Ursus arctos). Previous phylogeographic studies of the brown bear based on maternally inherited mitochondrial DNA have shown intraspecific genetic structuring around the northern hemisphere. The brown bears on Hokkaido Island, northern Japan, comprise three distinct maternal lineages that presumably immigrated to the island from the continent in three different periods. Here, we investigate the paternal genetic structure across northeastern Asia and assess the connectivity among and within intraspecific populations in terms of male-mediated gene flow.Sex-biased dispersal is widespread among mammals, including the brown bear , Sakhalin, and continental Eurasia . The Hokkaido brown bear population is paternally differentiated from, and lacked recent genetic connectivity with, the continental Eurasian and North American populations. We detected weak spatial genetic structuring of the paternal lineages on Hokkaido, which may have arisen through male-mediated gene flow among natal populations. In addition, our results suggest that the different dispersal patterns between male and female brown bears, combined with the founder effect and subsequent genetic drift, contributed to the makeup of the Etorofu Island population, in which the maternal and paternal lineages show different origins.Brown bears on Hokkaido and the adjacent southern Kuril Islands experienced different maternal and paternal evolutionary histories. Our results indicate that sex-biased dispersal has played a significant role in the evolutionary history of the brown bear in continental populations and in peripheral insular populations, such as on Hokkaido, the southern Kuril Islands, and Sakhalin.The online version of this article (10.1186/s40851-017-0084-5) contains supplementary material, which is available to authorized users. Homo sapiens) [Pan paniscus) [Pan troglodytes) [Pongo spp.) [Papio hamadryas hamadryas) [Canis lupus) [Ovis spp.) and horses  [Ursus arctos), which is widely distributed throughout the Holarctic region, shows strongly male-biased dispersal [Molecular studies based on both biparentally and uniparentally inherited, sex-specific genetic markers have shown empirically that sex-biased dispersal is widespread among mammals, but varies widely in direction and intensity –3. Most sapiens) , 8, bonoaniscus) , chimpanlodytes) , orangutgo spp.) , 12, hammadryas) , canids s lupus) , 15, andaballus) , 17. Theispersal –21, and ispersal .Some previous phylogeographic studies of the brown bear based on maternally inherited mitochondrial DNA (mtDNA) showed extensive intraspecific geographical genetic structuring in maternal lineages –27. The 2) is much smaller than the adjacent continent, the maternal genetic diversity is higher there than in northern continental Eurasia, where the brown bear predominantly shows a single mtDNA lineage [While Hokkaido Island , Sakhalin, and continental Eurasia . We discuss the effects of sex-biased migration on the evolutionary history of the insular and continental populations, and differences in the male genetic contribution to the population composition. Finally, we assess the strength of male-mediated gene flow among geographically adjacent insular populations in northeastern Asia.Muscle or liver samples from 55 male brown bears collected on Hokkaido Island were obtained from the Environmental and Geological Research Department, Hokkaido Research Organization Fig. . Tissue Female brown bear samples were included in each round of PCR amplification as a control to confirm male specificity. No amplification was observed from female samples in any round of amplification.The same sequencing primer sets and touchdown thermal cycling conditions as described in Bidon et al.  were useNine Y-linked microsatellite alleles  were determined by using two sets of multiplex PCRs, with the same primer sets as given in Bidon et al. . Each muπ), Watterson’s θW (per site), Tajima’s D, Fu and Li’s D and F, and Fu’s FS. Compound haplotypes were determined based on the combination of Y-linked SNPs in 3071 bp (3.1 kb data set) of Y-chromosomal DNA and six Y-linked microsatellite alleles. The genetic diversity of Y-chromosomal compound haplotypes was determined based on 214 brown bears, with the number of haplotypes (H), haplotype diversity (HD ± SD), and mean number of pairwise differences (MPD ± SD) calculated for each population. To assess the population size reductions, the modified Garza–Williamson (GW) index  was calculated using Arlequin ver. 3.5.2.2 [Data on brown bears, polar bears, and American black bears were added to our dataset from Bidon et al. . In Bido 3.5.2.2 , 45. Thehttp://www.fluxus-engineering.com). For network calculations, more quickly evolving microsatellite loci were weighted inversely to their variance in repeat length , and SNP loci were weighted 10 times the greatest microsatellite weight (Y-linked SNPs = 90) as per [A median-joining (MJ) network  of Y-chr) as per . MJ netw) as per . The nom) as per . Names o) as per  and Hira) as per .RST) for Y-chromosomal polymorphisms [Pairwise population differentiation values  and the times of population splitting were estimated by using the Bayesian-based coalescent approach implemented in the software BATWING . Time esribution  accordinribution , and a mribution . For eacribution . A generribution  to allowU. maritimus), and American black bears (U. americanus), and each of these species was reciprocally monophyletic for both the 5.3 kb and 3.1 kb data sets  and Watterson’s θW per site for all brown bears were (1.5 ± 0.1) × 10−4 and (3.0 ± 1.2) × 10−4, respectively. None of the four neutrality indices for the 5.3 kb data set was significantly different from the expectation under neutrality, nor did paternal brown bears experience a recent population expansion or contraction: Tajima’s D = −1.19 (P > 0.10), Fu and Li’s D = −1.95 (P > 0.05), Fu and Li’s F = −2 (P > 0.05), Fu’s Fs = −3.548 (P > 0.05).Nucleotide diversity  than when it was included in the North American  or Hokkaido  groups (Table P < 0.001), and the differentiation was more pronounced. The AMOVA results indicated that the Hokkaido group was highly differentiated from the Eurasian Continent (including Etorofu) and North American groups. There was no genetic connectivity between the Hokkaido group and the continental groups. In addition, there was no substantial population differentiation among the central, eastern, and southern Hokkaido populations.Hierarchical analyses by AMOVA were implemented for various geographical partitions of brown bear populations and groups , but least differentiated from the East Asia population (RST = 0.18) , whereas the latter two were not differentiated from one another (RST = 0.04).Although Etorofu Island is geographically close to Kunashiri and Hokkaido islands, it was significantly differentiated from the central, eastern, and southern Hokkaido populations (8) Table . The thrRST values between the North American and Eurasian Continental populations were lower than those between the North American and Hokkaido populations, or between the Eurasian Continental and Hokkaido populations.Excluding the Etorofu population from the Eurasian Continental group, Ne) for paternal lineages based on Y-linked SNPs in the 3.1 kb data set and six Y-linked microsatellites (Table BATWING was used to estimate the time to the most recent common ancestor (TMRCA) and effective population size  Table . In contWe detected one major haplotype BR1_05) throughout Hokkaido Island  from across Hokkaido was genotyped  apparently originated from eastern Hokkaido . Contrarped Fig.  and 3. Tped Fig. . In the Y-chromosomal DNA was genotyped for ten individuals from Etorofu, of which four had been included in an analysis of complete mtDNA sequences ; three oBrown bear populations in the Ural Mountains and Kamchatka Peninsula had higher haplotype variation than other populations, but neither population showed a clear relationship between genetic relatedness and geographical location. Western Asia, including the Ural Mountains, had the highest index values for paternal genetic diversity in our study Table , whereasBrown bears on Hokkaido and the adjacent southern Kuril Islands experienced different maternal and paternal evolutionary histories, demonstrating that the phylogeography in this region is considerably more complicated than would be expected from mtDNA studies alone. The weak spatial structuring of paternal lineages detected on Hokkaido appears to have resulted from extensive, continual gene flow via male dispersal among natal populations after the last population immigrated into Hokkaido from eastern Siberia via a land bridge during the last glacial period. The paternal genetic structure did not show distinct allopatric lineages, as have been observed for maternal (mtDNA) markers. While there were indications of heterogeneous male-mediated gene flow among populations on Hokkaido, it is unclear how much this influenced the connectivity and the maintenance of the local populations. Our results indicate that sex-biased dispersal has played a significant role in the evolutionary history of the brown bear in continental populations and in peripheral insular populations such as on Hokkaido, the southern Kuril Islands, and Sakhalin. Biparentally inherited autosomal DNA and whole-genomic data could further clarify the detailed demographic history and local adaptation of Asian brown bears.Additional file 1: Figure S1.Figure S2. Median-joining haplotype network for brown, polar, and American black bears, based on the 5.3 kb Y-linked data set. Figure S3. Median-joining haplotype network for brown bears, based on Y-chromosomal compound haplotypes combined with Y-linked SNPs from a 3.1 kb data set and Y-linked microsatellites. Haplotypes enclosed by a dashed line are from Hokkaido (including one Kunashiri brown bear). Figure S4. Map of Eurasia showing the geographical distribution of brown bear Y-chromosomal compound haplotypes. Each symbol represents one individual. Figure S5. Enlargement of the larger boxed area in Fig. S4, showing the geographical distribution of brown bear Y-chromosomal compound haplotypes around the Ural Mountains. (ZIP 4.95 mb)Median-joining haplotype network for brown, polar, and American black bears, based on the 3.1 kb Y-linked data set. Additional file 2:Table S1.Table S2. Analysis of molecular variance (AMOVA) for brown bear Y-chromosomal polymorphisms in various geographical partitions. Table S3. Analysis of molecular variance (AMOVA) for brown bear Y-chromosomal polymorphisms in various geographical partitions. Table S4. Analysis of molecular variance (AMOVA) for brown bear Y-chromosomal polymorphisms in various geographical partitions. (ZIP 63 kb)List of Y-chromosomal DNA compound haplotypes (YDNA haplotype), Y-chromosomal haplotypes based on only the 3.1 kb data set (YSNP_haplotype), Y-linked SNPs in the 3.1 kb data set, fragment sizes of six Y-linked microsatellites markers, and the number of individuals from each geographical regions having each Y-chromosomal DNA compound haplotypes.


