Mountains and near Point Conception, all of which lie in close proximity and showed relatively low differentiation (Table S3).Of the remaining clusters identified at als Fig. . One excals Fig. . At K ≥ Population structuring based on the microsatellites largely mirrored the diversity and distribution of mtDNA haplotypes Fig. . We recoNe) rather than amplification or other sample‐wide problems. Table S2 provides motif identities and summary statistics for all loci.All microsatellites conformed to mutational expectations as predicted by repeat motif, with the exception of allele 198 at RADR4‐09, which we determined was an out‐of‐phase indel . We detected possible null alleles and significant LD in two loci for the San Francisquito population only, but we suspect that these anomalies represent demographic artifacts  revealed no significant changes in AR , HO (P = 0.30), HE (P = 0.22), or relatedness (P = 0.49) for pre‐ and postfire samples.Diversity measurements by cluster based on the nested AR  and HO , and a significant negative relationship with respect to the average relatedness among individuals within a sample R . We did not detect any significant associations between these same indices and latitude. Ne(LD) estimates also suggested a longitudinal effect, where populations closer to the range edge tended to have smaller effective sizes (Table Ne(LD) values for some locations and independent onesamp runs produced very similar results (r = 0.92); however, the ordinal geographic pattern was not upheld in the former. Both Ne(LD) and Ne(ABC) showed decreasing trends across sampling years for San Francisquito. Error estimates for both approaches were overlapping across year classes, indicating that any temporal signal in the data was weak. The Ne(TM) estimate for San Francisquito  was in strong agreement with the ABC results for the 2002 and 2005–06 samples, and the slightly wider CIs also encompassed the Ne(LD)values.We found significant positive relationships between increased longitude of the sampling location and M‐ratios  than either the Aliso  or San Francisquito populations . These results were consistent with expectations based on the scenario comparisons from step 1. We note that the T1 parameter should be interpreted as the point in time in which the historical Ne was much larger than the current Ne; thus, the actual population bottleneck would have occurred at some time after T1. Even when considering the lower bounds of the 95% CIs, these estimates indicate that major size reductions in each population predate the surge of recent fires in southern California.Comparison of competing demographic scenarios showed that the model in which ses Fig. , whereasFST and RST among sampling sites were 0.26 and 0.31, respectively, and overall among site differentiation was significant . Pairwise FST and RST were significantly correlated based on a Mantel test ; thus, we discuss only the FST estimates .Average pairwise values for 6, r = 0.55, P ≤ 0.001) across the sampling range. Only a few sites along the southern slopes of the Santa Ynez Mountains and to the immediate west near Point Conception were not significantly differentiated. Differences among clusters identified at KMAX = 6 showed that pairwise FST estimates for interior samples ranged from 0.09 to 0.19, whereas the average among the three edge populations was 0.31. Comparison of residuals from the regression of pairwise FST values and corresponding geographic distances confirmed that genetic differentiation among sites increased toward the range edge , with larger differences observed as the sampling interval between pairwise comparisons increased . Inspection of allele frequencies across year groups showed that five low frequency alleles from three loci completely dropped out of the population over the 7 year time gap following the Copper Fire . Consistent with the comparison of diversity indices, the amova showed that only 2.1% of the total variation in pairwise FST estimates among year classes was explained by differences in the timing of the sampling.We conducted an additional set of pairwise R. draytonii populations forming the southern range edge and their interdigitation within major urban centers acts to suspend gene flow along the periphery of the distribution and raises conservation concerns for some of the most threatened populations in the species' range. On the one hand, the low diversity and isolation of sink populations at the range edge suggest that management efforts should focus on preserving high diversity, interior sources to the west/northwest  and range from 20 to 30 individuals per population – for study sites with more than 15 samples, our results are in general agreement with these data and add to growing evidence that effective sizes for North American ranids are typically on the order of tens of individuals, rather than hundreds or thousands  that cause species to move poleward often leave older populations with higher genetic diversity at the more southern latitudes  and do so with little regard for topography or vegetation. Thus, when pressed under certain conditions, such as low density or the disappearance of habitat, these frogs may have a greater tendency to behave outside the norm.Most sampling locations showed little evidence of recent gene flow, even in clusters with multiple sample site membership. This finding concurs with studies on other closely related ranid frogs, where populations tend to be highly structured by drainage basins  where frogs are more abundant and occur in less disturbed habitat and therefore have greater opportunity to receive immigrants from outside of the effected areas. While several of the more interior populations showed evidence of demographic