Populus tremuloides) suggested that Beringia is likely to be a refugium and the “ice-free corridor” in Alberta may represent a region where small populations persisted during the last glacial maximum (LGM). The purpose of this study was to ascertain whether the origins of trembling aspen in western North America are reflected in the patterns of neutral genetic diversity and population structure. A total of 28 sites were sampled covering the northwestern part of aspen’s distribution, from Saskatchewan to Alaska. Twelve microsatellite markers were used to describe patterns of genetic diversity. The genetic structure of trembling aspen populations was assessed by using multivariate analyses, Mantel correlograms, neighbor-joining trees and Bayesian analysis.In North America, the last ice age is the most recent event with severe consequences on boreal species’ ranges. Phylogeographic patterns of range expansion in trembling aspen  were observed. The most probable number of clusters detected by STRUCTURE was K = 3 (∆K = 5.9). The individuals in the populations of the 3 clusters share a common gene pool and showed a high level of admixture. No evidence was found that either Beringia or the “ice-free corridor” were refugia. Highest allelic richness (AR) and lowest heterozygosity (Ho) were observed in Alberta foothills of the Rocky Mountains.Microsatellite markers revealed little to no neutral genetic structure of P. tremuloides populations. Consequently, no divergent populations were observed near supposed refugia. The lack of detectable refugia in Beringia and in the “ice-free corridor” was due to high levels of gene flow between trembling apsen populations. More favorable environmental conditions for sexual reproduction and successful trembling aspen seedling establishment may have contributed to increase allelic richness through recombination in populations from the Albertan foothills of the Rocky Mountains.Contrary to our hypothesis, our results showed that microsatellite markers revealed little to no genetic structure in The online version of this article (doi:10.1186/s12862-016-0810-1) contains supplementary material, which is available to authorized users. Earth has experienced several episodes of severe climatic variation that have led to a succession of ice ages during the Quaternary (2.58 Mya until present) . During In North America, the last ice age is unquestionably the most recent event to have had severe consequences for boreal species genetic diversity . AccordiPopulus tremuloides Michaux), including: Beringia, the Grand Banks, the northeastern United States, the “Driftless Area” of the mid-western United States, the “ice-free corridor” along the and eastern slopes of the Alberta Rocky Mountains, e.g.,  Voss), black spruce (Picea mariana [Mill.] BSP), lodgepole pine (Pinus contorta ssp. latifolia [Engelm.] Critchfield), and P. tremuloides were usually located along the northern Pacific coast and in Beringia, as well as their presence south of the ice sheet [Populus in Alaska shortly after the beginning of the ice cap melting, suggesting that the genus has persisted in this area [Populus balsamifera L.), recent molecular evidence does not support Alaska as glacial refugium but does confirm the existence of two distinct groups in northwestern North America, i.e., a northern group in Alaska and Yukon, and a central group in central distribution area [P. balsamifera under Pleistocene range restrictions, with an expansion toward its margins during range expansion following LGM. For P. tremuloides, two models that were based on paleoecological data suggest the existence of refugial habitats in Beringia and was likely a true refugium for this species. Moreover, Callahan et al. [P. tremuloides to persist in this area during the LGM [Beringia has been suspected of being a refugium for mammals , herbacece sheet . Paleoechis area , 24–26. ion area . Keller ion area  concluden et al.  have sho the LGM .P. tremuloides. Yet, suitable habitat conditions for P. tremuloides during the LGM were not found in this area according to the simulations of by Roberts and Hamann [P. tremuloides in this region. This area appears to be important either as a potentially cryptic refugium or more likely as an admixture zone.The existence of an “ice-free corridor” between the Laurentian and Cordilleran ice sheets is debated . Since td Hamann . In contd Hamann  reportedThe purpose of this study was to ascertain whether the origin of trembling aspen in western North America is reflected in the patterns of neutral genetic diversity and population structure. In the present study, the glacial origin and post-glacial migration route in the northwestern part of the range was uncovered by studying the area analyzed by Callahan et al.  at a finSamples were collected from 28 geo-referenced sampling sites covering the northwestern part of trembling aspen’s distribution from Manitoba to Alaska  using the manufacturer’s protocol. To evaluate genetic diversity and structure, microsatellite markers were selected because they are rapidly evolving, powerful and economical tools for describing patterns of gene flow and diversity. Samples were amplified at 12 microsatellite loci: PTR1, PTR2, PTR3, PTR4, PTR6, PTR14, PMGC2571, WPMS14, WPMS15, WPMS16, WPMS17, and WPMS20  by removing loci with high presence of individuals with three alleles ; ii) by removing any sample with three alleles at one loci ; and iia), average number of effective alleles per locus (Ne), observed heterozygosity (Ho), and expected heterozygosity (He), were calculated using GenAlex v. 6.2 [e were computed, while Hardy-Weinberg (HW) equilibrium was assessed by calculating the inbreeding coefficients (Fis) and their corresponding P-values for all sampling sites. We also ran a global test of HW equilibrium for all the samples pooled together. Bonferroni correction was applied when testing the significance of heterozygosity deficit and heterozygosity excess. All of the HW equilibrium tests were performed in FSTAT [o were interpolated using the inverse distance weighting (IDW) method to create maps of each variable, using ArcMap .All descriptive genetic analyses were carried out with GenAlex v. 6.2 . Allele x v. 6.2 . To descx v. 6.2  with rarin FSTAT . Finallyade4 [mantel.rtest [st; Additional file mantel.correlog in the vegan package [P-value, after which Bonferroni corrections were applied .Mantel tests and correlograms, together with multivariate analysis of spatial patterns of genetic divergence (PCoA and RDA) were performed with the package ade4  in the Rade4 . A globael.rtest  to test  package . Particu package . Pairwisdudi.pco and pcaiv to calculate the respective PCoA and RDA ordinations [summary, provided the percentage of the unconstrained variation (PCoA axes representing the genetic differentiation) that was explained by the predictor (geographic location). The function randtest was used to evaluate the RDA significance by randomly permuting (Monte-Carlo test) the rows of the explanatory table [The results of the Mantel test and correlogram were confirmed by multivariate analysis, which was carried out using the functions inations . PCoA wainations . The outry table .To reveal genetic structure, and test whether the samples could be clustered according to their respective distribution zones, we used STRUCTURE v. 2.3.2 software . The anaFinally, we computed a neighbour-joining tree (NJT)  to see hMc) value for each population with the program M-crit, which was developed by JC Garza and EG Williamson [Mc and M-ratios we used a pre-bottleneck value  and the parameters were set as recommended by JC Garza and EG Williamson [−2 and 10−6 mutants/generation/locus; probability of changes greater than one step, pg = 0.12; and the size of non-one-step changes, Δg =2.8. Each set of simulations consisted of 10 000 iterations with the same values of θ for all sites under a two-phase mutation model (TPM). We considered that a M-ratio below the critical value Mc was indicative of a population decline. To test for heterozygosity excess, Bottleneck version 1.2.02 [M-ratios were estimated to detect historical bottlenecks at each site with the program MPval . To intelliamson . To calclliamson . The setn 1.2.02  was usedn 1.2.02 . Mode shn 1.2.02 . To deten 1.2.02 . In thisn 1.2.02 .a ranged from 4.5 (Peter Pond and High Level) to 9.5 (Steese Hwy), with an average of 6.4  and the highest being 4.3 (Kenai). Across all loci, only 34 individuals had one or more private alleles. Individuals with private alleles were spread across all sampled sites (data not shown). Ho had a mean value of 0.629 and was lowest in the Delta population (0.53) and highest in the Alders Flat population (0.76). The mean He was 0.625, ranging from 0.57 (Pass and Dawson Creek) to 0.66 .Over the entire population, the number of alleles that were observed per locus ranged from 10 (PTR2 and WPMS16) to 30 . The AMOVA (Table st = 0.031) was partitioned among populations and 96.9 % within populations (P < 0.001). No pairwise differences between populations, as estimated by Fst, appeared to be significant after applying a Bonferroni correction  and are displayed in blue, green and red Fig. .Fig. 4NeThe results of the NJT Fig. , which wMc  [P < 0.05), using TPM . Statistical significance to detected historical bottlenecks was maintained in 16 populations with higher values of θ (10), where M-ratio < tehorse) . Across PM Table . The graP. tremuloides populations and indicated little isolation by distance (IBD). Consequently, no divergent populations were observed near supposed refugia suggesting no evidence that Beringia or the “ice-free corridor” were refugia for trembling aspen. Finally, favorable conditions for sexual reproduction and successful trembling aspen seedling establishment could have contributed to the highest AR and lowest Ho that were observed in Alberta foothills of the Rocky Mountains.The purpose of this study was to ascertain whether the origin of trembling aspen in northwestern North America is reflected in the patterns of genetic diversity and population structure. Contrary to our hypothesis, microsatellite markers revealed little to no genetic structure in Our results support the findings of Callahan et al. , who fouP. glauca [P. glauca in Beringia during the LMG. Moreover, those simulations also suggested the presence of suitable habitats for P. contorta and P. tremuloides in Beringia [P. balsamifera, recent molecular evidence did not support Beringia as a glacial refugium, but confirmed the existence of two distinct clusters in our sample area of northwestern North America [The Rocky Mountains foothills of Alberta, which could have remained ice-free during the last glacial maximum , exhibi. glauca , 23 and . glauca , which sBeringia . For the America .The M-ratio bottleneck test proved to be sensitive to the choice of θ, with significant M-ratio values being obtained for all sites using values of θ = 1. The M-ratio detected persistent bottleneck signatures in 16 populations with θ = 10. Low M-ratios that were detected indicate that these 16 populations might have suffered from demographic declines, although they were not severely reduced in their genetic potential. We did not detect strong evidence for excess heterozygosity. Consistent results were obtained only for 1 population. Indeed, wild populations are rarely completely closed and even small numbers of migrants can mask the genetic signature of bottlenecks , 60. UndP. glauca, LL Anderson, FS Hu and KN Paige [−9 mutations per gene per generation). Their results with nuclear microsatellite markers support the idea that north-central Alaska served as a glacial refugium during the last glacial maximum for white spruce. Three genetic groups were detected: the first consisted of one population from north-central Alaska ; the second with one population from southern Manitoba; and the last group included the remaining 20 populations  forming the last group. These results revealed that there is not much structure and differentiation to be found for this boreal species, a result similar to what we found in trembling aspen. For P. tremuloides, Callahan et al. [Most of the studies that have detected phylogeographic patterns in boreal tree species in western North America (reviewed by ) have usKN Paige  suggesten et al.  found 2