Ucides cordatus is considered a key species for the ecological balance of mangrove forests and a major source of employment and income for traditional crab collectors in Brazil. Several studies evidenced weak genetic variation among populations due to an efficient larval transport. However, gene flow patterns of the species is poorly understood, with no information about migration rates. The influence of the two main Brazilian currents in larval dispersion is also not clear. In order to provide baseline information for conservation, planning and management of this important fishery resource, the present study aimed to estimate and evaluate spatial distribution of genetic diversity, migration rates and gene flow directivity among populations of U. cordatus in Brazil.The mangrove crab hA), isolation by distance tests, AMOVA, discriminant analysis of principal components (DAPC) and Bayesian clustering. We estimated the amount of ongoing gene flow between clusters using the coalescent-based method implemented in Migrate-N.Nine microsatellites were used to resolve population structure of 319 crabs collected from six sites located along the Brazilian coast. The degree of geographical differentiation included estimates of genetic diversity, population structure and gene flow models, with spatial analysis of shared alleles . FST and Jost’s D indexes were also estimated in pairwise comparisons and showed significant differences between most of the surveyed site pairs (P < 0.05). Structure evidenced a single genetic group among samples, however SAShA pointed to a non-panmictic condition (P = 0.011). AMOVA detected four statistical significant clusters with low level of differentiation . The gene flow model that best described the population connectivity was the island model, with ∼24 crabs being exchanged among localities per generation.Loci were highly polymorphic  evidencing high genetic variability. There was significant differentiation among localities, despite of the low value of U. cordatus. Despite the high gene flow and the weak population structure among samples, the significant genetic differences found suggest that gene flow alone does not bypass the effects of genetic drift, natural selection and/or human exploitation. These findings are vital for the establishment of a database to be used in the development of conservation programs.The high migration rates found among localities seem to be the main force acting to sustain the distribution of the genetic diversity of  Ucides cordatus is one of the most typical inhabitants of mangrove ecosystems in Central and South Americas  and has been extensively studied in Brazil. Its high fecundity are likely to cause direct impact on the species ecology and conservation. Recorded values of fecundity per female ranged from 36.081 to 250.566 eggs  while felinities . Studieslinities , which ilinities , triggerlinities . Adults linities .U. cordatus using molecular markers as Random Amplified Polymorphic DNA (RAPD), Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) . Even though, no quantitative information on the gene flow magnitude or its possible directivity were evaluated until now. Bayesian analysis with Markov chain Monte Carlo methods can be used to provide reliable gene flow estimates without the unrealistic assumption of symmetry of migration rates or equal population sizes  , controlCR-RFLP) , and IntCR-RFLP) . These sCR-RFLP) . More recordatus  evidenceon sizes . A methoU. cordatus genetic diversity along the Brazilian coastline, including estuaries under the influence of the NBC and Brazil Current (BC), two major oceanic circulation systems flowing in opposite directions, away from each other; (ii) test different models of migration to estimate larval dispersal directivity; and (iii) estimate the migration rates among localities. A new perfect tetranucleotide microsatellite locus is also reported for the assessment of genetic variability among U. cordatus populations.Due to the high ecological and social value of this species, a more extensive survey coverage also needs to be undertaken, particularly in estuaries under the possible influence of the North Brazil Current (NBC) . OliveirFrom 2006 until 2008 a total of 319 crabs were collected from six different estuaries as depicted in FJ483820–FJ483827)  extraction of Sodium Dodecyl Sulfate (SDS)/proteinase-K digested tissue of each individual . High moJ483827)  were useicro-Checker 2.2.3  and allelic richness per locus (RA) were estimated by Fstat 2.9.3 (ervus 3.0 (OH and EH) and exact tests for departure from Hardy–Weinberg expectations (pHWE) were performed using Genepop , θ (FST) and f (FIS) according to enAlEx 6.503 ) and coastline geographical distances   . Observerlequin 3.5  vs. NBC-2 (CUR and SOU).Patterns of population structure were first tested by AMOVA, using Aquin 3.5 , with 1,quin 3.5 . Thus, Famova 2.0 software . The optimal number of clusters (k-means) were estimated using different clustering solutions that were compared using Bayesian information criterion (BIC). The groups formed with samples from the different sites were then organized in a table whit rows corresponding to actual groups (“sample sites”), while columns correspond to inferred groups. Afterwards the “dapc” function was used and a new PCA was performed. At this time, the number of components representing at least 85% of the total variance of the data were chosen and the number of eigenvalues to be followed in the discriminant functions were defined. The results were showed graphically in a scatter plot with each point corresponding to a specimen.The R package Adegenet  was empligrate 4.2.14 (FCT results (see AMOVA above), where localities belonging to the same group (P < 0.05) were pooled together and analyzed as a single population. Afterwards, the following models of migration with their respective assumptions were tested:Directional migration 1: gene flow follows preferentially from North to South Brazilian coast;Directional migration 2: gene flow follows preferentially from South to North Brazilian coast;Directional migration 3: gene flow follows BC (southwards) and NBC (northwards) direction starting from the bifurcation of these main currents;Stepping stone model: there is symmetrical migration among neighbors and no direct migration among non-neighbor localities;Island model: there is a constant gene flow among each sample site;Panmixia: there is a single and huge population with random mate.Me 4.2.14  was usedigrate notation for each model is presented in results section following the number of groups established by AMOVA. Replicates were run using the microsatellite model in a single step method with constant mutation rate and starting parameters based on FST calculations. Prior distribution was set to be uniform for the mutation-scaled effective population size Θ (= 4eNμ) and mutation-scaled migration rates M (= m/μ). Runs were replicated with different random seeds four times to ensure convergence. The effective number of migrants (emN) was estimated by the relation emN = (Θ × M)/4. Calculation was performed with a burn-in of 10,000 iterations for each chain and one long chain with 100 increments and 10,000 recorded steps in each chain. Thermodynamic integration  was determined with Bezier approximated score after set a static heating scheme with four chains with temperatures 1.00, 1.50, 3.00 and 500,000.00. A matrix with the pairwise linear distance (in km) between each sample sites was used in the analysis, allowing migration rates to be scaled to by mutation rate and by these distances. Each proposed model was run ten times to check the standard deviations in marginal Likelihood and ensure reliability of estimates.The MNatural log Bayes factors (LBF) and the model with the best statistical support (higher probability) were calculated as suggested by U. cordatus ranged from 0.349 (locus UcSSR-04) to 0.946 (locus UcSSR-06), whereas expected heterozygosity ranged from 0.440 (locus UcSSR-01) to 0.930 (locus UcSSR-06). Overall mean observed heterozygosity for all nine loci (0.660) was lower than the mean expected heterozygosity (0.679). With the exception of locus UcSSR-04, all microsatellites conformed to Hardy–Weinberg expectations. The significant deviation from HWE (pHWE < 0.001) was interpreted as an indication of null alleles with an occurrence probability of 20.1% for this locus  alleles per locus . The PICis locus . When thFST) over all nine loci varied little with or without UcSSR-04 , showing low level of differentiation among localities. Estimates of endogamy overall samples (F) and within localities (f) were lower when this locus was excluded from analysis .Multilocus estimate for θ  .Pairwise = 0.021) . Mantel Regardless of these findings, pairwise comparisons of the genetic distances showed statistical significant differences among several localities. The southernmost sample (CAN) showed significant genetic differences from all other localities. As well, samples from FOR were statistically different from most other localities, with exception to its neighbor CAU. Neighboring localities CUR and SOU have also not shown significant differences . CAR samha, which has shown a non-panmictic condition (P = 0.011), albeit with a small difference between the observed mean distance distribution of alleles  when compared with the expected mean distance distribution .The assumption of panmixia events was statistically discarded after running SASFST = 0.019; P < 0.001). Considering the variation between BC and NBC groups, no differences were found , as well as for comparisons among BC vs NBC-1 vs NBC-2 . However, Samova results suggested an alternative grouping arrangement with significant differentiation for samples organized in four groups, named hereafter as: BC-1 (“CAN”); BC-2 (“CAR”); NBC-1 (“FOR and CAU”); NBC-2 (“CUR and SOU”). Following this indication FCT value remained low, but was significant .AMOVA has shown that only 0.71% of the total genetic variance is explained by differences among sample sites, although the variance was significant (tructure suggested lack of genetic differentiation among Brazilian samples with one single genetic group being detected (Posterior probabilities ≈ 100% for K = 1). However, another clustering solution suggested by DAPC analysis estimated the occurrence of six genetic groups  were used. After testing different migration models among these clusters, the most probable one was defined as the island model, with ≈ 100% of probability . Other t−5) and mutation-scaled historical migration rates (M) were 975.59 (s.d. = 0.974). Considering the island model, which assumes a symmetrical migration rate, the estimated indexes (and M) must be considered the same for all pair of population compared. Thus, a single value of effective number of migrants exchanged per locality was defined (23.94 crabs per generation).Mean estimate of Θ after ten runs was 0.098 . Among all loci, only UcSSR-04 was not in Hardy–Weinberg equilibrium (HWE), which may be due to the occurrence of null alleles and therefore, heterozygous individuals would have been scored as homozygous causing HWE deviations . In both situations estimates showed low levels of genetic differentiation among localities and no evidence of population structuring. Low levels of inbreeding in the species was detected after F and f calculations. Linearized pairwise FST and Jost’s D have also evidenced weak genetic differentiation among localities, in agreement with θ estimates and with previous results  when localities were arranged as BC-1, BC-2, NBC-1 and NBC-2, despite of the low FCT (0.037). SASha results also pointed to a non-panmictic situation for U. cordatus crabs, albeit with a small difference between the observed mean distance distribution of alleles (2055.0 km) and the expected mean distance distribution (2079.7 km).High gene flow between estuaries due to species pelagic larval export strategy is a plausible explanation for our findings. In numerous crab species with similar dispersal strategy larvae are thought to be transported up to 100 km seaward from its spawning location . Our stuigrate have also not suggested a scenario with completely free exchange of alleles among all sampling localities, and panmixia model was as well discarded. These two current branches run in opposite directions along the Brazilian coast (U. cordatus specimens) could potentially be transported over several 100 km in only one month. However, there is no evidence to support the main Brazilian currents (BC and NBC) as having a strong barrier effect on larval dispersion.The gene flow models run in Man coast , and laran coast  and NBC an coast , hence oU. cordatus migration patterns was the island model. The Bayesian approach of Migrate allows the estimation of gene flow without the assumption of symmetric migration rates or equal population sizes  rather than sample size (N)  . In addiD, FST and FCT) demonstrate that migration rates are actually not counterbalancing the loss of diversity due to genetic drift or selection events. Therefore, the reported levels of exploitation (anthropic selection) may promote a negative impact on populations  or human exploitation, which is excessive and should be taken into account for designing species conservation programs. Statistical models suggest that migration events occur following island model, with symmetrical rates of migration among locations. Thus, the connectivity observed between crab populations evidenced the importance of a cooperative work over a wide geographic range aiming at the conservation and management of this fishery resource.Thus, even with crab populations presenting homogeneous genetic distribution along the Brazilian coast and with high migration rates, the significant differences found among some localities cannot be rejected. 10.7717/peerj.4702/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.4702/supp-2Supplemental Information 2http://genepop.curtin.edu.au).The genotypes (raw data) used in the analyzes performed to calculate the estimates used in the manuscript. The data is distributed in the GENPOP software format (Click here for additional data file.