reduction, even a small number of immigrants from nearby sources could result in rapid genetic recovery during periods of low density  may not erase the signature of the initial expansion unless the population once again approaches drift‐mutation equilibrium, or the secondary decline is severe and protracted , we hypothesize that the bottleneck was caused by a founder event in San Francisquito Canyon, where frogs colonized the area from upstream reaches of the Santa Clara River that were unaffected by the disaster once habitat developed in situ after the dam collapsed. Under this scenario, our T1 and historical Ne estimates would reflect the status of this putative source population well before R. draytonii or the dam ever existed in San Francisquito Canyon.Because the current breeding pools owe their existence to remnants of the dam's infrastructure and plunge pool, and museum records indicate that the canyon did not support R. draytonii can be maintained while simultaneously maximizing the amount of genetic diversity in rescue populations. Translocation sites in the Santa Monica Mountains of coastal California are currently under consideration because of the availability of protected lands and the former presence of these frogs throughout this area. Today, the only surviving R. draytonii population in the Santa Monica Mountains occurs in East Las Virgenes Canyon.We outline a strategy for selecting source populations to help fulfill translocation and genetic rescue efforts by the National Park Service and the U. S. Fish and Wildlife Service . Our approach focuses on the ways in which the historical population structuring of R. draytonii with relatively long generation times. They also reveal the ways in which historical demographic events can increase extinction debt and limit the genetic resources of a population before more contemporary disturbances even occur – this problem may be particularly acute if the population is already naturally challenged due to its location at the edge of a species boundary.Our findings emphasize the need for continuous genetic monitoring over longer temporal scales following major contemporary disturbances to better understand the genetic consequences of these events, especially for species like 10.5061/dryad.g2b4h; www.datadryad.org), and DNA sequences are available on GenBank .Microsatellite data for this study are available on the Dryad database (DOI:Figure S1. Unrooted neighbor‐joining tree based on the pairwise Cavalli‐Sforza chord distances.Table S1. Locus labels, primer sequences, fluorescent dyes, and multiplex PCR mixes for the 15 microsatellite sequences used in this study.Table S2. Summary statistics for the microsatellites developed for R. draytonii.Table S3. Pairwise FST values.Data S1. Model codes for DIY‐ABC analyses.Click here for additional data file.


It is increasingly common for apex predators to face a multitude of complex conservation issues. In Australia, dingoes are the mainland apex predator and play an important role in ecological functioning. Currently, however, they are threatened by hybridization with modern domestic dogs in the wild. As a consequence, we explore how increasing our understanding of the evolutionary history of dingoes can inform management and conservation decisions. Previous research on whole mitochondrial genome and nuclear data from five geographical populations showed evidence of two distinct lineages of dingo. Here, we present data from a broader survey of dingoes around Australia using both mitochondrial and Y chromosome markers and investigate the timing of demographic expansions. Biogeographic data corroborate the presence of at least two geographically subdivided genetic populations, southeastern and northwestern. Demographic modeling suggests that dingoes have undergone population expansion in the last 5,000 years. It is not clear whether this stems from expansion into vacant niches after the extinction of thylacines on the mainland or indicates the arrival date of dingoes. Male dispersal is much more common than female, evidenced by more diffuse Y haplogroup distributions. There is also evidence of likely historical male biased introgression from domestic dogs into dingoes, predominately within southeastern Australia. These findings have critical practical implications for the management and conservation of dingoes in Australia; particularly a focus must be placed upon the threatened southeastern dingo population. The minAn alternative hypothesis is that dingoes and NGSD are part of an older dog radiation that immigrated into Australia via the land bridge between Australia and Papua New Guinea, which flooded 6,000–8,000 years BP  and northwestern (NW), in Australia with a pattern of geographical subdivision. Early studies using mitochondrial DNA were unable to elucidate continent‐wide biogeographic patterns, as they restricted genetic sampling to the mitochondrial control region  in length. The mitochondrial control region is 582 bp .2, 6.25 pmol of each primer, 7.5 mmol/L of dNTPs, 2.5 U of Taq DNA Polymerase , and 20–50 ng of DNA template. All PCR reactions were cycled using the following thermal profile: 98°C for 2 min, 95°C for 3 min (add Taq polymerase), then 95°C for 15 s, 52°C for 1 min, 65°C for 1 min for 10 cycles, then 95°C for 15 s, 52°C for 1 min, and 65°C for 1 min (increase time by 5 s each cycle) for 25 cycles followed by 65°C for 10 min.Qiagen DNeasy kits  were used to extract DNA, and mitochondrial loci were amplified using PCR Table . Briefly® . Purified templates underwent Sanger sequencing using standard BigDye terminator v3.1  technology. DNA sequence chromatograms were analyzed and aligned using Sequencher 5.1 .Prior to sequencing, PCR amplicons underwent