Interpretations of genetic data concerning the prehistory of Europe have long been a subject of great debate, but increasing amounts of ancient and modern DNA data are now providing new and more informative evidence. Y-chromosome resequencing studies in Europe have highlighted the prevalence of recent expansions of male lineages, and focused interest on the Bronze Age as a period of cultural and demographic change. These findings contrast with phylogeographic studies based on mitochondrial DNA (mtDNA), which have been interpreted as supporting expansions from glacial refugia. Here we have undertaken a population-based resequencing of complete mitochondrial genomes in Europe and the Middle East, in 340 samples from 17 populations for which Y-chromosome sequence data are also available. Demographic reconstructions show no signal of Bronze Age expansion, but evidence of Paleolithic expansions in all populations except the Saami, and with an absence of detectable geographical pattern. In agreement with previous inference from modern and ancient DNA data, the unbiased comparison between the mtDNA and Y-chromosome population datasets emphasizes the sex-biased nature of recent demographic transitions in Europe. Genome-wide ancient DNA (aDNA) data have shown a clear discontinuity between Paleolithic hunter-gatherers and Neolithic farmers5. Patterns of diversity suggested low Paleolithic population sizes, with regional differences among Western and Scandinavian groups6. This picture has been further refined by the study of the DNA of ancient Yamnaya herders from the region of the Pontic-Caspian steppe, the apparent source of Bronze Age migrations into Europe and Asia9, and a debated region of origin for Indo-European languages10. These studies highlight an introgression into Europe at around 4.5 thousand years ago (KYA) from the East, followed by development of genetic structure in Bronze Age Europe8.Our understanding of European prehistory has been revolutionized by the availability of new DNA sequencing technologies11. Analyses of aDNA show that today’s most frequent Y-chromosome haplogroup (R1b-M269) is very rare in Europe until 4.5 KYA5 (see summary elsewhere12), while it is present in all the Yamnaya samples9. This had initially suggested a major introgression of males from the Pontic-Caspian steppe; however, the R1b sublineage (R1b-L11) now common in Europe has not so far been found among Yamnaya sequences13. In contrast to Y data, patterns of ancient mtDNA reveal a period of widespread turnover of lineages in the Late Glacial period ~14.5 KYA15 and support the picture of a discontinuity between Paleolithic hunter-gatherers and Neolithic farmers seen in autosomal data, but contain no apparent signal of the Bronze Age steppe expansion16. Ancient DNA data on uniparentally-inherited markers therefore suggest a strong sex-bias in recent demographic changes in Europe.Ideas on European prehistory have been strongly influenced by studies not only of autosomal DNA, but also of uniparentally-inherited markers, which can provide information about sex-biased processes17 in modern European populations also emphasizes the importance of Bronze-Age transition20. Demographic reconstructions support an expansion starting ~2.1–4.2 KYA, and times-to-most-recent-common-ancestor (TMRCAs) of three major haplogroups, including R1b, are estimated between 3.5 and 7.3 KYA12. Many mtDNA studies have been undertaken in modern European samples, but most concentrate on particular segments of the mitochondrial genome21, consider the European continent as a single unit19, or have taken a phylogeographic approach, focusing on specific lineages of interest27. General conclusions have been that current mtDNA variation represents re-expansions from glacial refugia. However, population-based whole mtDNA resequencing studies in Europe are lacking, so a systematic comparison of the demographic histories of males and females has yet not been possible.Resequencing of the male-specific region of the Y chromosome (MSY)Here, we carry out a population-based study, resequencing mtDNA in a set of 17 populations for which MSY resequencing data are already available. The pattern observed in mtDNA is strikingly different from that in MSY, compatible with expansion after the Last Glacial Maximum, and emphasizing the male-specific nature of the Bronze Age expansion in Europe.12.To allow a comparison between female and male histories, we resequenced the mitochondrial genomes of 340 European and Middle Eastern individuals belonging to 17 populations Table  that wer21.We constructed a maximum parsimony (MP) tree Fig.  based on28 , U (17.9%), T (13.5%), J (9.1%), K (7.3%), and V (5.3%). The remaining 12.6% of the dataset is comprised of many minor haplogroups; these include  two examples of each of the lineages L2 and L3, typically found in sub-Saharan Africa29. In the remaining populations there is no obvious geographical pattern, in agreement with previous observations based on analyses of specific segments of the mitochondrial genome30.Visual inspection of the distributions of haplogroups Fig.  shows th12  for mtDNA.This phylogeography of mitochondrial genomes was compared with that of MSY, based on resequencing of 3.7 Mb of Y-DNA in the same set of samples12 Fig. . The geo12 Fig.  than for12 Fig. : for exa12 Fig. ; Table 112 in the same populations  based on mtDNA sequences Fig. . As expeD and FS for all populations except the Saami, which can indicate population growth; however, both values are significant for only twelve of the remaining populations. At a glance, there appears to be more diversity in southern than northern populations , and patchy population coverage.The study of mtDNA in Europe has a long history and has been influential in developing hypotheses about the origins of modern Europeans12 had previously been undertaken. The spectrum of haplogroups we observe , are in the Near and Middle East. This difference in timing of European female and male lineage expansions is mirrored in the Indian subcontinent, where a recent analysis33 shows that mtDNA expansions reflect processes in the pre-Holocene era, while MSY expansions are mostly in the last 10 KYA, with marked male-driven spread from Central Asia during the Bronze Age.Here, we have carried out whole mtDNA sequencing in a European and Middle Eastern population-based sample set of 340 individuals, in which MSY resequencingve Table , Fig. 1a34, it is worth noting that the 340 individuals carry only 318 distinct haplotypes based on complete mitochondrial genomes , Italian , Dutch [Frisia], Danish, Norwegian, Finnish [Saami], English42 , Orcadian42, Irish, Turkish and Palestinian. Twenty individuals from each of two additional HapMap40 population samples, CHB , and YRI  were included to provide variant validation data. All methods were carried out in accordance with relevant guidelines and regulations, and all experimental protocols were approved by the University of Leicester Research Ethics Committee. Informed consent was obtained from all subjects (University of Leicester Research Ethics Committee reference: maj4-cb66).DNA samples from 340 individuals belonging to 17 populations  across Europe and the Near East were used for analysis, as in our previous study of MSY diversityWe generated three datasets using parallel sequencing strategies  and bio43, using the Subtree-Pruning-Regrafting (SPR) algorithm44 with search level 0 in which the initial trees were obtained by the random addition of sequences (10 replicates). Branch lengths were calculated using the average pathway method44 and are proportional to the number of mutations. FigTree v1.4.045 was used for tree visualization. Haplogroups were predicted using HaploGrep246, and their phylogenetic coherence was verified using the tree, with manual examination of possible ‘phantom’ mutations, as inferred using Haplogrep246 (Table A maximum parsimony (MP) tree was constructed from coding-region sequences  via MEGA646 Table .47 based on either coding region or whole mtDNA sequences were constructed using Network 5.0.0.0, and represented using Network Publisher 2.1.1.2. Polymorphic sites were weighted according to their evolutionary rates using the parameters suggested in the literature48.Median-joining networks49 by using HaploGrep50 and their relative frequencies represented as pie-charts plotted on a geographical map.Haplogroups were defined according to Phylotree1651 using BEAST v1.8.0. MCMC samples were based on 50,000,000 generations, logging every 1000 steps, with the first 5,000,000 generations discarded as burn-in. Three runs were combined for analysis using LogCombiner. We used an exponential growth coalescent tree prior, HKY substitution model, and an uncorrelated relaxed clock with a lognormal distribution for mutation rate (2.21 ± 0.17 × 10−8 mutations/nucleotide/year52). TMRCAs were estimated in a single run including all 17 populations and assigning samples to specific clades in agreement with the MP tree shown in Fig. 52 in Soares et al. (2009) to infer the rate at each of the nodes of interest  and a generation time of 30 years54. For this analysis, the entire mitochondrial genome was considered: given the timeframe of interest (<40 KYA) the rate and its standard error were adjusted by using the calculator52 in Soares et al. (2009) to infer the rate at each of the nodes of interest (see Table BSPsS), nucleotide diversity, Tajima’s D55, and Fu’s FS56 were calculated57 using Arlequin 3.5. Correlations of S, and of nucleotide diversity, with latitude, longitude, and distances from glacial refugia were examined using the function cor.test of the package stats within R. The locations Anamur, Turkey  and Fleurac, France  were taken as proxies for the centres of the Near-Eastern and Franco-Cantabrian refugia respectively. Distances account for geographical barriers, and were estimated using the land transport distance tool at www.freemaptools.com.The number of polymorphic sites per population (All data generated during this study are included in this article (and its Supplementary Information files).Supplementary InformationSupplementary Dataset S1