FST distributions with those of the 1000 Genomes Project and commercial arrays. Although all arrays yielded similarly shaped (inverse J) FST distributions, DREAM’s autosomal and X-chromosomal distributions had the highest mean FST, attesting to its ability to discern subpopulations. DREAM performances are further illustrated in biogeographical, identical by descent, and copy number variation analyses. In summary, with approximately 800,000 markers spanning nearly 2,000 genes, DREAM is a useful tool for genetic anthropology, forensic, and personalized medicine studies.The human population displays wide variety in demographic history, ancestry, content of DNA derived from hominins or ancient populations, adaptation, traits, copy number variation, drug response, and more. These polymorphisms are of broad interest to population geneticists, forensics investigators, and medical professionals. Historically, much of that knowledge was gained from population survey projects. Although many commercial arrays exist for genome-wide single-nucleotide polymorphism genotyping, their design specifications are limited and they do not allow a full exploration of biodiversity. We thereby aimed to design the Diversity of REcent and Ancient huMan (DREAM)—an all-inclusive microarray that would allow both identification of known associations and exploration of standing questions in genetic anthropology, forensics, and personalized medicine. DREAM includes probes to interrogate ancestry informative markers obtained from over 450 human populations, over 200 ancient genomes, and 10 archaic hominins. DREAM can identify 94% and 61% of all known Y and mitochondrial haplogroups, respectively, and was vetted to avoid interrogation of clinically relevant markers. To demonstrate its capabilities, we compared its  The field of population genetics experienced astonishing changes over the past 50 years, generating a new understanding of variability at the molecular level that has allowed for the exploration of new biological paradigms. Over the past decade, this turmoil was driven by the wide availability of single-nucleotide polymorphism (SNP) microarray and next generation sequencing (NGS) data, which raised major questions concerning human early origins, interbreeding with archaic hominins, and the processes that shaped inter- and intrapopulation variability.Such questions are also the core components of forensic DNA phenotyping. Common forensic and mass disaster scenarios alongside accumulated criticism of eyewitness testimonies necessitated the development of more accurate and reliable DNA-based forensics tools based on short tandem repeats (STRs) extracted from minute DNA amounts . The a gInterest in human ancestry is not limited to genetic anthropologists, genealogists, and forensic experts. The relatedness of adaptations to diseases is instrumental to identify targets for drug treatment (Sheridan 2015). The appreciation that demographic histories, geographical origins, and migration patterns shaped the genetic risk to disorders and treatment response  underlieSince NGS technologies remain prohibitively expensive, microarray SNP technology became the “workhorse” for geneticists, although they are limited in a number of ways. First, genotyping data are susceptible to ascertainment bias due to the choice of SNPs . AlthougMotivated by progress in the studies of modern and ancient genetic diversity, adaptation mechanics, forensic phenotypes, and drug response, we aimed to design an affordable and all-inclusive microarray. Our goals were to: 1) design The Diversity of REcent and Ancient huMan (DREAM)—a state of the art SNP microarray dedicated to genetic anthropology and genealogy, forensics, and personalized medicine; 2) validate its accuracy; 3) evaluate its abilities to discern populations compared with alternative arrays; and 4) assess its performances on worldwide populations.Ancestry informative markers (AIMs) were obtained from the same 15 studies as listed in http://genome.ucsc.edu/; cited 2017 Jul 16) using the Burrows Wheeler Aligner (BWA version 0.7.15)  was then used for MarkDuplicates to remove reads with identical outer mapping coordinates (which are likely PCR artifacts). The Genome Analysis Toolkit RealignerTargetCreator module (GATK version 3.6) (Ancient DNA genomic data were obtained from 11 publications depicting 207 ancient genomes . In the  0.7.15) , allowin 0.7.15) . Picard ion 3.6)  was usedion 3.6)  were useion 3.6) . We usedion 3.6)  and Deniion 3.6) .To cross-validate DREAM’s genotypes, we genotyped 139 individuals from 17 worldwide 1000 GP populations including: Americans of Mexican ancestry (Los Angeles), Bengali (Bangladesh), British (England and Scotland), Caribbean Africans (Barbados), Colombians , Esan (Nigeria), Finnish (Finland), Gambian , Han Chinese , Iberian (Spain), Indian Telugu (UK), Italians , Kinh , Mende (Sierra Leone), Peruvians , Punjabi , and Yoruba . Genotypes were produced following the Axiom Best Practices Genotyping Analysis Workflow , which eMaternal and Paternal haplogroup calling was done using an internal haplogroup calling algorithm developed by the Genographic Project, as in FST based on 1000 GP phase 3 data  and e 3 data  providede 3 data . Calculae 3 data   correction for multiple tests  coverage obtained by each microarray. IBD varies by individual, population, proportion of rare alleles, and number of SNPs. For that, we assembled an autosomal data set by randomly selecting 30 individuals from three 1000 GP populations (phase 3) that have the same proportion of rare alleles (MAF < 0.5%) . Analyses were carried out using only the autosomal SNPs of each microarray. For each individual, we retained the average IBD with all individual of the same population. We then calculated the mean and standard deviation per population and divided them by the number of SNPs of the microarray.2r) using the PLINK (2r for each variant pair with two variants between them at most (ld-window = 2) without filtering low 2r values (window-r2 = 0).Finally, we compared the linkage disequilibrium (LD) patterns between the microarrays. For that, we used the 1000 GP (phase 3) data set. We randomly selected 30 individuals from four populations: Yoruba , Finnish (Finland), Japanese , and Puerto Ricans (Puerto Rico). We then analyzed the SNPs sequenced in those populations that were included in each of the five genotyping arrays: DREAM  HumanOmni5 , HumanOmni2.5 , Infinium Multi-Ethnic Global , and Human Origins . Lastly, we calculated the LD statistic . Using BCFtools , we mergWe applied Refined IBD implemented in Beagle version 4.1 (21Jan17.6 cc) with the reference human genome  to both reference populations known to have resided in a certain geographical region for a substantial period of time. GPS then converts the genetic distances between that individual and the nearest M = 10 reference populations into geographic distances. The reference populations can be thought of as “pulling” the individual toward their location in a strength proportional to the similarity of their admixture components until a “consensus” is reached  following  reached .k-means clustering and multiple pairwise F tests on populations with pN>4, where pN is the number of individuals within a population. For k = 2 to k =p N/2, we used k-means to identify k clusters and then the ANOVA F test to test whether cluster pairs are significantly different (P < 0.05). If the hypothesis is verified for all the pairs at iteration i, then another iteration follows until at least one pair violates the hypothesis and i-1k is the optimal number of clusters. Populations and subpopulations displaying only one individual were discarded from the data set. The final data set consisted of 587 individuals grouped into 123 subpopulations from 33 countries  graph G based on the 9 admixture components  as the basic clusters. If noise or overlap exists, outlier nodes are computed as a subset of the critical attack nodes, which form the cluster boundaries in G. We used integrity as the node-based resilience measure to cluster G due to its noted robustness when the number of ground truth clusters is not known a priori. Our integrity-based graph clustering results in eight clusters, each corresponding to a different color in the figure. The graphs are visualized using the Gephi 0.9.1 graph visualization program : BC(v) of a node v is the sum over all pairs of other nodes x, y, of the ratio of the number of x–y shortest paths that go through node v to the total number of x–y shortest paths. As BC(v) measures the extent to which v lies between other nodes (as well as between multiple clusters), larger nodes are intermediate to more pairs of graph nodes than smaller nodes in the visualization. Thus, the highest betweenness, larger nodes tend to lie on the boundaries between clusters in the NBR-Clust framework, representing outliers in terms of cluster overlap or noise. As such, we hypothesize that the largest, that is, highest betweenness centrality, nodes represent individuals with higher levels of admixture with respect to the clusters to which they are adjacent.The different sizes of the nodes (and node labels) were created using the To infer CNVs, we applied the Axiom CNV Summary Tool  to the 1PlotGenome script  was