purification by ExoSAP‐IT2.3650‐79.2b, 650‐79.3b, 990‐35, MS34CA, and MS41B as previously described  was used to genotype 29 single nucleotide polymorphisms (SNPs) from the nonrecombining Y chromosome (NRY) region as described in Sacks et al. . These 22.4D, Fu and Li's F*, and Fu and Li's D* . Mitochondrial networks were created for the concatenated diagnostic region and control region separately. Control region data were analyzed separately to allow incorporation of and comparison to the existing dingo control region dataset  haplotypes with three novel CR haplotypes in 124 dingoes (three dingoes were excluded due to PCR difficulties) and five NGSD Table . A singlA total of 39 mitochondrial diagnostic region haplotypes were observed in 124 dingoes and 5 NGSD . To further investigate the distribution of the Y chromosome haplogroups across Australia, we incorporated the dingo and NGSD data from Sacks et al. , and SE (H3/H1) (Ardalan et al., Our data suggest that the two divergent Y chromosome lineages observed in dingoes have different geographical origins and are plausibly the result of multiple immigrations into Australia, as postulated by Cairns and Wilton . NotablyThese data have intriguing implications for the movements of canids, and presumably humans, in Australasia. Cairns and Wilton  postulat4.2Demographic modeling and neutrality test results based on mitochondrial data should be treated cautiously but can give insight into modern and historical demographic patterns. Bayesian skyline plots based upon the individual mitochondrial lineages suggest that the SE population size has been stable until about 1,000 years ago, when it underwent rapid expansion Figure . The NW Biogeographic patterns within Australia provide insight into the modern dispersal and migration of dingoes. We observed that the geographical distribution of the two mitochondrial lineages, SE and NW, exhibits strong geographical subdivision Figure . Only a Contrary to demographic modeling, the neutrality test results indicate that the two dingo populations may be experiencing different demographic and/or selective pressures Table . These d4.3The H1 Y chromosome haplogroup is considered to be a European domestic dog haplogroup (Ardalan et al., The distribution of the H1 haplogroup in southeastern Australia further suggests it is likely the result of introgression Figure . First, 4.4The most important implication of these data is that conservation and management efforts should be focused on maintaining the existing dingo population structure and thus treating the two populations as distinct conservation units. Care should be taken not to deliberately translocate individuals between wild populations. Captive breeding programs may need to ensure that the two dingo populations are maintained separately, with mitochondrial and Y chromosome testing used to identify population ancestry. Fraser Island dingoes appear to share NW paternal lineage ancestry but carry SE mitochondrial types; if genetic rescue is attempted for this inbred island population, then individuals from appropriate genetic lineages should be located.Conservation groups have long described the presence of multiple morphological varieties of dingo, generally alpine, desert, and tropical; however, it is not clear whether this phenotypic variation is associated with the genetic subdivisions or phenotypic plasticity, although the boundaries do overlap. A future study of morphological and phenotypic variation as well as genetic variation may help answer this question. It is possible that the two dingo populations have different ecological or biological characteristics relevant to the conservation and management of the species or its role in specific ecosystems. Patterns of genetic subdivision in other large carnivores have been linked to ecologically relevant characteristics such as neonatal dispersal (Sacks, Bannasch, Chomel, & Ernest, The presence of the H1 haplogroup in southeastern Australia has important implications for conservation and future management strategies; namely, it highlights the importance of inhibiting further hybridization. Neutering male dogs and/or restricting them from reproducing with wild dingoes may help achieve this. Particularly, best practice should dictate that pet, livestock guardian, or working dogs in rural localities should be neutered or chemically castrated to avoid further risk of hybridization. Widespread lethal control measures are shown to also facilitate hybridization by breaking apart pack structures (Wallach et al., Knowledge concerning levels of genetic integrity in wild populations is necessary to inform management and conservation programs. The southeastern population of dingoes is under particular extinction pressure from both fatal management strategies and hybridization; steps should be taken to preserve this population before it is too late. A broad genetic survey of dingoes in National Parks and State Forests in southeastern states would be needed to pinpoint high dingo ancestry populations and thus where to focus conservation efforts. Currently state and federal legislation do not protect the dingo sufficiently and allow widespread fatal control measures Davis, , 2006. R5Distinct populations of apex consumers can exhibit different behaviors and prey on disparate trophic niches (Paiva, Fagundes, Romão, Gouveia, & Ramos, KMC completed experimental design, mitochondrial DNA data collection, analysis, and writing of manuscript. BNS and SKB provided advice concerning experimental design and analysis, collected the Y chromosome data, and edited the manuscript text. JWOB provided advice and comments concerning experimental design and manuscript text.The authors declare no conflict of interests.