Capra ibex) are wild goats that recovered from <100 individuals to ~50,000 within a century by population reintroductions. We analyzed the population genomic consequences of the Alpine ibex reintroduction strategy. We genotyped 101,822 genomewide single nucleotide polymorphism loci in 173 Alpine ibex, the closely related Iberian ibex (Capra pyrenaica) and domestic goat (Capra hircus). The source population of all Alpine ibex maintained genetic diversity comparable to Iberian ibex, which experienced less severe bottlenecks. All reintroduced Alpine ibex populations had individually and combined lower levels of genetic diversity than the source population. The reintroduction strategy consisted of primary reintroductions from captive breeding and secondary reintroductions from established populations. This stepwise reintroduction strategy left a strong genomic footprint of population differentiation, which increased with subsequent rounds of reintroductions. Furthermore, analyses of genomewide runs of homozygosity showed recent inbreeding primarily in individuals of reintroduced populations. We showed that despite the rapid census recovery, Alpine ibex carry a persistent genomic signature of their reintroduction history. We discuss how genomic monitoring can serve as an early indicator of inbreeding.Restoration of lost species ranges to their native distribution is key for the survival of endangered species. However, reintroductions often fail and long‐term genetic consequences are poorly understood. Alpine ibex ( Habitat protection and reintroductions are the main strategies for species conservation. The choice of the most suitable reintroduction strategy for a certain species is crucial for the future success and long‐term viability of populations. Despite a growing list of successful species reintroductions that re‐established historic species ranges , lack of gene flow with other populations, and inbreeding. The individuals’ ability to move in space and contribute to gene flow among populations is key for the long‐term survival of reintroduced populations. Hence, reintroductions in a continuous, connected landscape may be less likely to fail than reintroductions in a strongly subdivided landscape due to anthropogenic or topographical factors.The different stages of a species reintroduction are predicted to show different signatures of genetic subdivisions. At the early stage of species reintroductions, population differentiation is expected to be weak if reintroduced individuals were drawn at random from a source population. Subsequently, restricted gene flow in a fragmented habitat will lead to genetic drift and reduced genetic diversity. The effect of habitat fragmentation is an important factor for the long‐term viability of reintroduced populations. The most immediate consequence of reduced gene flow for a small population is an increase in inbreeding . Inbreeding can have harmful consequences on individual survival and reproductive success . However, traditional genetic markers such as microsatellites suffer from a number of biases and shortcomings. Microsatellite markers were generally developed to maximize polymorphism in a focal population, which may lead to ascertainment bias in other populations. Recently developed genomic tools based on high‐throughput sequencing such as restriction‐associated DNA sequencing (RAD‐seq) alleviate many of the shortcomings of microsatellite markers. The unbiased genomewide estimates of multilocus heterozygosity generally reflect more accurately levels of inbreeding  are an ideal system to test for the long‐term population genetic consequences of population reintroductions. Alpine ibex are a species of wild goat living at high elevation across the European Alps. The species was overhunted over centuries and went nearly extinct in the early 19th century. An extensive conservation program started in 1906 that reintroduced Alpine ibex to their original distribution range re‐establishing the census size from ca. 100 individuals to the current census of ca. 50,000 (Brambilla pers. comm.). The reintroduction of Alpine ibex to the European Alps was likely the most successful reintroduction of a large mammal. After recovery of the sole remaining population in the Gran Paradiso area of northern Italy to well over 1,000 individuals, 55 male and 45 female ibex were transferred from the Gran Paradiso area to two Swiss zoos to initiate a captive breeding program  is the closest relative to Alpine ibex  as a reference. We addressed the following questions: (i) What was the loss of genomewide diversity due to the reintroduction of Alpine ibex from a single source population? How do the levels of genetic diversity compare with the major Iberian ibex populations? (ii) Did the stepwise reintroduction strategy of primary and secondary populations lead to a stepwise increase in genetic subdivision with subsequent rounds of reintroductions? (iii) Did the reintroductions lead to genomewide signatures of recent inbreeding? Finally, we discuss how genomewide data can be used to assess the long‐term viability of reintroduced species.We aimed to identify the population genomic consequences of the Alpine ibex reintroduction strategy using Iberian ibex and domestic goat (22.1C. pyrenaica hispanica) populations, and four Swiss domestic goat breeds. Nine Alpine ibex populations were reintroduced to the Swiss Alps over the past century, and one was the population from the Gran Paradiso National Park in Italy. The sample size per population was 15 for the reintroduced Alpine ibex and Iberian ibex populations, 16 for the source population Gran Paradiso, and two to three per domestic goat breed . Adapters containing unique barcodes were ligated to the digested DNA. Ligated DNA was sheared using a COVARIS ultrasonicator and size‐selected at 300–700 bp on a CALIPER LabChip XT. After ligation of P2 adapters, a PCR was performed to amplify the library. Quality‐checked libraries were sequenced on an Illumina HiSeq 2000 platform to generate 100‐bp paired‐end reads. We obtained a total of 468 million high‐quality reads and a median of 2.3, 2.1, and 1.6 mio read pairs per Alpine ibex, Iberian ibex, and domestic goat individual, respectively . The RAD libraries were prepared as described in Grossen, Keller, Biebach, and Croll . In shorely Fig. .http://hannonlab.cshl.edu/fastx_toolkit/index.html) according to the P1 adapter barcode. Trimmomatic . Read alignments were used for multisample SNP genotyping as follows. Genotype calling was performed using HaplotypeCaller and GenotypeGVCFs according to best practices .Two maximum‐likelihood phylogenetic trees were built using RAXML  of all three species based on the genetic covariance matrix calculated from the genotypes  and SNPs were further filtered for a genotyping rate >80% and a minor allele frequency (MAF) of >5%. The function stat_ellipse in the R package {ggplot2} was used to draw a 95% confidence ellipse for each population assuming a multivariate K from K = 2 to K = 4 and ran for each K a burn‐in of 10,000 and 50,000 Monte Carlo replicates using an admixture model with correlated allele frequencies.For a complementary analysis of population structure, we used the Bayesian clustering method STRUCTURE .2.5restrict from EMBOSS  within 90 bp from a cut site. To calculate the SNP density (SNP/kb), the number of SNPs identified in each population  was divided by the median sequence length covered by at least seven reads in the population. As a cut site is always sequenced in both directions, the effective coverage at the restriction site was 14.The SNP density per population and per species, respectively, was calculated as the number of SNPs per kilobase sequenced with the SNP panel “general.” As variation in sequencing coverage among populations could lead to variation in the number of SNPs passing the filters described above, we decided to use SNP density instead of SNP number to control for variation in sequence coverage. RAD‐seq most consistently covers the region around the defined restriction enzyme cut sites. Hence, we restricted our genetic diversity analyses to these regions. Cut sites were identified in the goat genome by an in silico digest using n − 1 individuals with n being the sample size per group. For each random sample, we counted the number of SNPs with a minor allele count of ≥1. Then, the SNP count was divided by the median kilobase sequenced at a coverage of at least 7.As sample sizes differed among species and populations, we performed a resampling of the SNP density of randomly selected individuals. The different groups for which we performed a resampling were as follows: domestic goat, Iberian ibex from Maestrazgo, Iberian ibex from Sierra Nevada, reintroduced Alpine ibex, and Alpine ibex only from the autochthonous Gran Paradiso population. For each group, we randomly sampled 100 times without replacement subgroups of 1 to The expected multilocus heterozygosity per populations  are defined as tracts of homozygosity along physical positions of a chromosome. ROH arise if both parents share identical chromosomal segments  and Sierra Nevada Natural Space  as well as subsequently established secondary populations Figure  and S2. SD = 13%), 68% for Iberian ibex (SD = 13%), and 61% for domestic goat (SD = 9%). The number of SNPs (at cut sites) polymorphic within species was 20,744 in Alpine ibex (n = 135), 24,018 in Iberian ibex (n = 30), and 46,300 in domestic goat (n = 8). The median base pairs covered at restriction sites was 8.9, 8.3, and 7.6 Mb for Alpine ibex, Iberian ibex, and domestic goat, respectively. The coverage amounted to 0.28%–0.33% of the total genome. The median SNP density was 2.3, 2.9, and 6.0 SNPs per kilobase sequenced for Alpine ibex, Iberian ibex, and domestic goat, respectively.Using RAD‐seq, we obtained overall read alignment rates varying from 95% to 96% among species. The more stringent alignment rates  varied between 82% (domestic goat), 84% (Iberian ibex) and 85% (Alpine ibex). As the alignment rates obtained from samples of the different species were highly similar, we expected no or only minimal ascertainment bias in genotype calling among species. All mapped reads were used for SNP calling, and we obtained a total of 101,822 autosomal SNPs (at cut sites), which had the rare allele shared among at least two individuals and an overall genotyping rate of >45%. Our paired‐end RAD‐seq approach enabled the identification of optical and PCR duplicates. The proportion of reads detected as optical duplicates ranged from 0.228 to 0.583 among individuals and the proportion of reads detected as PCR duplicates ranged from 0.232 to 0.312. To test whether the removal of all duplicates  had an impact on population differentiation parameters, we performed an additional principal component analysis. The differentiation of ibex populations was not discernably different if only reads devoid of duplicates were used see Fig. . The mea3.1As expected, maximum‐likelihood phylogenetic analyses showed that Alpine and Iberian ibex were closely related Figure b.A principal component analysis clearly separated all three species Fig. A. The fiFST = 0.39). PC1 (35.1%) clearly separated the two populations and therefore explained considerably more variation than PC2 (4.7%). Individuals from the Sierra Nevada population were mostly differentiated by PC1, while individuals from the Maestrazgo population were differentiated by both PC1 and PC2. A PC analysis for domestic goats alone showed a clustering according to breeds  to 0.08 .The extant differentiation among reintroduced Alpine ibex populations should reflect both the genetic diversity of founder individuals and demographic effects experienced by the populations since the reintroduction. Hence, we analyzed the population structure of the Gran Paradiso source population, and the primary and secondary reintroduced populations. The PCA of Alpine ibex populations showed a pronounced substructure with three differentiated axes Figure a. The GrK was K = 2 according to the delta K method  was found between source and reintroduced populations.The hierarchical AMOVA mirrored the results of PCA and STRUCTURE analysis and found significant differentiation among the groups (comprising the respective primary and secondary reintroduced populations) and among all individual populations Table . However3.3The Gran Paradiso Alpine ibex population and the Iberian ibex populations give insights into levels of genetic diversity in populations of ibex that have experienced bottlenecks but not reintroductions. Comparing the genetic diversity of the Gran Paradiso population with the reintroduced populations gives insights into the impact of reintroduction bottlenecks. SNP density (SNPs per kilobase sequenced) was similar in the Gran Paradiso population and the two Iberian ibex populations. However, SNP density was lower in all reintroduced Alpine ibex populations Figure . As estiExpected heterozygosity was highest in the two Iberian ibex populations , intermediate in Gran Paradiso (0.032) and lowest in the reintroduced populations  thresholds used for this analysis