designed as an Applied Biosystems Axiom custom array. The Axiom genotyping platform utilizes a two-color ligation-based assay using 30-mer Oligonucleotide probes synthesized in situ onto a microarray substrate. There are ∼1.38 million features (or cells) with each SNP feature containing a unique 30mer oligonucleotide sequence complementary to the sequence flanking the polymorphic site on either the forward or the reverse strand. Depending on the 3′ (SNP-site) base , solution probes bearing attachment sites for one of two dyes are hybridized to the target complex, followed by ligation for specificity. DREAM was designed with 809,781 oligonucleotide sequences complementary to the forward or reverse strands (probesets) that interrogate 799,120 markers (SNPs or InDels). The following sections detail how markers were selected to enable ancestry, genealogy, forensics, and personalized medicine applications.AIMs are invaluable tools in population genetics and genetic anthropology as they allow the identification of populations that vary in substructure, quantification of the degree of admixture, and detection of subtle population subdivisions using a limited number of markers . We collAncient DNA from sequence or genotype data allows direct observations of past admixture and migration events and is often the only evidence that allows the examination of historical hypotheses. As such, ancient DNA studies have provided insights into human evolution and migration . We curated genetic data from over 200 ancient genomes  online. To facilitate studies on the extent of gene flow from Neanderthal and Denisovan populations to modern humans, we included SNPs from multiple low-coverage genomes while restricting the selection to markers validated by the 1000 GP. As such, we randomly selected 1,000 and 3,000 SNPs for Denisovans 4 and 8, respectively, and 5,000 SNPs from six Neanderthals . Overall, we selected 78,724 markers , 12,550 of which were culled from archaic hominin genomes.FADS1 and FADS2 . Following previous mapping efforts and based on the literature published over the past 5 years, we constructed a comprehensive list of adaptive traits and curated variants and genes that are significantly associated with those traits. Genes significantly associated with adaptations of interest were recorded and included in the design  for DNA phenotyping. Following previous studies  and baseCNVs have contributed significantly to hominid evolution , biodiveL > 10,000 bp) or shown to differentiate populations , Hypothyroidism , cholesterol , platelets, and red and white blood cells , whose data are associated with human genetic variation in drug responses  ∼75% of, whose god cells  were alsand BEST  and nearand BEST .Studies of sex bias in human admixture, migrations, and kinship analyses typically require a high coverage of the X chromosome. We thereby enriched the X chromosomes with SNPs selected uniformly throughout the genome. We prioritized SNPs that had Applied Biosystems Axiom confirmed probes and those that are targeted by Illumina’s HumanOmni5 array. Overall, 50,265 SNPs were selected.Of particular importance is the major histocompatibility complex (MHC) locus involved in autoimmune and infectious diseases. The MHC region is the most gene-dense region in the human genome. However, the high density in polymorphisms and linkage disequilibrium have limited our understanding of its role. To facilitate further research of this locus, we included SNPs for which Applied Biosystems Axiom had confirmed probesets and that reside within the 4 M bp of the MHC. Overall 16,434 SNPs were selected.To enable further research into traits of interests, we targeted SNPs that reside within or in the 100 kilobases flanking regions of the genes of interest. We used STRING  to find figs. 2 andTo enable cross-platform kinship analyses, we selected ∼230,000 SNPs distributed uniformly throughout the genome that had Applied Biosystems Axiom confirmed probesets.Finally, we interrogating over nearly 14,000 markers to identify SNPs defining Y and mtDNA haplogroups  online.We excluded most of the SNPs that required four probesets or more unless they were vital to call haplogroups. To improve coverage, we prioritized SNPs that required a single probset over those that required two. We also filtered out all the markers that were recorded in the ClinVar database   X, 13,576 Y-chromosomal, and 1,172 mitochondrial markers without clinical relevance. The design spans over 1,903 genes  online eGenotype accuracy was assessed by genotyping 139 individuals from 17 worldwide populations found in the 1000 GP data  and cross-validating them with the 1000 GP data. About 100% (139/139) of the samples passed sample QC, and 97.5%  of the markers passed marker QC. For autosomes that passed marker QC, the concordance rate was 99.70%  and the total marker call rate was 99.70% . For the nonPAR X markers, the concordance between the genotypes from the 46,020 markers  and the 1000 GP phase 3 genotypes was calculated as 99.76% . For the Y chromosome, the concordance between the genotypes from the 7,745 markers (part of the 1000 GP phase 3) and the 1000 GP phase 3 genotypes was 99.59% . For the mtDNA markers, the concordance between the genotypes from the 859 markers (included and part of the 1000 GP phase 3) and the 1000 GP phase 3 genotypes was calculated as 99.84% . Overall, we confirmed that nearly all the genotypes captured by the DREAM array are accurate.The SNP density across all chromosomes is shown in DREAM’s potential to assist in ancient DNA studies was evaluated by calculating the number of ancient DNA genotypes for each ancient genome  online. DREAM’s ability to infer uniparental haplogroups was computationally assessed against the respective trees. DREAM markers identified 94% and 61% parental and maternal haplogroups, respectively . All theCompared with whole genome data, allele frequencies (AF) in microarrays are typically shifted toward intermediate frequency levels , which lFST is a measure of differentiation whereby the genetic variation of the subpopulation is measured relative to the total population  than DREAM’s due to the high fraction of rare SNPs in these arrays. The magnitude of the differences between the FST values of these arrays was also large for autosomal  and X-chromosomal SNPs . These results suggest a reduced ability of the competing arrays to elucidate ancient demographic processes  would consist of a large number of SNPs in high LD. A comparison of the LD patterns of SNPs from the four 1000 GP populations, which overlapped with each of the five microarrays showed, that the Human Origins microarray had the smallest fraction of high LD markers followed closely by DREAM  online. NeanderthalN = 450, Neanderthalr = 0.75, DenisovanN = 450, Denisovanr = 0.91) and exhibit similar between-population patterns in the two data sets.DREAM’s ability to infer IBD with archaic hominins was evaluated by comparing the total IBD between worldwide individuals, Neanderthal, and Denisovan calculated using the complete 36 million SNPs (1000 GP data set) and DREAM SNPs, representing 1.86% of the complete data set  online. reference populations known to have resided in a certain geographical region for a substantial period of time  tool, which matches the admixture proportions of a test individual with those of  of time . The effG (betweenness centrality represent genetic mixtures with populations graphically adjacent to them, we created a Ppopulation graph G (G. Here too, nodes with notably high betweenness are the Bermudian, Tatarstan-Russian, Puerto Rican, Lima-Peruvian, North-Northeast Indian, and Antananarivo-Madagascan populations. These enlarged nodes lie on cluster boundaries. For example, the Madagascan node with high betweenness is adjacent to the Oceanic, East Asian, and his own African cluster, in support of recent reports of shared ancestry (Individual clustering by admixture proportions is an effective way to describe population structure  and evalG  online wgraph GP  online bancestry . North Iancestry . Our finFST distributions of the DREAM array to those of the 1000 GP and commercially available arrays, we demonstrated DREAM’s ability to differentiate populations within global data sets. Lastly, we demonstrated the biogeographical accuracy of DREAM and its potential ability to infer CNVs. We expect that the use of the DREAM in genealogy and research will expand our knowledge of our species. We designed, developed, validated, and assessed the DREAM microarray, an all-inclusive SNP genotyping chip dedicated to genetic anthropology and genealogy, forensics, and personalized medicine. DREAM can be used to study the genetic relationships between ancient humans, archaic hominins, and modern humans as well as to improve our understanding of human migratory history, adaptations, and the molecular mechanisms that regulate forensic-relevant traits. By comparing the MAF and Genome Biology and Evolution online.E.E. initiated the study and designed DREAM with Y.L. and T.W. M.L.B. supervised the work. E.E., L.Y., A.A., M.H., D.A., G.V., G.E., and U.E. carried out the analysis. E.E. and L.Y. wrote the manuscript. All authors approved the manuscript.Supplementary Figures and TablesClick here for additional data file.