The domestic water buffalo is native to the Asian continent but through historical migrations and recent importations, nowadays has a worldwide distribution. The two types of water buffalo, i.e., river and swamp, display distinct morphological and behavioral traits, different karyotypes and also have different purposes and geographical distributions. River buffaloes from Pakistan, Iran, Turkey, Egypt, Romania, Bulgaria, Italy, Mozambique, Brazil and Colombia, and swamp buffaloes from China, Thailand, Philippines, Indonesia and Brazil were genotyped with a species-specific medium-density 90K SNP panel. We estimated the levels of molecular diversity and described population structure, which revealed historical relationships between populations and migration events. Three distinct gene pools were identified in pure river as well as in pure swamp buffalo populations. Genomic admixture was seen in the Philippines and in Brazil, resulting from importations of animals for breed improvement. Our results were largely consistent with previous archeological, historical and molecular-based evidence for two independent domestication events for river- and swamp-type buffaloes, which occurred in the Indo-Pakistani region and close to the China/Indochina border, respectively. Based on a geographical analysis of the distribution of diversity, our evidence also indicated that the water buffalo spread out of the domestication centers followed two major divergent migration directions: river buffaloes migrated west from the Indian sub-continent while swamp buffaloes migrated from northern Indochina via an east-south-eastern route. These data suggest that the current distribution of water buffalo diversity has been shaped by the combined effects of multiple migration events occurred at different stages of the post-domestication history of the species. Bubalus bubalis  is native to the Asian continent. Through historical migration events and recent importations, the species reached a worldwide distribution during the last century  or subspecies . Besides displaying distinct morphological, cytogenetic  and behavioral traits, the two types also have traditionally had different purposes and geographical distributions  in north-western India  estimate the levels of molecular diversity, (ii) describe population structure, and (iii) identify historical relationships between populations and migration events.The collection of samples used for the present study was carried out during years 2011 and 2012, before Directive 2010/63/EU came into force . Thus all experimental procedures were compliant with the former EU Directive 86/609/EEC, according to which no approval from dedicated animal welfare/ethics committee was needed for this study. The permission to carry out the sampling at each farm was obtained directly from the owners. All the samples were collected during routine veterinary checks and in compliance with local/national laws and ethical rules in force at the time of sampling in the countries participating to the International Water Buffalo Genome Consortium (IWBGC).ex-situ populations reared in the Philippines.A total of 333 individuals were sampled from 31 populations covering a large part of the worldwide geographical distribution of water buffalo  with the Axiom® Buffalo Genotyping Array 90K from AffymetrixSince the Axiom® Buffalo SNP panel has been developed starting from a set of river-type buffalo breeds . To check the effectiveness of this strategy in reducing the impact of AB, we compared the average values of observed heterozygosity obtained within this dataset to those obtained from a second version of the dataset which included all SNP markers that resulted polymorphic overall, named poly-ALL hereunder. Poly-ALL dataset has been used only for the purpose of this comparison and no further analyses were performed on it.Thus, to reduce the impact of AB, the main dataset was built by including individuals from both river and swamp-type populations and only those SNP markers that were polymorphic in swamp buffalo  procedures performed with the R package stats R package. The number of most informative dimensions was identified from the bar plot of their eigenvalues.To evaluate the relationships between individual multilocus genotypes, Multi-dimensional Scaling (MDS) plots based on the IBS distances were obtained with the Arlequin version 3.5.2.2  and expected heterozygosity (HE); (ii) compute Wright's FST fixation index  within the tree-like graph, and estimates the proportion of admixture displayed by the receiving groups. In order to avoid issues related to missing values, all marker positions displaying missing data were removed. Furthermore, to assess the robustness of the graph underlying the modeled migrations, we adopted the following bootstrap-based procedure implemented in BITE package  and with a number of SNPs per block equal to 50. The most meaningful number of migrations, mbest, was identified based on the variance explained, the log likelihood and p values associated with each m, and the biological meaning of the migrations themselves. Then 100 bootstrap replicated runs of the analysis with mbest migrations were performed, and a consensus tree was built with the consense executable implemented in PHYLIP ver. 3.696 . Thus, the working version of the dataset included 20463 SNPs, 327 individuals and 30 populations. Population size ranged from three to 15, with an average of 10.90. Table During QC procedures to create the poly-ALL) contained 52637 SNPs, 335 individuals and 31 populations.The dataset version based on markers polymorphic overall  and 0.067 (SWATH_THT), but was never statistically significant  to 0.448 (SWAID_JAV vs. RIVMZ) overall, from 0.006 (RIVPK_AZK vs. RIVPH_IN_MUR) to 0.199 (RIVIR_MAZ vs. RIVMZ) among the river buffalo group, from 0.004 (SWACN_GUI vs. SWACN_YIB) to 0.232 (SWAID_NUT vs. SWABR_CAR) among the swamp buffalo group, and from 0.247 (SWATH_THS vs. RIVCO) to 0.448 (SWAID_JAV vs. RIVMZ) between river and swamp populations.Fjaatha software, the number of migrants varied between 0.010 and 75.000 .The MDS plot Figure  allowed Notably, the position of the swamp Carabao breed on the second axis corresponds to that of the river population from Romania.Similarly, the third dimension  are placed in a basal position, while the Brazilian Carabao branch forks at a distance from the network formed by the remaining swamp populations.ADMIXTURE software analysis, the first subdivision highlighted at K = 2 is between river- and swamp groups of populations  and of the Philippines (SWAPH). At K = 3  was identified as the number of migrations of choice to run the subsequent bootstrap-based analysis. The consensus tree obtained from the 100 replicates  are adopted when dealing with swamp-type buffaloes. In fact, due to the over-representation of river buffalo breeds in the SNP discovery panel, the array is affected by a moderate-to-high degree of AB if used in swamp buffalo, as described by Iamartino et al.  and furtThe strategy