can have an impact on the estimates of Ne. The analyses above were performed with MAF > 0.1 (see Table Ne estimates based on different MAF thresholds). Note that Waples and Do .Estimates of effective population sizes revealed that all secondary reintroduced Alpine ibex populations had a e Figure . The thrs and Do  showed tNe estimates of the secondary reintroduced populations with observed levels of genetic differentiation among populations suggest that the point estimates of Ne are reasonable. Assuming no differentiation at the time of reintroduction , pairwise FST is predicted to increase over time according to 1−FST=1−12Net . Although this assumption is unlikely to be true, harmonic mean Ne estimates based on pairwise FST values indicated a Ne of 120 in primary reintroduced populations (lower than estimated from LD) and a Ne of 60 in secondary reintroduced populations.Although the upper bounds of the estimates were often infinite, comparisons of the  Wright, , where t3.5n = 3) and 179 Mb, respectively. In comparison, the total length of ROH (>5 Mb) was less than 10 Mb in all domestic goat. Reintroduced populations had the longest total length of ROH  along the genome Figure . Using sROH Fig. B, and ROROH Fig. . In domeROH Fig. .10,000 is defined as the total length of ROH longer than 10 Mb divided by the autosomal genome length   Fig. . Biebach92) Fig.  used pop4Sustainable species conservation depends on understanding the genetic consequences of management strategies. Here, we described a systematic genomic survey and population genetic analysis of a species that was successfully reintroduced into a fragmented alpine habitat. We compared the autochthonous source population of all reintroduced Alpine ibex populations, several reintroduced Alpine ibex populations, two autochthonous populations of a closely related species and several breeds of the related domestic goat. The systematic sampling and high‐density markers allowed investigating the effects of two critical stages of a species reintroduction  on genomewide polymorphism.4.1Our analyses were based on high‐throughput RAD‐seq genotyping. High‐throughput genotyping such as RAD‐seq and other barcoded next‐generation sequencing techniques can introduce potential biases in the genotypic dataset. We performed a series of analyses to identify issues arising specifically from barcoded sequencing libraries that combine a large number of individuals of potentially different DNA quality. We found that uneven coverage among individuals (and populations) can introduce biases if these are not properly controlled for. For example, sequencing coverage was inversely correlated with genotype missingness (data not shown). Hence, we required a minimum coverage for any individual to be included in the analyses. We chose SNP density as a measure of genetic diversity rather than raw SNP counts to control for remaining variation in per‐individual coverage. Variation in the proportion of genotyped markers  can influence measures of heterozygosity and population differentiation . We found strong evidence for within‐species substructure that reflects the reintroduction strategy. At the time of the first reintroductions in the early 20th century, there was likely virtually no substructure among Alpine ibex as the reintroductions were initiated from a single source population. Hence, the observed genetic differentiation among reintroduced populations was the likely result of genetic drift over the past 8–10 generations. The three primary reintroduced populations in Switzerland showed considerable genetic differentiation. The repeated founding of secondary populations mostly from a single primary population exacerbated the genetic differentiation from the Gran Paradiso source population. Estimates for genetic structure among reintroduced populations based on microsatellite markers *Ht). Although there was considerable variation in estimates of Ne based on loss of expected heterozygosity, FST and LD, all methods suggest that the secondary reintroduced populations had effective population sizes of 120 individuals or fewer. Frankham, Bradshaw, and Brook . The two Iberian ibex populations experienced each two distinct bottlenecks, and the two populations have been isolated for about 25 generations since the drastic census reductions in the early 19th century .Alpine ibex suffered a unique population bottleneck compared to other ibex species due to the near extinction in the 19th century. However, the Iberian ibex occupy an alpine habitat that is similarly fragmented by topography. Both autochthonous populations of Iberian ibex and the source population of Alpine ibex were confronted with strong barriers to dispersal. At high population densities, some barriers might be overcome due to high dispersal pressure. Such dispersal may lead to the colonization of new territories or to population expansion , may provide further insights into the origins of the domestic goat diversity.Domestic goat exhibited the highest levels of genetic diversity. This may seem surprising because most domesticated plant and animal species suffered from considerable losses in genetic diversity  had consistently higher genetic diversity than reintroduced populations. One Gran Paradiso individual showed extraordinarily high levels of heterozygosity  or multilocus heterozygosity. The affordability of RAD‐seq or genotyping by sequencing led to the implementation of genomewide inference in a number of recent conservation studies despite considerable challenges  up to 0.1. Values in this range were shown to correspond to pedigree inbreeding estimates of matings among half sibs  risks due to the reintroduction history persist. Therefore, future reintroductions should be genetically monitored even after the successful establishment of a population, with a particular focus on early signs of inbreeding. Such monitoring would ensure the early detection of genetic problems of endangered species and would allow initiating the necessary management actions.https://doi.org/10.5061/dryad.n276d.The SNP Data set is available from the Dryad Digital Repository:  Click here for additional data file.


The origin and diversification of Sino-Tibetan speaking populations have been long-standing hot debates. However, the limited genetic information of Tibetan populations keeps this topic far from clear. In the present study, we genotyped 15 forensic autosomal short tandem repeats (STRs) from 803 unrelated Tibetan individuals from Gansu Province (635 from Gannan and 168 from Tianzhu) in northwest China. We combined these data with published dataset to infer a detailed population affinities and genetic substructure of Sino-Tibetan populations. Our results revealed Tibetan populations in Gannan and Tianzhu are genetically very similar with Tibetans from other regions. The Tibetans in Tianzhu have received more genetic influence from surrounding lowland populations. The genetic structure of Sino-Tibetan populations was strongly correlated with linguistic affiliations. Although the among-population variances are relatively small, the genetic components for Tibetan, Lolo-Burmese, and Han Chinese were quite distinctive, especially for the Deng, Nu, and Derung of Lolo-Burmese. Han Chinese but not Tibetans are suggested to share substantial genetic component with southern natives, such as Tai-Kadai and Hmong-Mien speaking populations, and with other lowland East Asian populations, which implies there might be extensive gene flow between those lowland groups and Han Chinese after Han Chinese were separated from Tibetans. The dataset generated in present study is also valuable for forensic identification and paternity tests in China. The Sino-Tibetan languages, spoken by over a billion people all over East Asia and Southeast Asia, have been classified into two subfamilies, namely Chinese and Tibeto-BurmanDuring the past two decades, genetic evidence, especially from the maternal mitochondrial DNA (mtDNA) and the paternal Y chromosome, has shed more light on the history of Sino-Tibetan populations. MtDNA evidence reveals a northern Asian origin of Tibetans, due to the high frequencies of northern Asian specific haplogroup A, D, G, and M8346796910From previous studies, the origin of Sino-Tibetan populations seems to involve substantial genetic admixture with surrounding populations. However, the limited markers of mtDNA and Y chromosome and small sample sizes and insufficient sampling of genome-wide study are far from enough to give a comprehensive understanding about the genetic history and admixture process of Sino-Tibetan populations. In addition, Tibetan populations of Gansu province, the key area for the diversification of Amdo Tibetans, have seldom been studied genetically. Therefore, we analyze 15 autosomal STRs in 635 and 168 unrelated individuals from two Tibetan populations in Gannan and Tianzhu of Gansu province to explore the genetic structure of Tibetan populations in northwest China and to test population affinities and the level of admixture of Sino-Tibetan populations with surrounding populations.We collected blood samples of 635 and 168 unrelated individuals from two Tibetan populations in Gannan and Tianzhu, Gansu province. Our study was approved by the Ethnic Committee of Gansu Institute of Political Science and Law. The study was conducted in accordance with the human and ethical research principles of Gansu Institute of Political Science and Law. All individuals were adequately informed and signed their informed content before their participation. For each sample, genomic DNA was extracted according to the Chelex-100 method and proteinase K protocolAllele frequency, heterozygosity, polymorphism information content (PIC), discrimination power (DP), probability of paternity exclusion (PPE) were calculated using PowerStatesV12 (http://www.promega.com/). Tests for Hardy–Weinberg equilibrium were performed in Arlequin v3.5.1.31420212223242526272829303132333435363738394041424344454647484951Fifteen STR loci were genotyped in two populations sampled from Gannan and Tianzhu of Gansu province and their allele frequencies along with a number of genetic and forensic parameters of interest are provided in We performed various parameters of genetic diversity and distances to infer population structure between Tibetans in Gannan and Tianzhu, as well as compared them with previously studied populations. The Tibetans in Gannan and Tianzhu fall into the general profile of Tibetan groups, showing extremely small genetic distances with other Tibetan populations. The within-population component of genetic variation, estimated here as 99.14% , accountWe then applied a model based clustering algorithm in Structure to infer the detailed genetic ancestry at individual level. This approach will place individuals into K clusters, where K is set in advance but can be varied. The results for K = 2 to 7 are shown in The origin and diversification of Sino-Tibetan populations have become long-standing hot topics among linguists, population geneticists, anthropologists, and archaeologists. However, the limited genetic information of Tibetan populations in northwest China has made this topic far from clear. Here, we genotyped 15 forensic autosomal STRs from 635 and 168 unrelated Tibetan individuals from Gannan and Tianzhu of Gansu province, together with published forensic dataset to infer a detailed genetic structure of Sino-Tibetan populations. The Tibetans in Gannan share a very similar genetic makeup with other Tibetan populations from Tibet, Qinghai, and Yunnan. While Tibetans in Tianzhu County seem like to share more genetic component with lowland East Asians, such as Han Chinese, Muslim and Korean populations, which is understandable as those Tibetans are surrounded by Han Chinese and Chinese Muslims. The genetic structure of studied Sino-Tibetan populations is strongly correlated with linguistic affiliations, as we can detect three distinctive genetic components for Tibetan, Lolo-Burmese, and Han Chinese although the among-population variances are relatively small. The Yi of Yunnan province, one of Lolo-Burmese speaking populations, is found out to be an admixture between Tibetan, Han Chinese, and southern natives (Tai-Kadai and Hmong-Mien speaking groups). However, other Lolo-Burmese populations, such as Deng, Nu, and Derung, form a distinctive cluster, which is probably due to long-term isolations and genetic drift as those populations are all small and living on hunting and gatheringPrevious studies, especially using mtDNA and Y chromosome, had suggested the North Asian origin of Tibetan populations5514The genetic makeups of the Tai-Kadai and Hmong-Mien speaking populations in south China are similar with Han Chinese rather than with Tibetan groups. The Muslim populations in northwest China and Korean people also exhibit the similar component cluster pattern. The possible scenario for this observation is that those lowland populations might have extensive gene exchange with Han Chinese after Han Chinese were separated from Tibetans. The autosomal STR results are consistent with uniparental Y chromosomal and mtDNA evidence that southern natives made a greater contribution to the maternal lineages of southern Han ChineseHow to cite this article: Yao, H.