Bactrocera correcta is one of the most destructive pests of horticultural crops in tropical and subtropical regions. Despite the economic risk, the population genetics of this pest have remained relatively unexplored. This study explores population genetic structure and contemporary gene flow in B. correcta in Chinese Yunnan Province and attempts to place observed patterns within the broader geographical context of the species’ total range. Based on combined data from mtDNA cox1 sequences and 12 microsatellite loci obtained from 793 individuals located in 7 countries, overall genetic structuring was low. The expansion history of this species, including likely human-mediated dispersal, may have played a role in shaping the observed weak structure. The study suggested a close relationship between Yunnan Province and adjacent countries, with evidence for Western and/or Southern Yunnan as the invasive origin of B. correcta within Yunnan Province. The information gleaned from this analysis of gene flow and population structure has broad implications for quarantine, trade and management of this pest, especially in China where it is expanding northward. Future studies should concentrate effort on sampling South Asian populations, which would enable better inferences of the ancestral location of B. correcta and its invasion history into and throughout Asia. Bactrocera correcta (Bezzi) (Diptera: Tephritidae), the guava fruit fly, is one of the most destructive pests of many tropical and subtropical fruits and vegetables, such as guavas, mangoes, citruses, melons and chili peppers, causing production losses and quality degradation131Bactrocera correcta was first reported in Bihar, India in 1916791011B. correcta was first discovered in the Yunnan Province (Yuanjiang and Mosha) in 1982B. correcta (Bezzi), which first entered China through YunnanB. correcta is capable of establishment across the whole of ChinaB. correcta in China, its economic importance, and the risk of this species being introduced, establishing and invading other regions of China argue for greater understanding of population relationships and invasion routes.Yunnan is located in southwest China, adjacent to Myanmar, Laos and Vietnam, from which entry of exotic species into Yunnan has been facilitated by similar environmental conditionscox1) gene is easily amplified162023Bactrocera fruit fly species162526Information regarding genetic diversity, genetic structure and gene flow are key issues when developing management strategies19B. correcta B. correcta is expanding northward in China via gradual movement from adjacent populations or if new, adventive northern populations are derived from further away. Likewise, we seek to determine whether any locations within Yunnan Province show signs of having been the original invasive location in China and from which the invasion progressed. Additionally, we incorporate limited public data from South Asia  to address hypotheses concerning this region as the putative ancestral location of the species. Taken together, this study provides foundational data for understanding the population dynamics and genetic structure of B. correcta, which will contribute greatly toward development of control measures for this fly in Yunnan.Here, we assess population genetic structure within Yunnan Province via dense geographical sampling to determine patterns of gene flow and dispersal within China. We then place these patterns in a broader geographical context by sampling other locations across the species’ distribution. In doing so, we build on previous localized evidence concerning the genetic structure and invasion history of B. correcta were collected from 18 sites in China (Yunnan), Laos, Myanmar, Thailand, and Vietnam from 2008 to 2013cox1 sequences from GenBank for flies from India (accession numbers GU323781 and GU323782) and Sri Lanka Specimens of cox1 fragment was amplified and sequenced according to the method of Liu et al.cox1 sequence from each individual were assembled using DNAMAN 5.2 . To delete low-quality sections, all sequences were aligned with the standard sequences of B. correcta from BOLD using Clustal Xcox1 sequences. Unique sequences were deposited in GenBank with accession numbers KU669296–KU670076. Information about the twelve microsatellite loci and amplifying methods analyzed hereGenomic DNA was extracted from each specimen using the Tissue/Cell DNA Mini Kit . A 658 bp cox1 data, the nucleotide composition and variable positions were visualized using MEGA 6π), haplotype diversity (Hd) and number of haplotypes for each geographical population of B. correcta were estimated using DNASP 5.1For NA), number of effective alleles (NE), observed heterozygosity (HO), expected heterozygosity (HE) were calculated each population using POPGENE 1.32For microsatellite data, the number of alleles TM Earth 5.2B. correcta, the correlation analysis results were plotted in SPSS v16.0 .Pairwise cox1 haplotypes were inferred using a haplotype network, constructed under the median-joining (MJ) method in NETWORK 4.6B. correcta. We set the number of clusters (K) from 1 to 10 and conducted 10 independent runs for each value of K. Each run consisted of a burn-in period of 50,000 steps, followed by 100,000 Markov chain Monte Carlo (MCMC) repetitions with a model allowing admixture. ΔK valuesFct value associated with the subdivision scheme by repeating the analysis with K ranging from 2 to 9. Hierarchical analysis of molecular variance (AMOVA) was performed using Arlequin 3.5 to evaluate the distribution of molecular variance among groups, populations and individuals for both types of markers based on the grouping strategy from STRUCTURE and SAMOVA.Evolutionary relationships among cox1 sequences from 20 populations (including India and Sri Lanka) were examined using mismatch distribution and neutrality tests in Arlequin 3.5 with 1000 bootstrap replicates. Six parameters were calculated: effective population size before expansion (θ0), effective population size after expansion (θ1), Time of populations expansion (T), Tajima’s D, Fu’s FS and sum of square deviation (SSD) between expected and observed mismatch distribution.The demographic history analysis of all B. correcta, this program computes the probability that it belongs only to that population, the probability of being a migrant from each of the other populations and the probability of being a migrant to other populations16The GENECLASS v2.0 programB. correcta from 18 sites across China and Southeast Asia. Inclusion of 12 additional cox1 sequences from GenBank from India and Sri Lanka, produced a final alignment of 600 bp for 793 individuals. The A + T content was 63% (28.8% A and 34.2% T), higher than the G + C content (16.5% G and 20.5% C). This nucleotide composition is similar to B. dorsalisB. cucurbitaeB. correcta. Of these, 30 haplotypes were shared by at least two populations (47.62%), with the most frequent haplotype H5 present in 17 populations. The number of haplotypes for per population ranged from 1 to 22  while VIET was the lowest (Hs = 0.444) followed by YNHH (Hs = 0.617)  .FST values . Microsatellites by contrast suggested that most pairwise comparisons were significantly different; however, actual FST values were generally lower than 0.1, suggesting low population structure. An exception was the YNHH population, which was supported as more different to all other sites, with FST estimates ranging from 0.1 to 0.149, average 0.122. Mantel tests showed no significant relationship between genetic (FST/(1-FST)) and geographical distances. , indicating the absence of IBD    . AMOVA a < 0.01) .cox1 dataset produced significant negative Tajima’s D and Fu’s FS values (SSD > 0.05). Ratios between estimated effective population size after expansion (θ1) and effective population size before expansion (θ0), which can serve as an estimate of the extent of population growth, indicated that B. correcta exhibited a certain degree of population growth in all the populations  to 0.727 (TCHM into VIET). In these analyses, migration rate estimates below 0.100 imply restricted gene flow, whereas moderate to high gene flow is suggested by values greater than 0.100. Interestingly, the migration rates from the YNHH and VIET populations to other populations were all below 0.100, except for YNHH into VIET (m = 0.122). Meanwhile, estimated migration rates were also very low between BURM and other populations. The probability with which individuals were assigned to their own populations varied from 0.344 (YNWS) to 0.501 (BURM)  .B. correcta in Yunnan Province, China, and neighboring countries. Our aim was to examine population structure and gene flow within Yunnan and to place Chinese diversity and structure in a regional context.Understanding population structure and gene flow among regionsare very important aspects in the management of quarantine fruit flies. In this study, we obtained data from both mitochondrial and nuclear DNA markers of an extensive sampling of The clearest signal of population structure across the sampled locations supported the population YNHH, from eastern Yunnan, as being significantly structured from all other populations. This pattern was exemplified by low levels of estimated gene flow between this site and all others, and manifests in this site being supported as a separate genetic entity in Bayesian clustering analysis of microsatellite genotypes. Yunnan Province is characterized geologically as a longitudinal range-gorge region, where the mountain chains tend to run south to north, potentially blocking the spread of mobile insects eastward4B. dorsalis16Elsewhere across the sampled distribution, there were a small number of locations that were supported as significantly structured. Firstly, Myanmar, situated southwest of Yunnan and west of Southeast Asia also exhibited some evidence for significant genetic differentiation from other populations. This pattern may be associated with significant mountain ranges and/or reduced trade with other Asian countries that act to limit direct and indirect gene flow between Myanmar and surrounding regions. Myanmar displayed a close relationship with adjacent western Yunnan and Thailand, and high gene flow was inferred from these locations into Myanmar, in contrast to minimal migration in the opposite direction. This pattern has also been described in the highly invasive species Fs indices, unimodle mismatch distributions and non-significant SSD values all supported the hypothesis of a sudden population expansion model. As B. correcta is a phytophagous and highly polyphagous fruit fly, presence of abundant hosts may aid its regional dispersal. Combined with highly frequent fruit trade between Yunnan and other Asian countries, this likely presents advantageous conditions for the long-distance dispersal of this fly355More broadly, resolved patterns are consistent with recent demographic expansion in this species. Significant negative Tajima’s D and Fu’s B. correcta was first recorded in southern Yunnan Province in 19825760B. correcta having established first in western Yunnan before expanding into other regions in China.Within China, B. correcta in the region. In particular, populations in southern and eastern Yunnan displayed lower genetic diversity than western populations. This suggests that these populations may be the result of gradual eastward migration from an initial invasion into western Yunnan. This is contrary to previous hypotheses that the region was an original entry point for B. correcta into China, which were based on its geographical proximity and horticultural trade relationships with neighbouring countries, along with the first detection of B. correcta in China occurring in southern Yunnan. Instead, this scenario finds little support in our data, although it cannot be conclusively excluded.Patterns across the rest of Yunnan Province shed some light on movement among populations and the potential invasion history of cox1 sequence data allows some comment on the hypothesis that Bactrocera species may have originated in India7cox1 network for South Asian locations. In marked contrast: and despite low sample size, there was high haplotype diversity (7 haplotypes from 12 sequences across both sites), with these being mostly internal to the network and often shared between many other locations. Thus, these data provide some evidence for these populations being more ‘ancestral’ than Southeast Asian populations, indicatingthat we cannot reject the idea that Bactrocera originated in India. The limited data for India and Sri Lanka restricts our deductive capacity. Further sampling will be required to determine the geographical origin of B. correcta and resolve patterns of gene flow and dispersal between India and other Asian countries.Our inclusion of Indian and Sri Lankan FST indices) were significant for microsatellites than for cox1. We argue that this is most likely driven by the faster mutation rate of the former, although it may possibly represent some degree of female-biased dispersal.We applied two sets of molecular markers to avoid any bias due to the use of only a single marker. Mitochondrial DNA corresponds to the maternal lineage, making it sensitive to selective neutrality, loss of mutation-drift equilibrium and male-to-female sex ratio balanceB. correcta, with exceptions in eastern Yunnan, Myanmar and Vietnam. We provide support for hypotheses of first entry of this species into China occurring in western Yunnan, with gradual dispersal eastward. Given current global warming trends, which will render more of northern China ecologically suitable for B. correctaIn conclusion, we provide evidence based on combined independent molecular markers that there is high gene flow among most Southeast Asian populations of How to cite this article: Qin, Y.