adopted here  allowed a significant reduction of the impact of AB  different from the description of diversity, the development of a more balanced or swamp-type specific array would be advisable.O = 0.417; RIVPH_IN_MUR, HO = 0.412) may have been influenced by AB, since these breeds were among those included in the SNP discovery panel .Among river buffaloes, our evidence indicating the breeds from the Indian sub-continent of the Murrah-Nili Ravi-Kundi group as the most variable also agrees with previous research based on microsatellite , in line with those of the neighboring populations . This discrepancy between SNP- and microsatellite-based heterozygosity estimates could be due to marker selection, since in the aforementioned study only three microsatellite loci were typed.A general agreement between SNP- and microsatellite-based heterozygosity estimates emerges from the comparison of our results with the literature, with the sole exception of the Egyptian animals. In fact, contrary to previously reported microsatellite-based estimates of HO described above is mostly confirmed by the corrected HE values, which also indicated the river populations from Colombia and the Murrah from Brazil as highly heterozygous . In both cases, high HE values likely mirror the effect of the Indian Murrah ancestry of the southern American populations combined with limited but detectable crossbreeding with Mediterranean water buffaloes.The trend of HO = 0.413 likely derives from crossbreeding with river gene pool, the highest HO values were observed in Thailand  to slightly negative , but they were never statistically significant , and to a lesser extent also the population from the Philippines (SWAPH), showed clear signals of a river-type genomic contribution that, according to our results Figures .These findings agree with bibliographic records that accounted for the establishment of crossbreeding programs in several countries to increase milk production in swamp populations  and the Indian Murrah reared in the Philippines (RIVPH_IN_MUR) are characterized by the highest figures for corrected Ho Table , and alsh Figure .O and HE values and also bear signs of the combined effects of a long-time isolation and human-mediated selection, as highlighted by their outlier behavior in the MDS , Turkey (RIVTR_ANA) and Iran .Conversely, the Mediterranean breeds RIVIT_MED, RIVMZ, and RIVRO display the lowest HS Figure  and in th Figure . The disRegarding the Iranian breeds, a previous study based on mitochondrial DNA . Besides displaying the highest Hes Table , in both Figures , 5 they  Figures .The other populations of the Indonesian islands  bear signs of geographical isolation, as testified by the small area in a peripheral position that they occupy in Figure E values after grouping the populations based on their geographical provenance  ADMIXTURE software membership coefficient at K = 6 averaged over geographical areas; (ii) the outcomes of previous molecular-based research, and (iii) historical and archeological evidence, the emerging picture allowed us to formulate the following scenario: after domestication in the Indian sub-continent, early domestic river buffalo populations spread westwards through south-western Asia, with a probable migration wave that led the proto-Mediterranean populations into southern Europe and whose traces are still recognizable in the distinctiveness of the present-day Mediterranean buffalo gene pool. According to the historical records .The genotype data used in the present study are available from the Dryad Repository (doi: LCo wrote the manuscript; LCo, DI, AVal, JW, and PA-M: conceived the study; EN and DI: contributed to data production, and quality control; MMi, EV, MD, FP, LB, and LCo: performed data analysis; LCo, MMi, EV, MD, and PA-M: contributed to the interpretation of the results; SA, JH, LCr, SZ, AL, GH, LY, XH, FZ, S-JL, SW, RL, YG, MMo, YM, FG, AVla, BV, LR, GC, AA, IS, EÜ, MK-C, JG, YU, PB, MA, RP, MGD, PG, JB, EH, YY, CS, BM, AVal, AS, JW, and PA-M: provided samples or funded part of the analyses; All authors read, made corrections, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Anopheles nili group—a large taxonomic group that diverged ~3 Myr ago. Using 4,343 single nucleotide polymorphisms (SNPs), we detected strong population structure characterized by high‐FST values between multiple divergent populations adapted to different habitats within the Central African rainforest. Delineating the cryptic species within the Anopheles nili group is challenging due to incongruence between morphology, ribosomal DNA, and SNP markers consistent with incomplete lineage sorting and/or interspecific gene flow. A very high proportion of loci are fixed (FST = 1) within the genome of putative species, which suggests that ecological and/or reproductive barriers are maintained by strong selection on a substantial number of genes.Ongoing speciation in the most important African malaria vectors gives rise to cryptic populations, which differ remarkably in their behavior, ecology, and capacity to vector malaria parasites. Understanding the population structure and the drivers of genetic differentiation among mosquitoes is crucial for effective disease control because heterogeneity within vector species contributes to variability in malaria cases and allow fractions of populations to escape control efforts. To examine population structure and the potential impacts of recent large‐scale control interventions, we have investigated the genomic patterns of differentiation in mosquitoes belonging to the  We also examined ancestry proportions and admixtures between populations in Admixture v1.23  across SNPs using the populations program of the Stacks pipeline. Mean FST values were also used to quantify pairwise divergence between populations. To infer the demographic history of different populations, we used the diffusion approximation method implemented in the package ∂a∂i v 1.6.3 . We assembled 197,724 RAD loci that mapped to unique positions throughout the reference genome. After applying stringent filtering rules, 408 loci present in all populations and in at least 50% of individuals in each population were retained. Within these loci, we identified 4,343 high‐quality biallelic markers that were used to analyze population structure and genetic differentiation.We collected mosquitoes from four locations out of 28 sampling sites Figure  and sequ3.2An. nili and An. ovengensis . These five subpopulations are strongly correlated with the different sampling sites suggesting local adaptation of divergent populations. Importantly, Structure and Admixture analyses reveal that, at k = 2, one population identified by morphology and the diagnostic PCR as An. nili has almost the same ancestry pattern as the largest An. ovengensis cluster  for 145 CF, CK, and BJW conceived and designed the experiments. CF, CK, SG, and BJW performed the experiments. CF and CK analyzed the data. CF, CK, and BJW wrote the manuscript. Click here for additional data file.