-B. et al. Genetic structure of Tibetan populations in Gansu revealed by forensic STR loci. Sci. Rep.7, 41195; doi: 10.1038/srep41195 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Proceratophrys moratoi, is a criticallyendangered toad species with a marked population decline in southern BrazilianCerrado. Despite this, new populations are being discovered, primarily in thenorthern part of the distribution range, which raises a number of questions withregard to the conservation status of the species. The present study analyzed thegenetic diversity of the species based on microsatellite markers. Our findingspermitted the identification of two distinct management units. We found profoundgenetic structuring between the southern populations, on the left margin of the TietêRiver, and all other populations. A marked reduction was observed in the contemporarygene flow among the central populations that are most affected by anthropogenicimpacts, such as extensive sugar cane plantations, which presumably decreases habitatconnectivity. The results indicated reduced diversity in the southern populationswhich, combined with a smaller effective population size, may make these populationsmore susceptible to extinction. We recommend the reclassification of P.moratoi as vulnerable and the establishment of a special protectionprogram for the southern populations. Our results provide important insights aboutthe local extinction of southern populations of this toad.The Morato's Digger Toad,  The understanding of genetic diversity, population structure, gene flow, and thedispersal capacity of endangered species are fundamental to conservation genetics . StudiesProceratophrys moratoi  estimatethe genetic diversity, inbreeding levels, and effective size of populations; ii)characterize the spatial genetic structure and gene flow between populations; and iii)integrate the results into management recommendations for the conservation of thisspecies.To complement these findings, we analyzed the genetic characteristics of P. moratoi populations were recorded prior to2013 were surveyed  and according to P.moratoi populations. PCR assays prepared and amplifications were donefollowing the recommendations of these authors. Successfully amplified PCR productswere analyzed in a Dual Dedicated Height vertical electrophoresis sequencing system(CBS Scientific) using a denaturing 6% polyacrylamide gel and stained with silvernitrate (The 22 microsatellite loci developed by nitrate .Only the markers that presented a clear profile (without unspecific or stutter bands)and had been amplified in at least one individual from each population  were used for the population-level analyses presented here.The markers were evaluated in MICRO-CHECKER 2.2.3 . Given to)and expected heterozygosity (He), the percentage of polymorphic loci (P),number of alleles (Na), and the effective number of alleles(Ne) (Fis) was calculated in FSTAT 2.9.3.2 (Intra-population variability was quantified according to the observed (Hles(Ne) , allof  2.9.3.2 , as werei.e., non-outliers, were included inall the subsequent analyses.Inferences on population structure may be affected by deviations from neutrality,such as outlier markers that are suffering selection pressure, resulting in certainbiases in the analyses. The LOSITAN  was determined by a Bayesian approach using STRUCTURE 2.3.4(k) and the mostprobable value being selected by the Evanno method in STRUCTURE HARVESTER 0.6.93 inferred from the analysis, with 200,000sweeps, burn-in of 40,000, and the spatial interaction parameter (ψ) fixed at 0.6, asrecommended by the authors. To identify the number of sample clusters present in thedata, we plotted the deviance information criterion (DIC) produced for the comparisonwith Kmax and selected the value of Kmax when the DIC stabilized (RST index) and geographic (Euclidean) distance among the sample points, with10,000 randomizations run in IBD 1.52 , run in TESS 2.3.1 (abilized  was usedIBD 1.52 . The promc), allele frequencies (A), and endogamy (f) wereadjusted  to ensure that around20–60% of the total changes would be accepted, as recommended by i.e. no persistent hills and valleys) indicates goodmixing and effective sampling from the posterior distribution.Estimates of contemporary gene flow were obtained in BAYESASS+ 3.0.3 . Five ine in the past . A mutation rate of 1.27 x10-3 per generation was considered for the calculation ofmh and Ne, based on the estimates of mc) was compared with historical levels (mh) using the Mantel test run in Ztmantel  were estimated in MIGRATE-n 3.6 , which wthe past . This raZtmantel , runninge generations  in only two populations. The highest recapture rate was recorded in Bauru,with three individuals being recaptured , with 24 samples beingused for the analyses. In contrast, only a single pair of identical genotypes wasdetected in the São Carlos population, with only 40 of the 41 samples beinganalyzed.The combined analysis in CERVUS identified identical genotypes  presented an excess of unspecific bands, nd afurther two (Pmoratoiμ23 and Pmoratoiμ25) hadstutter bands, which hampered the interpretation of the genotypes in somepopulations. In order to avoid biases in the estimates of genetic diversity andpopulation structure, these loci were excluded from the population analyses.A total of 22 polymorphic loci were evaluated, although one locus,Pmoratoiμ27 / r = 0.078;Pmoratoiμ29 / r = 0.095) and one in thepopulation from São Carlos (Pmoratoiμ29 / r =0.179). Evidence of stuttering was found in one locus from Bauru(Pmoratoiμ13 / r = 0.103). Overall, significantevidence of genotyping errors was found in only four (7.84%) of the 51 tests (3populations x 17 loci). The global FST values were calculated in the FreeNA application, with the unadjusted value(0.196) being highly similar to the adjusted value (0.194). This led us to maintainthese loci in the population analyses, given that small differences support theassumption that the estimates of genetic diversity and population differentiationwould not be affected significantly by this degree of genotyping error. Nosignificant linkage disequilibrium was detected between the pairs of loci, based onthe analysis in GENEPOP . Given thesefindings, all 17 independent loci were included in the population analyses.The 17 remaining loci were also amplified in the samples from the Botucatupopulation. When analyzed in MICRO-CHECKER, no evidence of large allele dropout wasfound in any of the loci, whereas null alleles were identified in two loci in theBrotas population , the highest levels of genetic diversity were recorded for thenorthern  and central (São Carlos e Brotas)populations. By contrast, the southern populations  presented the lowest levels of genetic diversity. Based on thisfinding, allelic richness was compared between the two main groups: group 1 =northern + central populations, and group 2 = southern populations (except Botucatu).This was done in FSTAT using the randomization tests with 1,500 permutation atsignificance level α = 0.05. Significantly higher genetic diversity was identified ingroup 1 (p = 0.018 / G1 > G2). While it was not possible to amplify the genotypesin the vast majority of the Botucatu samples, the few recovered genotypes permittedthe detection of high levels of endogamy in this extinct population. Based on the stepwisemutation model, LOSITAN identified the marker Pmoratoiμ6 (p = 0.978)as the most likely to have been affected by positive, i.e.,directional selection. The permutation test run in SPAGeDI detected a significantcontribution  of the stepwise mutations to populationstructuring, indicating that the RST provided the best fit for the data. Based on this result, the most adequateLOSITAN output was that which determined that the locus Pmoratoiμ6is possibly under selection, and for this reason it was excluded from all subsequentanalyses.The LOSITAN analysis based on the infinite alleles model identified balancingselection in locus RST index of population differentiation (RST = 0.912 / p = 0.00), Botucatu and Paranaiguara (RST = 0.670 / p = 0.01), and Botucatu and Uberlândia (RST = 0.636 / p = 0.00). While many of the comparisons with the population fromLençóis Paulista had high RST values , the results were notsignificant (p = 0.99) due to reduced sample size.In the pairwise comparisons using the ntiation  at signiOverall, 44 of the 122 distinct alleles produced by the neutral markers wereexclusive to one of the populations. The southern populations were characterized byreduced numbers of private alleles, with four being recorded in Bauru, two in Avaréand Botucatu, and one in Lençóis Paulista. In the northern portion, by contrast, nineprivate alleles were recorded in Brotas, eight in Uberlândia, and seven inParanaiguara and São Carlos.P. moratoi populations .The STRUCTURE analysis identified two clusters representing the highest hierarchicallevel in the genetic structure of the k = 2) indicates a profounddegree of structuring between the central (São Carlos and Brotas) + northernpopulations  and the Bauru population, to thesouth, located on the left margin of the Tietê River (downstream flow). The othersouthern populations  presented a mixeddistribution overlapping the two clusters (Q < 0.7). While the Botucatu populationis characterized by an excess of empty genotypes, with few or no data being availablefor most individuals, it presented the same pattern of mixture with thegeographically closest populations, that is, Avaré and Lençóis Paulista, whichindicates an adequate amount of informative data. At k = 4, lessprofound structuring was detected, with the populations from Minas Gerais and Goiásforming a single cluster, three others clusters on the populations from São Carlos,Brotas, and Bauru, and the southern populations  once again presenting a mixed pattern, with a slight tendency to group withthe Bauru population , the exception being the individuals from theBotucatu population. At k = 4 (H' = 0.754), high Q values wereobserved in all the individuals from the Paranaiguara, Ituiutaba, and Uberlândiapopulations, all from the same cluster, Bauru and Avaré ,and the population from Brotas. Mid-range Q values (0.5–0.7) were obtained for theSão Carlos and Lençóis Paulista, and also for the Botucatu population, but inindividuals representing distinct clusters. This analysis revealed an almost perfectdifferentiation between the population groups located on the left (southern) marginof the Tietê