-J. et al. Genetic diversity and population structure in Bactrocera correcta (Diptera: Tephritidae) inferred from mtDNA cox1 and microsatellite markers. Sci. Rep.6, 38476; doi: 10.1038/srep38476 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Haemonchus contortus populations can inform some aspects of this parasite’s population genetics and epidemiology. However, almost nothing is known about such variation in China.Studying genetic variation within and among H. contortus (n = 184) representing seven distinct populations in China were collected, and genetic variation within and among these populations was explored using eight distinct microsatellite markers.Adult males of FIS) indicated that all eight microsatellites were highly polymorphic. Various analyses  revealed high within-population variation, low population genetic differentiation and high gene flow for H. contortus in China.Genetic parameters, such as heterozygosity and inbreeding coefficient  contains supplementary material, which is available to authorized users. Haemonchus contortus, Haemonchus placei, Ostertagia ostertagi and Teladorsagia circumcincta ) and 30–50 ng of DNA template (except for no-template controls). The cycling protocol was: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 1 min, with a final extension of 72 °C for 5 min. Amplicons were examined on agarose gels (1.5%) to verify that they represented single bands and then column-purified  and sequenced directly (using primer NC2) in an automated DNA sequencer (ABI 3730).To confirm the identity of the nematodes as G CT-3′) . Briefly2, 250 μM of each dNTP, 100 pmol of each primer, 1 U of Taq DNA polymerase (TaKaRa) and 30–50 ng of DNA template (except for no-template controls). The cycling protocol was: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 50–60 °C  were selected based on previous publications , 18. PCRee refs. , 18) foree refs. , and anyHo and He), the number of alleles (A) and population inbreeding coefficients (FIS) for individual loci using the program GenALEx [Genetic characteristics were estimated by calculating observed and expected heterozygosities . All of these markers displayed a high degree of polymorphism, with the number of ‘alleles’ per locus ranging from 6 to 15 (Table Ho) ranged from 0.103 to 0.923, and the expected heterozygosity (He) from 0.237 to 0.848 .All 184 adult specimens of H. contortus populations. For some marker/population combinations, there was no significant departure from HWE after sequential Bonferroni correction, and FIS values were low (see Table Ho) was less than the expected heterozygosity (He), and FIS values were high  was undertaken to assess the suitability of the eight selected markers to assess genetic variability in the seven FST values were calculated to estimate the degree of genetic differentiation among the seven H. contortus populations using the eight microsatellite markers , ranging from 0.0024 to 0.0640, the population from Inner Mongolia showed the highest level of genetic divergence from other populations, with FST values ranging from 0.0352 to 0.0640  was greatest, and the peak of ΔK was highest  among populations was conducted , group 2  and group 3 (Inner Mongolia). Most (96.1%) of the genetic variation was distributed within populations: 3.3% of variance components among groups (FCT) and 0.6% of variance components among populations within groups (FSC). These findings suggest a very low genetic sub-structuring of H. contortus populations in China.Bayesian clustering analysis showed no obvious subdivision at the population level (results not shown). We explored K values between 1 and 10. For Evidence of sub-structuring was assessed by Principal coordinate analysis (PCoA) analysis Fig. . The twoH. contortus populations indicated that the genetic differentiation of these populations did not follow a pattern of isolation by distance   Fig. , indicatH. contortus populations showed that the populations from Inner Mongolia and Yunnan exhibited heterozygosity-excess, according to the IAM and TPM/SMM models of Sign test and the Wilcoxon signed-rank test  nucleotide sequence data [H. contortus populations in China.In the present study, we explored the population genetic structure of seven nce data  and reveH. contortus populations is in accordance with the previous studies of H. contortus [Teladorsagia circumcincta and T. tenuis [Deviation from HWE was observed for five of eight markers for some populations  from China, following Bonferroni correction, which is likely due to the presence of null alleles. As the repeated “genotyping” from the same samples gave consistent results, and individual samples that failed to amplify for one locus could be genotyped robustly for other loci, the null alleles might not relate to allelic drop-out or poor template quality; otherwise, the apparent ‘null alleles’ might relate to sequence variation at primer annealing sites and/or due to selection, mutation or genetic drift [ontortus  and othe. tenuis , 7, 35.H. contortus was supported by AMOVA analysis results, which showed that the majority (96.1%) of genetic variation was within H. contortus populations, suggesting a high level of gene flow among populations, and PCoA results indicated that no discernable geographical sub-structuring was evident over large distances in China ..H. contoH. contortus populations from sheep and goats were not apparent, showing that there is no reproductive isolation [H. contortus populations in China, however, the population from Inner Mongolia showed a higher FST value  of China, the main climatic characteristics are warm temperatures and high rainfall in summer. Otherwise, winters are cold (-20 to -30 °C), and summers are hot (20 to 30 °C) and dry, with limited rainfall throughout much of the year in temperate zones . Previous studies , 39 havepastures . Consequ to year . The Aut to year . The ecoH. contortus populations in China by microsatellite analysis were essentially in agreement with those of a previous study based on mitochondrial nad4 gene sequence data [H. contortus populations might be ascribed to a large effective population size and low rates of genetic drift [H. contortus populations on a global scale, supported by the studies employing both microsatellite markers and the nad4 gene sequence data [H. contortus populations.The results of the low level of genetic differentiation among the seven nce data . These fic drift , 4. Howence data , 18. AppH. contortus in China using microsatellite markers. The findings provide a snapshot of the genetic make-up of H. contortus populations in parts of China using these polymorphic markers, revealing high within-population variation, low population genetic differentiation and high gene flow for H. contortus. This study should provide a reference for assessing genetic change in H. contortus over space and time during or following the implementation of particular treatment or control strategies, or as a consequence of environmental, management and climatic factors.This is the first population genetic study of


AbstractPampusargenteus is an economically important fish that is often erroneously identified as Pampusechinogaster. No population genetic analyses have been performed on the true P.argenteus species. Here, the mitochondrial control region (CR) was used to evaluate the population genetics and elaborate the historical demography of the Silver pomfret collected from six geographical locations in China, Pakistan, and Kuwait. A high level of genetic diversity was demonstrated in this species. Analysis of molecular variance (AMOVA) revealed that the genetic divergence was mainly derived from within the populations (P < 0.05). A historical demographic analysis indicated that the Silver pomfret experienced a recent population expansion during the late Pleistocene. The phylogeographical structure revealed two obvious lineages that diverged in the late Pleistocene, during which the Silver pomfret populations historically experienced exotic divergence and mixed again with differentiated populations. Currently, Silver pomfret populations have insufficient time to attain migration-drift equilibrium. Population genetic data of the Silver pomfret can provide preliminary genetic knowledge for its fishery management. Pampusargenteus  is an economically important species that plays a vital role in commercial fisheries  are typically mistaken as the same species  and that P.argenteus was absent from these regions. Finally, P.echinogaster, which were significantly different from those of P.argenteus.The Silver pomfret isheries . Pampus species . HoweverP.argenteus is absent from the Yellow Sea, Bohai Sea, and Northern East China Sea and that the so-called “P.argenteus” referenced in previous studies  were analyzed.To date, no population genetic analyses have been reported based on the true bination . In thisIn total, 114 Silver pomfret individuals were collected from the northern waters in Kuwait, Sonmiani Bay, Ormara, Pasni, Xiamen, and Taiwan between 2010 and 2014 Figure  to ensurK digestion and extracted with Qiagen DNeasy kit. The extracted DNA was assessed by 1.5% agarose gel electrophoresis and stored at –20 °C for PCR amplification. The mtDNA CR was amplified with the primers F-gao: 5'-GAAGTTAAAATCTTCCCTTTTGC-3' (forward), and R-gao: 5'-GGCCCTGAAGTAGGAACCAAA-3' (reverse). Each PCR was performed in a 25 μL reaction mixture containing 17.5 μL of ultrapure water, 2.5 μL of 10× PCR buffer, 2 μL of dNTPs, 1 μL of each primer (5 μM), 0.15 μL of Taq polymerase, and 1 μL of DNA template. PCR amplification was performed in a Biometra thermal cycler under the following conditions: 5 min of initial denaturation at 95 °C; 30 cycles of 45 s at 94 °C for denaturation, 45 s at 50 °C for annealing, and 45 s at 72 °C for extension; and a final extension at 72 °C for 10 min. PCR products were purified, and both strands were sequenced. The newly isolated nucleotide sequences were deposited in GenBank under accession numbers MF402948–MF402998. Two CR sequences of Pampuschinensis  were used as the out-group.Genomic DNA was isolated from muscle tissue by proteinase CR sequences were edited and aligned using DNASTAR software. Polymorphic sites, haplotype number, and molecular diversity indices for each population were calculated using ARLEQUIN version 3.5 (NJ) method implemented with 1000 replicates in MEGA 5.0 (AMOVA) was performed using ARLEQUIN to investigate the partition of genetic variation among the populations. An unrooted minimum spanning tree (MST) was constructed via the MINSPNET algorithm as implemented in ARLEQUIN to show the relationship among haplotypes and subsequently drawn by hand , based on three parameters: 0, θ1θ (θ before and after population growth) and τ   . The valrgenteus . Bayesiargenteus . In the rgenteus  was applPageBreakCR fragment sequences were 450~453 bp in length, including a 70-bp partial fragment of the tRNApro, and no variable site was detected in the tRNA fragment. After deleting the 70-bp fragment, the obtained target fragment was 380~383 bp in