Aedes aegypti is essential to understanding their population dynamics as well as for the analysis of factors responsible for their resistance and ecological adaptation. The use of molecular markers in identifying differences amongst populations of Ae. aegypti in different geographical areas as well as the temporal variation of the vector populations has contributed to the improvement of vector control strategies. The present study aims to determine the genetic variability of Ae. aegypti populations in a small geographical area  by means of inter-simple sequence repeat (ISSR) and single nucleotide polymorphism (SNP) molecular markers.The study of the genetic structure of HE) ranging from 0.261 ± 0.03 to 0.120 ± 0.032, while a similar trend was detected through SNPs across populations with an HE between 0.375 ± 0.054 and 0.269 ± 0.042. The population’s genetic differentiation assessed with ISSR and SNP markers indicated a very low structuring among the populations with the highest diversity observed within the populations 72 % (ISSR) and 92 % (SNP). Clustering analysis also suggested little variation among populations: the seven populations were grouped into only three ISSR clusters and a single panmictic group based on SNP markers. The present study identified a close relationship between the populations, which probably results mainly from passive gene flow between mosquitoes from distinct geographic regions, influenced by humans commuting along roads.ISSR markers revealed a more heterogeneous pattern of genetic diversity among the populations with an expected heterozygosity  contains supplementary material, which is available to authorized users. Aedes aegypti is a major vector of several human arboviruses including Yellow fever, Dengue fever, Chikungunya and Zika virus in several tropical and sub-tropical countries. In Brazil, the mosquito has been reported in all states and the number of cities where it is found has increased with time .The inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) technique amplifies regions between microsatellites, using core microsatellite sequences as primers. Two important characteristics are observed when using ISSR-PCR: (i) high levels of polymorphism, which is particularly important for geographically small-scale intraspecific studies that aim to genetically identify different populations of a species; and (ii) the increased annealing temperature of the primers, which leads to high reproducibility of the DNA band pattern .Ae. aegypti is essential in order to gain an in-depth understanding of population dynamics, factors responsible for resistance to insecticides and ecological adaptation  method and results compiled using CLUMPP [The index of genetic diversity and the molecular variance (AMOVA) were carried out using GenAlEx . GeneticGenAlEx . In thisg CLUMPP . Populatg CLUMPP . To iden8RY, which varied between 450 and 2230 base pairs (bp) and 13 bands for the primer (GA)8RY, which varied between 350 and 1700 bp, resulting in a total of 30 bands pgk, nak and ferh which were monomorphic in CA, PI and NEO, respectively  in PI where a distinct pattern of HE was detected between populations , ferh (PI), nak and chym (NEO) and ef2 and aelmuc1 (UMB). In all 6 loci, a deficit of heterozygosity was observed  equilibrium, significant deviation from HW equilibrium was not detected, with The populations’ genetic differentiation was assessed using ISSR and SNP markers across all 7 populations. The genetic distances among populations ranged from 0.033 (NEO and MA) to 0.120 (ARA and UMB) for ISSR markers  were not in HW equilibrium in one population each. Since HW disequilibrium did not occur in all population/locus, the possibility of null alleles due to problems in the annealing sites of the initiators used was discarded [nak loci in Neopolis and monomorphism in Pinhão is especially relevant as it is located in the Sodium channel gene, which is the target-site gene for pyrethroids and organochlorines insecticides [Anopheles and Culex populations under insecticide selection pressure [Even though most of the locus/population were under HW equilibrium, six markers (iscarded . Nonethecticides . The linpressure –39.vs codominant) and level of polymorphism and mutation rates  [Both ISSR and SNP markers also indicated a high level of genetic similarity among populations, with the ISSR of seven populations grouped into three clusters whereas for SNPs, a single panmictic population was detected. Although both markers are in agreement with low differentiation among populations, the discordance between the number of clusters could result from inherent differences (dominant ocesses) . Thus, tocesses) . SecondlAe. aegypti in northeastern Argentina and Uruguay. Around 89 % of the diversity observed within populations in those studies was based on the same markers applied here [Ae. aegypti circulating in Brazil [The low differentiation in Sergipe’s cities observed by molecular variance analysis (AMOVA) for the ISSR and SNP markers, with most of the variation detected within the populations, was also reported in ied here . Furthern Brazil , 43. Nevn Brazil , 44.Ae. aegypti with little genetic variation in Brazil as suggested by mitochondrial markers [In addition to the likely impact of the introduction of  markers , it is aAe. aegypti populations when compared to areas that are more isolated. Additionally, Damal et al. [Paduan et al.  showed tl et al.  suggest l et al. , as seenAe. aegypti population, possibly reflecting intense gene flow mediated by the passive dispersion of mosquitoes, due to the intense traffic of vehicles among the cities. The results of this work could provide important assistance in the performance assessment of strategies for the control and handling of Ae. aegypti. Such actions have been the basis for the prevention of Dengue fever, Zika virus and Chikungunya epidemics with a focus on the control of both larval and adult stages chiefly with insecticide. That practice has generated resistance processes, compromising mosquito control and increasing the risk of epidemics every year. Thus, knowledge of the genetic structuring and population dynamics of the species is crucial as these genetically different populations may present differences concerning their vectorial capacity and vector competence.Our study showed that there is little differentiation among Sergipe’s