and the right (northern) margin of this river (k = 2 and k = 4) and Lençóis Paulista(k = 4) populations which had individuals that were assigned todifferent clusters.Based on the initial STRUCTURE analyses, barriers to gene flow were added to the datainput to represent the location of the Tietê River, which apparently represents themajor barrier to the dispersal of nd k = 4  identifiis river A, B. Excr = 0.638; p <0.002). However, when only the populations in the vicinity of the Tietê River (withina radius of 80 km) are included in the analysis, that is, excluding the populationsfrom Minas Gerais and Goiás, the isolation by distance was not significant.The analysis of isolation by distance, based on the pairwise comparison of all thepopulations detected a significant tendency (TableS1) are low (mc < 0.10), except for three significant one-way routes betweenUberlândia and Ituiutaba (15% of migrants between populations 119 km apart),Uberlândia and Paranaiguara , and Bauru andAvaré . The highest rates of self-recruitmentwere found in the central populations, São Carlos and Brotas.The delta values of the BAYESASS runs were within the range expected for a pattern ofconvergence. Plots produced by TRACER indicated that the chain was well mixed(results not shown), confirming that the convergence of runs has been reached andthat the estimates of gene flow were probably reliable. In general, contemporarymigration rates  forParanaiguara, 224 (CI: 0 – 551) for Ituiutaba, 108 (CI: 0 – 433) for Uberlândia, 211(CI: 0 – 524) for São Carlos, 216 (CI: 0 – 537) for Brotas, 118 (CI: 0 – 420) forBauru, 96 (CI: 0 – 420) for Avaré, 211 (CI: 0 – 485) for Lençóis Paulista, and 146(CI: 0 – 472) for Botucatu.As for historic migration using the 95% confidence intervals (CI), the mostsignificant routes (> 1%) were between Brotas and Bauru , Brotas and Uberlândia  and Brotas and São Carlos, and between São Carlos and Brotas . The largest population-scaled mutation rate was recorded for the northernpopulation from Ituiutaba , and the smallest, in thesouthern population from Avaré . Based on the formula Θ =4Nmc and mh values for the populations and routes analyzed as a whole , although there was a marked tendency for positive values except for the Brotas to São Carlos, São Carlos to Brotas, and Brotas toBauru routes (mh > mc). Similarly, no significant difference was found between contemporary andhistoric migration patterns in the northern  and southern populations , although migration rates were significantly higher in the past in thecentral populations .No significant divergence was detected between the No evidence of recent decline was detected in any of the populations using either theone-tailed Wilcoxon test or the shifted mode, with the exception of the now extinctpopulation from Botucatu. Significant results were obtained for both tests, using theTPM model with 70% and 90% SMM , and no significant bottleneck one-tailed Wilcoxon: p = 0.15) was detectedin the case of the SMM model.P. moratoi, that is, on the left margin of the Tietê River,presented lower levels of genetic diversity in comparison with the other populations.While most of these southern populations are represented by small samples, which mayexplain part of this difference, it is important to note that the Bauru population,represented by more than 20 individuals, was characterized by low levels of diversity,which is typical of the region. In addition, despite the small size of the samples fromthe northern localities of Paranaiguara (n = 3) and Ituiutaba (n = 4), the fact thatthese populations presented high levels of diversity contradicted possible samplingeffects.The populations located in the southern portion of the geographic distribution ofe.g. Avaréand Lençóis Paulista, pers. obs.) show signs of drastic decline, while others have become extinct. This suggests that the reducedindices of diversity recorded in this region may be related to small populations sizes,a characteristic also observed in the estimates of historic population size. Inferenceson immigration rates obtained in MIGRATE are generally not influenced by sample size,although the confidence interval tends to be larger in samples with fewer individuals may have been related to the small sample size , whichprobably affected the reliability of the estimates provided by MIGRATE. Smallerpopulations tend to be more susceptible to extinction resulting from stochasticprocesses, which may explain the extinctions observed in the southern sector.Differences in inter-population diversity have also been recorded in the Europeantree-frog, Hyla arborea, which is not considered endangered insoutheastern Europe, whereas the populations in the western portion of the distributionappear to be more vulnerable, with marked population decline and low levels of geneticdiversity , which does support the reliability of the few genotypes analyzed.In addition to its low genetic diversity, the Botucatu population also presented highlevels of endogamy. Evidence of a recent bottleneck was also found in this population.ollution , all of Pmoratoiμ5 was not amplified in the southern populations reinforcesthe differences encountered in the other analyses . The Tietê River effectively separates the clusters (see TESSdata), and its role in the structuring of the populations is reinforced by thenon-significant isolation by distance found among the populations located in thevicinity of this river. An analysis of the morphological characteristics of theP. moratoi populations found little divergence, except for subtledifferences in the coloration of the specimens  found among nearby populations wasexpected given the philopatry of amphibians to their breeding sites and reduced vagility, despite the reduced distancebetween them (22 km) and the absence of riverine barriers. The opposite pattern wasfound between other populations (mh < mc). In the specific case of São Carlos, anthropogenic impact is the highest of anyof the populations analyzed, and is characterized by a mosaic of sugarcane plantations(22% soil coverage) and areas of regenerated forest interspersed with small remnantislands of native vegetation  presented the highest rates ofself-recruitment and significantly lower contemporary gene flow in comparison withhistoric migrations . On the other hand, it is also possible to infer that, as the central andnorthern populations are larger, they are less vulnerable to extinction resulting fromshort-term stochastic processes, in contrast with the Botucatu population, which is nowextinct. Levels of genetic diversity appear to persist due to the relatively recentprocess of population fragmentation, although with ongoing isolation and resultingincrease in genetic drift over the long term, a loss of genetic diversity and increasein endogamy will be expected, especially in the central populations.Our study indicates that P. moratoi, and our findings on effective population size and the number of adult individuals (< 250) ineach population, we recommend the reclassification of the conservation status of thespecies according to the relevant P. moratoi as Vulnerable (VU),in accordance with criteria A2a and C2a (i). In this case, while less preoccupying,the species is still considered to be under some threat of extinction, given theevidence of decline in the southern populations and the reduced numbers ofindividuals.Based on the extension of the geographic distribution of P. moratoi, whererelatively high levels of genetic diversity were found.The anuran fauna of the Cerrado is still very poorly known , which re-emphasizes the need for the separate managementof these two units  the definition of two management units, one consisting ofthe southern populations, on the left margin of the Tietê River in São Paulo, and theother of all the other populations. This primary division was reinforced by STRUCTURE duex situ populations with individuals from the southern localities with the aim of preservingthese unique genetic stocks. The southern populations may be at the limit ofviability due to the marked effects of drift, the reduced capacity to respond toselective pressures, and the low probability of any increase in genetic variabilitydue to a rescue effect from other populations.As the present analyses do not provide estimates of the divergence times of thedifferent management units, we would recommend the application of a more systematicphylogeographic approach based on additional molecular markers  for a more reliable definition of the patterns and the timing of thediversification of the different lineages. We would also recommend the establishmentof


It is unclear whether Indo-European languages in Europe spread from the Pontic steppes in the late Neolithic, or from Anatolia in the Early Neolithic. Under the former hypothesis, people of the Globular Amphorae culture (GAC) would be descended from Eastern ancestors, likely representing the Yamnaya culture. However, nuclear  and mitochondrial (11 complete sequences) DNA from the GAC appear closer to those of earlier Neolithic groups than to the DNA of all other populations related to the Pontic steppe migration. Explicit comparisons of alternative demographic models via approximate Bayesian computation confirmed this pattern. These results are not in contrast to Late Neolithic gene flow from the Pontic steppes into Central Europe. However, they add nuance to this model, showing that the eastern affinities of the GAC in the archaeological record reflect cultural influences from other groups from the East, rather than the movement of people. However, the place of origin of the first IE speakers has not been identified, nor has any consensus emerged about the time and the mechanisms by which IE languages spread over Western Eurasia. Based on the linguistic and archaeological evidence, the earliest speakers of a proto-IE language have often been identified with people living in the Pontic steppes about 6000 years ago , with thThe majority of linguists currently support the hypothesis of a late, Pontic spread, here referred to as the Kurgan hypothesis , althougf3- and f4-statistics  plots, clustering, and fitting of admixture graphs and clade tests based on cs e.g. ,20,21). ,21. f3- cs (e.g.  argued t2.(a)The samples analysed in this study come from a Megalithic barrow, an elongated oval of almost 22 m in length and up to 6 m in breadth, in Kierzkowo, Żnin district, northwestern Poland , a typic(b)(i)AP, including both ancient and present-day individuals and A, including only ancient individuals.To analyse the GAC individuals in the context of ancient and present-day genetic diversity, we merged them with 249 ancient individuals AP dataset using the snpgdsPCA function in the SNPRelate package [AP, using the software 4P [We ran PCA on the  package . For dettware 4P , considetware 4P .K= 2 and K= 10, using 10 replicates per K with different random seeds. The optimal value of K was evaluated through a cross-validation procedure, thus identifying the number of ancestral populations for which the model had the best predictive accuracy. To summarize the degree of genetic relatedness between the GAC samples and the populations of the A dataset, we estimated outgroup f3-statistics using the ADMIXTOOLS program qp3Pop [f3 . We also estimated the f3 statistic considering as target the individuals of the Corded Ware population, in the form f3 . Maximum-likelihood trees summarizing variation in the AP dataset were inferred by TreeMix [Population structure was inferred from both datasets using ADMIXTURE ; we assum qp3Pop , in the  TreeMix , adding  TreeMix  (see Dat(c)(i)http://www.fluxus-technology.com).Seventeen specimens, bones and teeth, were selected  and their mtDNAs were analysed independently in two different laboratories . We collected from the literature ancient mtDNA data from the same populations described in the nuclear data section ((ii)abcrf and the predict functions provided in the abcrf R package. To evaluate the ability of the ABC-rf procedure to distinguish among the models tested, we calculated the classification error using as PODs each dataset of our reference table. The ability of the selected models to actually generate the observed variation was assessed using linear discriminant analysis (LDA) and PCA. To estimate the models' parameters we selected the best 5000 simulations out of 1 million for each model selected. Posterior probabilities for models and parameters were calculated using R scripts from http://code.google.com/p/popabc/source/browse/#svn%2Ftrunk%2Fscripts, modified by S.G.We compared different demographic models, each characterized by a different set of migration events, via approximate Bayesian computation via random forest (ABC-rf) . In all cases, we assumed some level of genetic continuity across three geographical regions, namely Eastern Europe, Central Europe and the Near East; the models then differed as for the presence of one or two migration events, connecting different regions. The analysis required two main steps; in the first one we estimated the number and the extent of migration waves from the Pontic steppes (arrows 1 and 2 in 3.