length, which corresponded to the 15,699–16,080 bp region of the complete mitogenome of P.argenteus (KJ569773). Thirty-eight variable sites and 23 parsimony-informative sites were assessed within the target fragment. There were 23 transitions, five transversions, and five insertions/deletions. The ratio of transitions to transversions was 4.6, indicating that the mutations in the CR sequence of P.argenteus had not reached saturation. The A+T content (70.33%) was significantly higher than the G+C content, indicating a significant AT preference.After a manual correction, the h) were detected within each population, demonstrating a high level of genetic diversity in this species. In contrast, low levels of nucleotide diversity (π) were observed. Overall, the average values of the h and π were 0.932 ± 0.013 and 0.018 ± 0.010, respectively  divergence rate, the time since the population divergence occurred was estimated to be 0.06–0.12 million years ago, dating back to the late Pleistocene.FST values between six populations were low (from 0.012 to 0.062) and statistically non-significant, except for those from the Xiamen and Kuwait populations  as one gene pool, and the divergence was attributable to 66.72% of the genetic variation among the populations. To further investigate the possible effects, the populations were partitioned into two and three gene pools, and both pools yielded a significant divergence within the populations (P < 0.001) (Table The ns Table . All res1) Table . TherefoFS test was significantly negative for both lineages (P < 0.05), whereas the D test was not significantly negative (P > 0.05). However, both the SSD and HRI tests were not statistically significant (P > 0.05), suggesting that the two lineages were consistent with the null hypothesis of the sudden expansion model. Thus, the studied Silver pomfret populations experienced a recent expansion event.The observed mismatch distributions of Silver pomfret were established for the two lineages Figure . The mis5–7.8 × 105 and 1.4 × 105–2.8 × 105 years ago based on the divergence rate of the mitochonPageBreakdrial CR (5–10%/MY) and τ values, respectively. The ratio values (1θ/0θ), which estimated the effective female population sizes after and before the expansion, were 2000 for lineage A and infinite for lineage B  approximately 3.2 × 105 years before the present, and the effective population size of lineage B increased sharply from 1.7 × 105 years ago , L.crocea  and Trichiurushaumela , if attention is not paid to the conservation PageBreakof resources. In fact, a decline in the Silver pomfret resources has been reported in some waters due to over-fishing and the devastation of marine ecology. Therefore, fishery management measures regarding the Silver pomfret must be implemented in a timely manner.It is necessary to assess the genetic population diversity and genetic structure of marine fish for fisheries management and conservation. The contemporary genetic structure of the Silver pomfret revealed in this study can preliminarily improve genetic knowledge and provide a firm basis of fishery stocks in the Indo-Pacific Oceans. Although the Silver pomfret currently exhibits a relatively high genetic diversity, it is likely to experience a disaster similar to that experienced by traditional economic fish, e.g.,


Solidago virgaurea complex along an elevational gradient is caused by genetic differentiation. A common garden experiment was based on seeds collected from nine populations of the S. virgaurea complex growing at elevations from 1,597 m to 2,779 m a.s.l. on Mt. Norikura in central Japan. Population genetic analyses with microsatellite markers were used to infer the genetic structure and levels of gene flow between populations. Leaf mass per area was lower, while leaf nitrogen and chlorophyll concentrations were greater for higher elevations at which seeds were originally collected. For reproductive traits, plants derived from higher elevations had larger flower heads on shorter stems and flowering started earlier. These elevational changes in morphology were consistent with the clines in the field, indicating that phenotypic variation along the elevational gradient would have been caused by genetic differentiation. However, population genetic analysis using 16 microsatellite loci suggested an extremely low level of genetic differentiation of neutral genes among the nine populations. Analysis of molecular variance also indicated that most genetic variation was partitioned into individuals within a population, and the genetic differentiation among the populations was not significant. This study suggests that genome regions responsible for adaptive traits may differ among the populations despite the existence of gene flow and that phenotypic variation of the S. virgaurea complex along the elevational gradient is maintained by strong selection pressure.Plant species distributed along wide elevational or latitudinal gradients show phenotypic variation due to their heterogeneous habitats. This study investigated whether phenotypic variation in populations of the  Seeds were placed on two filter papers in a petri dish, which were kept wet with de‐ionized water. The petri dishes were placed in an incubator . Temperature and light conditions in the germination experiment were according to Kondo : Tempera2.3A common garden experiment was conducted for two growing seasons. In May 2013, germinated individuals obtained in the germination experiment were planted into pots (7.5 cm in diameter) filled with potting compost . Pots were placed in a thermostatic chamber at 20.0°C for 16 hr of daylight duration.S. virgaurea complex at the common garden reflected those in natural populations . This subset of plants was placed at a common garden in the campus of Shinshu University  in Matsumoto, Japan. The mean annual temperature was 11.8°C at Matsumoto Weather Station (610 m a.s.l.) between 1981 and 2010. The mean temperatures of August and January were 24.7 and −0.4°C, respectively. The annual mean precipitation was 1,031 mm, with most precipitation in summer. Although the elevation of our common garden was 650 m, it was ideal to set up common gardens at the same elevational range where samples were taken . However, as a practical problem, it was impossible to make such ideal common gardens, and we had to make a common garden in the university campus (650 m a.s.l.). However, the elevational differences in the phenological patterns of the ions see . Therefohttp://rsbweb.nih.gov/ij/index.html). Laminae and petioles of each individual plant were weighed after oven‐drying at 80°C for 48 hr. The LMA of each individual plant was calculated as lamina dry mass divided by the lamina area. The above‐ground dry mass of each individual plant in the first growing season was calculated as the sum of the total lamina dry mass and petiole dry mass.The plants were watered once a day or once every 2 days, were fertilized once a week , and were relocated every week to reduce possible positioning effects during the first and second growing seasons. No bolting individuals appeared by the end of the first growing season . In September 2013 (147th day after sowing), the rosette diameter in two perpendicular directions (one was the maximum width) was measured for each individual plant, and the above‐ground part was harvested. The rosette area was estimated as an ellipse. Each rosette leaf was separated into lamina and petiole, and the laminae of each individual plant were scanned. We measured the total lamina area of each plant using free software ImageJ 1.47  was measured using FLASH2000 . Leaf chlorophyll was extracted using dimethylformamide (4 ml). The absorbance of samples extracted from leaf samples at 663.8 and 646.8 nm was measured by using a spectrophotometer  and was substituted into Porra's equations  and tubular flowers . In the investigation of flowering phenology, the flower head was regarded as flowering when at least one tubular flower of the flower head was developed. Similarly, the flower head was regarded at the finish of flowering when all florets in a flower head had wilted and were discolored. We defined the following flowering phenological stages for each individual plant: (1) day of bud formation (the day when at least one bud appeared), (2) onset day of flowering (the day when at least one flower head flowered), (3) peak day of flowering (the day when most flower heads flowered), (4) finish day of flowering . The flowering period was defined as between the onset and finish days of flowering for each individual. Ten flower heads were chosen randomly for all flowering individual plants, and their involucral length and diameter were measured by using a caliper.During the second growing season in May 2014, all survived individual plants were transplanted to new pots (25.0 cm in diameter) filled with potting compost. All individual plants formed scapes in the second growing season. Growth traits , reproductive traits, and flowering phenology) were measured in the second growing season. We observed the presence or absence of flower buds, and we counted the number of flower heads and the number of flower heads that finished flowering for each individual plant every day. The number of flower heads per individual plant, the number of flower heads per day, and day of each phenological stage  were determined. Plants from the S. virgaurea complex tend to disperse from the plant by wind soon after seed maturation, and involucres also tend to drop from the plant after the release of seeds. The dispersed seeds and dropped involucres were not included in measuring plant biomass. Therefore, the sum of stem mass and leaf mass, except for seeds and involucres, was regarded as the above‐ground biomass in this study. The below‐ground parts  were also dug up after the harvest of the above‐ground part. Soil on roots was washed out. The below‐ground biomass was weighed after oven‐drying at 80°C for 48 hr.Stem height was measured for all bolting individuals every week. The above‐ground part of each individual was harvested after the finish day of flowering. The above‐ground part of each individual was separated into stem, leaf, and reproductive organs, and each organ was stored in a separate envelope. Each organ was oven‐dried at 80°C for 48 hr and weighed. Seeds of the 2.4S. virgaurea complex were additionally sampled from each of the nine study populations in the field. The leaf sample of each population was stored in an envelope and was kept at 3°C until DNA extraction.In June 2014 (second growing season of the common garden experiment), several leaves were sampled from each plant in the common garden for molecular analysis. In September 2014, 11–18 leaves of the S. virgaurea complex and Solidago altissima, respectively. Here, 18 primers were selected . Sakaguchi and Ito . Sakaguchi and Ito  and Saka and Ito  and Saka and Ito .PCR amplification was done in a 6 μl volume containing approximately 10 ng DNA, 2× Multiplex PCR Master Mix , 0.01 μmol/L of forward primer, 0.02 μmol/L of reverse primer, 0.1 μmol/L of M13 primer . The PCR thermal profile included denaturation at 94°C for 9 min, followed by 35 cycles of 94°C for 30 s, 57°C for 1 min, 68°C for 1 min, and a final 20‐min extension step at 68°C. The PCR products (1 μl) were separated by electrophoresis by using an automated sequencer  with 0.4 μl of CEQ DNA Size Standard‐600  and 20 μl of Sample Loading Solution . The fragment size and allele identification were determined using software Fragment Analysis version 8.0 .2.52.5.1a + b concentrations, chlorophyll a/b ratio, number of flower heads per individual plant. Relationships of four phenological stages  against the elevation of provenance sites were also analyzed using GLMM. Statistical significance of each regression model was assessed using a likelihood ratio test. Distribution of likelihood ratio in a null model was computed as the chi‐square distribution approximation.A generalized linear mixed model (GLMM) was used to analyze morphological and physiological traits and flowering phenology in the common garden among the nine populations with different elevations of provenance sites at which seeds were originally collected. The germination rate per population and mean seed mass per maternal plant were regressed against the elevation of provenance sites by GLMM. Maternal plants were treated as a random effect because the germination rate and seed mass may