Archaeological, palaeontological and geological evidence shows that post-glacial warming released human populations from their various climate-bound refugia. Yet specific connections between these refugia and the timing and routes of post-glacial migrations that ultimately established modern patterns of genetic variation remain elusive. Here, we use Y-chromosome markers combined with autosomal data to reconstruct population expansions from regional refugia in Southwest Asia. Populations from three regions in particular possess distinctive autosomal genetic signatures indicative of likely refugia: one, in the north, centered around the eastern coast of the Black Sea, the second, with a more Levantine focus, and the third in the southern Arabian Peninsula. Modern populations from these three regions carry the widest diversity and may indeed represent the most likely descendants of the populations responsible for the Neolithic cultures of Southwest Asia. We reveal the distinct and datable expansion routes of populations from these three refugia throughout Southwest Asia and into Europe and North Africa and discuss the possible correlations of these migrations to various cultural and climatic events evident in the archaeological record of the past 15,000 years. Climate shifts dramatically influenced the occupation of Southwest Asia by anatomically modern humans since their first occupation at the sites of Skhul and Qafzeh in Israel some 80 to 130 ka641012Autosomal analyses have identified a cryptic population, “Basal Eurasians,” that injected significant genetics into the European population distinct from the Southwest Asian agricultural revolution signature16Lineages that evolved in relatively isolated refugia populations could carry genetic evidence of that isolation and subsequent expansion. Such signals identified source populations for admixture events marking European settlement1516188Here, we take the approach of identifying and dating population isolation prior to expansion, and tracing routes of dispersal throughout Southwest Asia and into Europe and Africa. We analyzed a comprehensive autosomal and Y-chromosome dataset of Eurasian and African populations identifying genetic signals of regional LGP population isolation, and contrasted expansion time estimates and dispersal routes in the region with archaeological44A total of 8,515 samples were analyzed  for theiOur previously described samples (n = 2047)23282328In addition, Y- Hg and haplotype data were also incorporated from prior studies , includiAutosomal data were obtained from a prior studyThe initial populations were defined primarily based on modern national boundaries or sub-regions. Pooled populations were identified through preliminary BATWING see  analysisWe sought to analyze the data without preconceptions of the history of southwest Asian populations, allowing the data to govern their own analysis. Our data preparations sought to ensure the STR loci and haplogroup derivations were uniform across populations and haplogroups, requiring factoring to a lowest common denominator across all haplogroup and population studies.,) as two topologies instead of one, counting an  split prior vs. subsequent to the  split as two distinct topologies, inducing a spurious interaction between  and  split times, even though those splitting events are independent. Running BATWING with two conformations ), and , CD), where AB represents A and B pooled, and CD represents C and D pooled removes the interaction. The two estimates of the first split provide a check for consistency. This process also establishes pooled populations. We adapted the pooled regions for the second phase of analysis when pooling of similar regions following similar expansion routes were shared by multiple haplogroups. The resulting pooled regional designations are Arabia , Yemen (by itself), Armenia and Turkey (one region), Caucasus (Georgia), Mesopotamia , Cyprus, Southern Levant , Northern Levant , Egypt, North Africa , and Ethiopia .BATWING analyzes population splits of the form Since BATWING input only involved genetic data and population assignments, results might have been geographically random. Instead, the modal tree population splits easily lie on a map. The combinatorial enumeration of trees vs geography is non-trivial. However, simple limits may be estimated by considering the chances that an east-west split would have accurately classified Northern and Southern Levant (4 populations) on one side and Mesopotamia and Arabia (7 populations) by chance on the other, for example, which has a p-value = 0.003 by a Fisher exact test.FST computations, MDS, and complete linkage agglomerative clustering were not sufficient to resolve regions in a meaningful way. AMOVA  haplogroups. Details on these methods are in the Principal component analysis (PCA) was applied to relative haplogroup frequencies. Multidimensional scaling (MDS), was applied to NETWORK was used to compute Reduced Median (RM) networks  for haplBATWING parameters are described in the BATWING population splitting was applied to individual haplogroups drawn from both whole-population and haplogroup-specific studies. BATWING samples STR haplotype phylogenies and population split phylogenies, and Bayesian prior distributions to estimate effective population sizes, and mutation rates, using a Metropolis-Hastings Markov-Chain Monte-Carlo simulation. BATWING assumes a single-step mutation model, and coalescence governed by an effective population size, and it models population splitting by dividing the total parent population effective population size among the child populations. The population splitting assumption is the weakest link in application to individual haplogroups. BATWING provides a fixed then expanding effective population size model, allowing for substantial flexibility in population coalescences that the expanding haplogroups will have evolved within as they migrated. Detailed considerations are offered in the FSTs. Divergence dates were estimated from FST estimates  using published genome-wide marker data2930FSTs were computedFSTs described above). MDS was also applied to the pairwise FST’s. We selected a number of MDS dimensions large enough so that the histograms of reconstructed distances became stable. Agglomerative clustering using Ward’s method and Neighbor-Joining (NJ) was applied to the distances reconstructed from the three leading dimensions produced by MDS  according to the individuals’ population assignments.We tested whether or not the populations were genetically differentiated. Pair-wise d by MDS . While aon tests  contrastRST distances analyses compared to RST distances analyses applied to all haplogroups  in the Caucasus and for J2 in Armenia, decreasing south and east, although high diversities do not entirely correlate with high relative frequencies , all well into the LGP.We performed an ADMIXTURE analysis to identify and quantify ancestral components in Southwest Asians across all chromosomes using 188,974 SNPs . ADMIXTUanalysis  most cleFSTs  than the ancestral differentiation dates suggested by the ADMIXTURE derived FST ancestral date estimates of 37.4 ka and 27.4 ka.The unrooted NJ trees show strikingly varying branch lengths, and remarkably differentiated substructure within the major putative northern and southern refugia, as well as some suggestion of a Levantine refugium. For those populations expanding from the north, the trees reveal a similar east/west split as identified for the J1/J2 expansions. We note the pairwise et al.Our Y chromosome and autosomal analysis identified genetic signatures of three likely centers of isolated evolution followed by population expansions. These modern centers correspond to archaeologically known LGP refugia494512BATWING characterized three features in the genetic record during the expansion of J1 and J2. The earliest dates, with the oldest differentiations for J2 at 8.4 ka, and J1 at 8.9 ka, show an early divergence between the Caucasus from the rest of the populations. BATWING does not model admixture; population split estimates dates to the time admixture ceases. In practice, very minor admixture can cause split times to be underestimated. The branches, to the east down through Mesopotamia and to the west down through the Levant, maintained mutual isolation. Those earliest dispersions show post-last-glacial-period dates but could have been earlier than BATWING estimated if admixture persisted. Later, a significant number of branches occurred within a very narrow window of time, nearly polytomously, followed by relatively stability, marking the emergence of stronger regional isolation suggesting sedentism. That window marks a period near the end of the Holocene Climatic Optimum (HCO - 9 ka–5 ka). Generally, the expansions show movement from northern regions until the HCO, with subsequent isolation and differentiation in the arable regions  during aridification.Subsequent expansions of J1 show the Mesopotamian branch extending to Arabia and Yemen at about 4 ka. The signal of the original North African J1 expansion is overwhelmed in Egypt by more recent Levantine J1s . J2 showY-M35 (E1b1b1) dominates North Africa much more than Southwest Asia, with a Neolithic split at 11.6 ka between eastern and western North Africa. BATWING will interpret unique haplotypes among rare E1b1b1 samples as older isolation. Most of the populations show apparent isolation dating to the HCO with some associations showing as recent links between central Asians and the Southern Levant (2.9 ka), and the Caucasus with Mesopotamia 4.6 ka .FSTs FST derived dates. The larger scale ADMIXTURE results however, correspond more closely with the earlier isolations observed in Y STR estimates of African population J and E (e.g. E-M81 is estimated at 14.2 kya reported at YTree https://www.yfull.com/tree/E-M81/) haplogroups. This contrast suggests ongoing mobility and admixture within Southwest Asia subsequent to the expansion into Africa, since BATWING identifies splitting times since the end of admixture, or else the BATWING dates within Southwest Asia refer to a more recent expansion that completely subsumed an earlier expansion that reached into Africa. This mobility and admixture was greatly reduced following post HCO agricultural settlement.The Levantine autosomal expansion appears, to parallel the Natufian expansion .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.