(a)(i)AP dataset. The newly reported GAC individuals fell within a cluster comprising most Early and Middle Neolithic individuals . As previously observed [To explore the genetic affinities among ancient and modern-day individuals, we examined a PCA plot of the observed , a clearobserved ,20,35 .We also computed a matrix of genetic distances between pairs of individuals in the A, dataset , revealed three ancestral components, clearly separating three groups. The orange component is found at high frequencies in hunter-gathering populations such as the Holocene samples from Hungary and in the Motala samples from Sweden. All Early Neolithic populations are characterized by having a large proportion of their genotypes represented by a yellow component, which, strikingly, is also strongly represented in Chalcolithic samples from Iberia consistent with a common origin from first farmers of Anatolia [Clustering by ADMIXTURE  of the gAnatolia . By contExcept for one of our Kierzkowo samples (in which it represented about 15% of the genotype), the Steppe-related component was absent altogether in the GAC population. All other GAC samples showed, instead, a mix between a major Early Neolithic component (up to 83%), and the component found at high frequencies among hunter-gatherers (up to 30%). When the ADMIXTURE is asked to cluster the samples into four rather than three groups, the Early Neolithic cluster fissions into two, and the new component (green) is present in the GAC, as well as in several other Western European populations. This second Neolithic component, here referred to as Western Europe Neolithic, accounts for a large share of the ancestry of individuals such as those from Iberia (Iberia_CA), La Mina (LaMina_MN) and Els Trocs (Els_Trocs_EN).AP dataset  confirms the pattern of relationship between the studied populations. Additional genomic components become apparent, an expected consequence of demographic changes occurred in later prehistoric and in historic periods. However, considering six groups, we could reproduce the clusters previously described in the ancient samples: Early Neolithic (red), hunter-gatherer (yellow), Steppe-related (green) and Western Europe Neolithic (orange). As was the case in the analysis conducted only on ancient samples, the GAC population showed both the Western Europe Neolithic and the Early Neolithic component, with negligible, if any, the presence of the component so strongly associated with the Kurgan migration.The ADMIXTURE analysis on the (ii)f3-statistics, in the form , where X represents, in turn, each ancient population in our dataset. Once again, the GAC people appeared to have more in common with the other Middle Neolithic samples, in particular from Hungary, Iberia and Sweden, than with geographically closer samples , to verify whether we could detect signals of introgression from the Pontic steppes in the later, Corded Ware population, as already observed by Haak et al. [et al. [Moving from individual to population comparisons, we summarized levels of shared genetic ancestry between pairs of populations since their divergence from an African outgroup calculating sets of  samples . A genetk et al.  and Alle [et al. ; a clear(iii)These evolutionary links represent departures from a simplistic, tree-like model of population split, followed by divergence in isolation. To account for population contacts after their initial separation, we then added a number of putative migration events to the maximum-likelihood population tree inferred from our data . The inia–c and d–g). The GAC population followed this trend, showing only an increased gene flow with the contemporary Middle Neolithic samples from Sweden . The only evidence for reduced gene flow is an apparent barrier surrounding Hungary in the Copper Age map.Finally, by a graphic method, EEMS , we iden(b)(i)Results obtained from mitochondrial DNA enrichment are summarized in electronic supplementary material, table S4. Samples 6.3 and 7.5 had mean coverage <1 fold and were excluded from further analyses. Among the other samples, the average mtDNA coverage ranged between 6.3 and 244.6, and the average fragment length was between 50.9 and 67.6 bp. As already mentioned, radiocarbon dating of some bone remains found outside the burial chamber indicates they belong to historical times . Average fragment lengths were the same for the samples found inside (Neolithic period) and outside  the burial chamber. By contrast, the two groups of samples differed for the deamination rate at read termini: the proportion of C to T misincorporations is between 30% and 40% in the Neolithic individuals, and only 11% and 12%, respectively, in historical samples 8.8 and 8.9, possibly correlated with their different ages . Sample (ii)et al. [For 11 out of 17 GAC individuals, mitochondrial DNA was typed by a capture NGS run. The samples were analysed with the NGS pipeline described in Modi et al. . Three iϕST pairwise distances estimated for this subset, the Early Bronze Age people, represented by the Srubnaya culture, appear connected with the eastern Corded Ware peoples, and also close to the Yamna. The GAC samples are clearly separated from those populations, and show instead a closer relationship with the western, Late Neolithic, Bell Beaker population .To formally test the Steppe migration hypothesis, we selected a subset of the mtDNA data including the nine GAC individuals and 56 samples from five populations , and we ran some preliminary analyses on it. In the neighbour joining (NJ) tree inferred from the The median-joining network  (electro(iii)ABC-rf , or a migration from the Pontic steppes before the onset of the GAC (MIG1) found very limited support in the ABC analysis. The best fit in the first round of simulations was obtained including only a later migration from Yamna, i.e. one not affecting the GAC individuals for which we have information (MIG2), followed by the model in which two subsequent migrations are considered . Adding, in a second round of simulations, a further episode of gene flow, this time eastwards , improved the fit, but not dramatically so. The proportion of mtDNA lineages possibly derived from those of the Yamna people is less than 50%.In short, simulation-based tests on mtDNA variation do not suggest that the GAC people of this study have special links with migrants from the Pontic steppes, but show a direct connection between the Yamna and later Central Europe cultures (Corded Ware and Bell beaker), who derived almost half of their mitochondrial variation from them. The emerging picture is thus one in which migrations from the Pontic steppes into Central Europe left a trace in the genomes of the Corded Ware culture, but not in those of the GAC.At this stage, it is hard to say whether and to what extent the finding that migrations from the Pontic steppes had little or no demographic impact on the GAC also has implications for the Kurgan hypothesis. There is little doubt that in the Late Neolithic there were indeed migration processes from the Pontic steppes into Central Europe, documented by the archaeological and genetic links between the Yamna and Corded Ware cultures. However, depending on the number of people involved, migrations may or may not leave a recognizable trace in the genetic makeup of a population. The data we provide show that such a trace is not apparent in the genomes of the GAC people. Rather, evolutionary connections are evident between the GAC and other European groups, both at the nuclear and mitochondrial level. In short not all population relationships in the Central European late Neolithic correspond to those proposed in the original Kurgan model. Of course, there is also the possibility that GAC sites other than those investigated in this study might show different genomic features, but at this stage, this is only a matter of speculation. Therefore, either the GAC people pre-existed and were extraneous to the Pontic Steppe migration process envisaged by Gimbutas, or the Pontic steppes migrants' contribution was represented by few individuals, too few indeed to leave a trace in the genetic makeup of the GAC population.et al. unpublished data [et al. [To get a deeper insight into the linguistic changes prompted by Neolithic migration, one should have an idea of the languages spoken in that period, which is currently out of reach. Therefore, our work leaves open the possibility that the GAC was in contact with Yamna, but in this case, the interaction was mostly at the cultural level, entailing very limited migration, if any, contrary to the predictions of Gimbutas' Kurgan hypothesis. One alternative is that that the Pontic steppe migration did not profoundly affect mitochondrial variation, as implied by several studies showing an excess of migrating males in expanding pastoral economies (see Saag hed data  and Kris [et al. , and ref


We identified a total of 11 single nucleotide polymorphisms (SNPs) and 12 haplotypes (Ha1–Ha12). Low genetic diversity  was detected for COX-III, and haplotype diversity of Tibetan chicken populations (0.750 ± 0.018) was markedly higher than lowland chicken populations (0.570 ± 0.028). Obvious genetic differentiation (nucleotide divergence = 0.092~0.339) and conspicuous gene communication (gene flow = 0.33~32.22) among 12 populations suggested that Tianfu black-bone fowl (white feather) was possibly introduced from Tibetan chicken. SNP m.10587 T>C affects the specific functions of the COX enzyme. Haplotype Ha3 was found in Tibetan chickens, and SNP m.10115G>A caused an amino acid substitution  associated with phospholipid binding, while mutations m.10017C>A and m.10555G>A and the previously reported SNP m.10065T>C reduced the hydropathy index to some extent. Together, this indicates that the mitochondrial membrane is more hydrophobic in Tibetan chickens.To obtain a full understanding of the genetic diversity of the cytochrome oxidase III gene Gallus gallus gallus), making it particularly good at flying and foraging on the plateau alpine region [Domestic chickens fulfill various roles ranging from food and entertainment to religion and ornamentation . Tibetane region –5. The bAvian species living at high altitudes are characterized by the high oxygen affinity of their haemoglobin , 7. Tibe COX-I gene is used as a standard marker for DNA bar coding to enable species identification in animals [ COX-III gene of bar-headed geese contained a nonsynonymous substitution (Trp-116→Arg) that resulted in a major functional change of amino acid class. This mutation was predicted by structural modeling to alter the interaction between COX-III and COX-I, which contributed to adaptation in mitochondrial enzyme kinetics and O2 transport capacity and may finally contribute to the exceptional ability of bar-headed geese to fly at extreme heights [Mitochondrial DNA (mtDNA) sequences are widely used in molecular evolutionary studies. These sequences are useful for estimating times of species and population divergences, comparisons of relative rates of evolution, and phylogenetic inferences within and between vertebrate species . The com animals , 13. The animals . The COX heights .COX has been investigated in several biological studies [ COX-III have rarely been reported in chickens. However, COX-III is an important component of the respiratory chain and is very conserved among species. Adaptive changes in COX activity can alter the ATP supply derived from oxidative phosphorylation during hypoxia [ COX-III SNPs in 12 chicken populations (five lowland and seven highland populations) to better understand the COX-III genetic diversity and to determine the contribution of specific SNPs to high altitude adaptations in Tibetan chickens. studies , 16, 17, hypoxia . The quaIn all experimental populations, 5 populations  belonged to lowland chickens; the other 7 populations  which belonged to Tibetan chickens were collected . Blood sµl volume with 100–150 ng of genomic DNA, 25 mM MgCl2, 2.5 mM of dNTP mixture, 2 mM each primer, 5 µl of 10x buffer, and 1.25 U LA Taq polymerase  under the following conditions: denaturation at 94°C for 5 min, then 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 60 s, followed by a final extension at 72°C for 7 min [We extracted mtDNA by salt extraction method . PCR useor 7 min . PCR pro (Hd), nucleotide diversity (Pi), nucleotide divergence (Dxy), net genetic distance (Da), coefficient of differentiation (Gst), and gene flow (Nm), Tajima's D value neutral test, were defined using DnaSP V5 software [http://www.fluxus-engineering.com/sharenet.htm). The complete mitochondrial genome sequence of the red jungle fowl was used as the reference sequence (GenBank accession number: NC_001323).Raw sequences were aligned and edited by DNAstar software . We exported all sequences as an aligned FASTA file. Sequence variations were identified using MEGA 6.0 software . Standarsoftware , whereashttp://beast.bio.ed.ac.uk/). Samples from the posterior were summarized on the maximum clade credibility tree using the program TreeAnnotator v1.4.8 (distributed with BEAST) and visualized using the program FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). Statistical differences in COX-III haplotype frequencies between Tibetan chickens and lowland chickens were analyzed using Fisher's exact test; odds ratios (OR) and 95% confidence intervals (95% CIs) were also calculated. P value < 0.05 was taken into account as statistical significance. The bioinformatics platform MitoTool (http://www.mitotool.org/) was used to analyze haplotype distribution frequencies between Tibetan chickens and lowland chickens [P < 0.05 were taken to be statistically significant.Analysis of molecular variance (AMOVA) was estimated using Arlequin 3.0 software . Bayesiachickens . P < 0.0http://wlab.ethz.ch/protter/start/). Hydropathy plot of the COX-III protein was predicted using the Tmpred Program [http://www.ch.embnet.org/software/TMPRED_form.html).Protter  was used Program  . We calculated the overall base composition of COX-III from the 12 chicken populations using MEGA 6.0. Cytosine (C) was shown to be the rarest nucleotide (16%) with guanine (G) to the most common (32%). The A + T% was around half of COX-III (52%).The length of 300 COX-III sequence from 300 individuals.  Hd, Pi, and other information within each group. The number of variable sites in each population varies from 1 in Diqing and Shigatse to 7 in Haiyan, and the sequences from Doilungdêqên, Ganzi, Nyingchi, Shannan, were 5, 5, 3, and 2, respectively. The highest haplotype diversity was found in Haiyan and Doilungdêqên. While the average number of nucleotide differences in Emei from lowland chickens was greater than others. Taken together, a total of 8 haplotypes were identified in lowland chickens, and the overall haplotype diversity, nucleotide diversity, and average nucleotide differences were 0.570 ± 0.028, 0.00155, and 1.216, respectively. In Tibetan chickens, a total of 9 haplotypes were identified, and the overall haplotype diversity, nucleotide diversity, and average nucleotide differences were 0.750 ± 0.018, 0.0016, and 1.250, respectively. The result showed that the genetic diversity of the Tibetan chicken was noticeably higher than that of lowland chickens. After Tajima's D value neutral test, all P values were greater than 0.1; therefore, the 12 populations belong to neutral mutations.A total of 11 SNPs (including six singleton sites and five parsimony-informative sites (with no insertions/deletions)) accounted for 1.403% of the total 784 bp (Dxy) and net genetic distance in nucleotides (Da). Within the 12 chicken populations, the average Dxy was 0.062% (range 0.092%–0.339%) and the average Da was 0.196% (range 0%–0.216%) ( Da (0.216%) was found between Diqing and Jiuyuan black chickens (between Tibetan chickens and lowland chickens), and the smallest Da (0%) was observed between Haiyan and Doilungdêqên chickens; the smallest Dxy (0.092%) was observed between Black-Tianfu and Muchuan black chickens (within lowland chickens) and the largest Dxy (0.339%) was found between Diqing and Emei chickens (between Tibetan chickens and lowland chickens).Nucleotide polymorphism among populations can be represented by nucleotide divergence–0.216%) . The lar (Da) to demonstrate that Haiyan and Doilungdêqên are most similar in highland chickens  and that Muchuan black-bone fowl and Tianfu black-bone fowl (black feather) are most similar in lowland chickens . Conversely, Tibetan chickens (Diqing) appear more distantly related to Jiuyuan black fowl  and Emei black fowl .To pinpoint the most and least closely related populations, we used net genetic distance in nucleotides (Da) are substantially consistent with the outcome of nucleotide differences (Dxy).These findings are conclusive of definite genetic differentiation between different groups of chickens, with the strongest differentiation seen between Tibetan chickens and lowland chickens. Net genetic distances in nucleotides (Gst) can reveal the extent of gene flow and genetic drift to some extent, while gene flow can uncover possible gene infiltration among populations. In lowland and Tibetan chicken populations, distinct gene exchange  was detected between White-Tianfu (lowland chickens) and Haiyan and Doilungdêqên (Tibetan chickens). However, at the same altitude, obvious genetic differentiation was found in lowland geographical populations (P < 0.05) while conspicuous gene communication (Gst  =  0.01  Nm  =  29.34) was only detected in Muchuan and Black-Tianfu chickens. Obvious genetic differentiation appears to have occurred in Tibetan chickens (The coefficient of differentiationchickens . This in∗∗P < 0.01) which implied that the genetic divergence within populations was significant. The results indicate that the twelve geographic populations do not produce largely genetic differentiation, while the genetic diversity in COX-III gene mainly comes from within the populations (AMOVA showed that the percentage of variation within populations (70.43%) was greater than that between populations (29.57%). Fst value was 0.2957 (ulations . COX-III. ∗P < 0.05) in Tibetan chicken, respectively. Similarly, in lowland chicken, the SNP m.10081A>G was significantly different from Tibetan chicken with the allele frequency of 57.2%. The four SNPs  were only distributed in Tibetan chicken breeds with the allele frequency of only 0.6% and 7.7%, while the other three SNPs  were only distributed in lowland chicken with the allele frequency of 0.7%. We consider that the seven SNPs showed nonsignificant difference between Tibetan chicken and lowland chicken breeds (P > 0.05).We detected seven synonymous mutations  and four nonsynonymous substitutions  inWe identified 12 haplotypes (Ha1–Ha12) in 300 chickens from the 12 different populations . The med Meleagris gallopavo as an outside group to construct a phylogenetic tree using the Bayesian method. The correlation of the 12 haplotypes is shown in We also used theP value, 1.911 × 10−14∗; OR, 30.375, 95% CI, 7.332–125.837). Haplotypes Ha1 and Ha5 also appear to be significantly associated with lowland adaptation , whereas haplotype Ha6 does not seem significantly associated with altitude adaptation at the 0.05 level   . COX-III variants m.10017C>A and m.10555G>A are located in the transmembrane helical structure, while SNP m.10115G>A is located in the outer surfaces of the inner mitochondrial membrane  than lowland chickens (0.570 ± 0.028). For the results observed, we think that artificial selection leads to reduced nucleotide diversity in lowland chickens. Although 11 SNP sites were identified in COX-III from 12 geographic populations, the average genetic distance was only 0.196%, revealing a low level of genomic polymorphisms in all populations. This low level of genetic diversity indicates that this gene is functionally important and hence has an evolutionary constraint. The 12 populations belong to neutral mutations (P > 0.1). This suggests that these populations are stable and have not undergone a population expansion in the past few years.By analyzing population haplotype diversity and the structure of chicken breeds in southwestern China based onGst  =  0.01, Nm  =  25.21; Gst  =  0.05, Nm  =  4.9), indicating that the Tianfu white-bone fowl was introduced from high altitude area. Moreover, Tibetan chickens from Haiyan and Doilungdêqên regions were most similar  suggesting that the genetic distance of the two regions is very close, and gene exchange is very rich .The analysis of the coefficient of differentiation and gene flow between populations suggests that some chicken populations have undergone genetic differentiation at places of equal altitude. This may reflect the introduction of varieties of different regions. For instance, the Tianfu white-bone fowl from a lower altitude has a close population genetic relationship with the Tibetan chicken  between an expanded sample of 56 Tibetan chickens and 152 lowland birds [ COX-III SNPs to explore the different haplotypes detected a novel mutation associated with high altitude adaptations. AMOVA showed that COX-III variation mainly existed within a population. Of the 12 defined haplotypes, the existence of Ha4, Ha7, Ha8, and Ha10 only in highland chickens and Ha9, Ha11, and Ha12 only in lowland chickens indicates different degrees of genetic divergence between Tibetan chickens and lowland chickens. Ha1, Ha2, and Ha4 were found to be the earliest ancestors , with the C allele at m.10587 T>C was found to have a probability of being over 30.375 times better adapted to hypoxia than the T allele. We propose that m.10587 T>C affects the functions of the COX enzyme in a similar way to the effect of m.10081A>G [The regulating mechanism innd birds . Anothernd birds  eight SNncestors , while h10081A>G  on high ∗P < 0.05), shared by populations Ganzhi and Diqing, caused the amino acid mutation Val62Ile  were peculiar to highland chickens. Mutation m.10115G>A (with the allele frequency of 9.7%, Val62Ile . A previIn conclusion, we found largest genetic differentiation between Tibetan and lowland breeds and identified haplotype Ha2 is associated with Tibetan chicken populations. The possible association between increased hydrophobicity/reduced hydrophilic characteristics of the mitochondrial membrane and high altitude adaptation could provide a theoretical reference for poultry genetics. Therefore, our results provide a theoretical basis for future research into fowl breeding.