differ between maternal plants due to genetic differences, even at the same elevation. The GLMM was also used to analyze relationships of the elevation of provenance sites with morphological and physiological traits, that is, stem height, above‐ground biomass, below‐ground biomass, rosette area, ratio of below‐ground biomass to total biomass (above‐ and below‐ground biomass), LMA, leaf nitrogen and chlorophyll Mean involucre length and diameter were compared among the nine elevations of provenance sites using the nonparametric Kruskal–Wallis test. The nonparametric Steel–Dwass multiple comparison test was used to compare each pair of populations.2.5.2S. virgaurea complex along an elevational gradient, we analyzed the genotyping data obtained with the 18 microsatellite markers. Deviation from the Hardy–Weinberg equilibrium was estimated within a population by using the probability test for Genepop 4.4.3 , effective number of alleles (AE), observed heterozygosity (HO), expected heterozygosity (HE) were calculated by GenAlEx 6.502  with geographic distances and with elevational distances using the Mantel test. A partial Mantel test was used because the geographic distance correlated to the elevational distance in this study. All statistical analyses were conducted using the free software R version 3.1.2 . Seed mass was also greater for seeds of higher elevations of provenance sites .The germination rate showed a sigmoid curve against elevations of provenance sites at which the seeds were originally collected  and LMA  were significantly lower for higher elevations of provenance sites. Leaf nitrogen, chlorophyll a+b concentrations, and chlorophyll a/b ratio significantly increased with elevation of provenance sites  than for low elevations  Figure . The flo3.3p < .05). However, these loci were not excluded from the further analyses because results did not change after their removal.Two loci deviated from the Hardy–Weinberg equilibrium in more than half of the populations (six populations for Sol_2003631 and eight populations for Sol_2005892). These two loci were removed from further analyses because they likely harbor null alleles. The Bayescan showed that loci Sol_2015992 and Sol_2066912 of the 16 loci deviated significantly from neutral evolution (A) was 4.44–5.00, the effective number of alleles per population (AE) was 2.24–2.57, the observed heterozygosity (HO) was 0.38–0.46, and the expected heterozygosity (HE) was 0.44–0.48 . The range of pairwise FST values was considerably narrow (between 0 and 0.027). Of the 38 pairs, only 10 pairs showed FST values significantly different from zero . The principal coordinate analysis also showed no genetic differentiation among the nine populations, that is, individuals from different elevations forming one group  was correlated significantly with geographic distances . However, the pattern of isolation by elevation was not found if the partial Mantel test regulates the effect of the geographic distance . Therefore, these results indicate that the significant correlation between the elevational distance and pairwise FST values may be caused by high correlation between geographic distance and elevational distance.The genetic differentiation between populations . Flowers of the 4.3S. virgaurea complex at higher elevations in the field. Early flowering is adaptive to high elevations because plants must produce mature seeds by the end of the short growing period  started some months earlier than natural populations of the S. virgaurea complex in the subalpine zone , suggesting that plants grown in the common garden attained the effective accumulated temperature necessary to flower earlier than natural populations in the subalpine zone because the common garden location was warmer than the subalpine zone. Therefore, the elevational differences in phenological patterns in the common garden reflected those in natural populations; nevertheless, there were elevational differences between the common garden and natural populations of the S. virgaurea complex.Assuming the time of flowering is regulated by the effective accumulated temperature, 4.4Arabidopsis halleri subsp. gemmifera shows elevational phenotypic variation, like the S. virgaurea complex, and clinal changes have been detected in adaptive gene alleles in its populations along elevational gradients under gene flow , which in turn causes phenotypic variations observed in the common garden experiment.Although flowering of the 5S. virgaurea complex populations along an elevational gradient were shown to be linked by substantial gene flow between neighboring populations by molecular analysis using neutral microsatellite markers. However, many morphological and physiological traits and flowering phenology showed genetic differentiation along elevations of provenance sites in the common garden experiment. These results suggest that only the genome regions of adaptive traits may display differentiation due to strong selection pressures despite the existence of gene flow. Our findings provide an example of plant micro evolution that genetically maintains adaptive traits to their local environments, even in narrow geographical ranges and under gene flow that could homogenize local adaptation.In this study, S. virgaurea subsp. asiatica and S. virgaurea subsp. leiocarpa are distributed below and above ca. 1,950 m a.s.l., respectively, based on morphological characteristics of flower heads  in multiple mountain ranges is needed to understand the origins of alpine subspecies of subsp. leiocarpa in Japan.In our study area (Mt. Norikura), a et al.  reportedNone declared.


Prunus sibirica L.), a temperate deciduous species distributed in this arid belt, using two chloroplast DNA (cpDNA) sequences, seven chloroplast microsatellite loci (cpSSRs), and 31 nuclear microsatellite loci (nSSRs), to study its phylogeography. Chloroplast data showed the complete fixation of two different genetic groups: the eastern and western groups. Genetic differentiation between the two groups was significant . This pronounced phylogeographic break was also indicated by nSSR data, but there were disparities regarding individual populations. An asymmetric gene flow via pollen and seeds likely resulted in discordance between the present-day geography of nuclear and chloroplast lineages. There was a distinct boundary between the two large groups, which were fixed for two of the most ancestral chlorotypes. Two populations with the highest chloroplast genetic diversity were located in the Yanshan Mountains and Jinzhou, considered to be the glacial refugia. The split of chloroplasts between the eastern and western groups was estimated to have occurred ~0.1795 Ma, whereas nuclear divergence occurred approximately 13,260 years ago. Linear regression analysis showed that climatic factors  had a significant correlation with mean ancestry value (P < 0.05) indicated that they were potential factors for the formation of the two groups. In addition, this boundary was a contact zone between two groups from different refugia.There is evidence that a band of dry climate separated plants in East Asia into distinct northern and southern groups. However, few studies have focused on the arid belt in this region, especially with regard to plants. We analyzed genetic variation in 22 populations of Siberian apricot ( Phylogeographic studies have been used to investigate the effects of past climatic changes on the genetic structure of plant species, and allow inferences to be made about species evolution. The distribution ranges of plants and the genetic diversity within and among present-day populations of such organisms in the Northern Hemisphere have been affected deeply by climatic changes over the last 2 million years Hewitt, . It has Instead of a physical barrier , an east-west arid belt which was between 35 and 45°N, has been thought to act as a climate barrier between the two regions . Northern China (including north and northeast China) is covered with diverse plant biomes that range from tropical to cold forests and taiga , covering the whole range of East Asia, to study the role of the climate barrier in diversification. Both nuclear and chloroplast data showed that the Yanshan Mountains contained a dividing line, separating the northeast and southwest lineages. Similar results have been described in other species, such as, walnut  is one of them. This temperate, deciduous, and wind-pollinated species is distributed widely across the mountainous areas of north and northeast China. It can also be found in eastern Siberia and Mongolia  than for nuclear DNA Ennos, . Indeed,In this study, we re-ran the STRUCTURE analysis using nSSR data we obtained, and used a set of chloroplast simple sequence repeats (cpSSRs) and cpDNA sequences to analyze Siberian apricot populations in China. The major aims of this study were to address the following questions: (1) How did nuclear DNA and cpDNA lineages of Siberian apricot distribute? (2) Does the Siberian apricot show any concordance between the geographical distribution of cpDNA and nuclear DNA lineages? (3) Did refugia exist in the Siberian apricot distribution regions? (4) What is the potential factor(s) of the formation of different lineages?In total, 672 Siberian apricot individuals were collected from 22 populations covering the entire range of the natural distribution in China  for 1 min, and 72°C for 1 min, followed by 8 cycles of 94°C for 30 s, 53°C for 40 s, and 72°C for 45 s, with a final synthesis step at 72°C for 10 min. The products were separated with an ABI 3730XL DNA Analyzer using GeneScan-500LIZ as an internal marker . The fragments were sized using the Gene-Marker 1.75 software .We selected 19 cpSSR markers after an initial screening from 40 candidate cpSSR markers . Amplification was carried out under the following conditions: 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, Tm for 1 min, a 2-min extension at 72°C, and a final synthesis step at 72°C for 10 min. PCR products were purified using a TIANquick Midi Purification Kit following the manufacturer's protocol (Tiangen). Purified PCR products were sequenced directly in both directions using the PCR primers on an ABI 3730XL DNA Analyzer (Applied Biosystems).Using universal primers, we conducted an initial screening for sequence variability of various chloroplast markers against 15 samples. The cpSSR haplotypes of the 15 samples were different from each other based on the result of completed cpSSR analysis. The intergenic spacers (IGSs) of the following pairs of genes were amplified using previously published primers: T, average within-population diversity HS, and two differentiation measures  were estimated for haplotypes and compared via a test with 1,000 permutations using Permut 2.0 software , the effective number of haplotypes (Ne), the number of private haplotypes (Np), Nei's haplotype diversity (H), and the unbiased haplotype diversity (He) Nei, . Total gatpB-rbcL and trnQ-rps16 sequences were combined into a complete sequence. Multiple alignments were performed with the ClustalX program  were estimated with the same parameters as cpSSR analysis. All populations were grouped by performing spatial analysis of molecular variance (SAMOVA) using the SAMOVA software (ver. 1.0)  for the “best” grouping of populations. Then, the observed genetic variation among and within the populations and groups was characterized by an analysis of molecular variance (AMOVA) using ARLEQUIN (ver. 3.5) software  reported for the angiosperm species 1–8.24 × 10−9 s s−1 y−1 to estimate the timescale of divergence  steps. A uniform prior probability distribution was used to accommodate the uncertainty of the prior knowledge. We compared the outcomes of all five clock models with the Yule speciation process by using the TRACER program (ver. 1.6)  software were implemented to estimate phylogenetic relationships and divergence times between cpDNA haplotypes  software  program was used to simplify the comparison of clustering results from the STRUCTURE programs across all 20 repetitions of K and to determine the most likely number of clusters.In this study, we used the correlated allele frequencies model and the admixture model .An approximate Bayesian computation (ABC) approach was used to infer the recent colonization history of the Siberian apricot. The STRUCTURE analysis revealed two clusters that corresponded to the western group (WG) and northeastern group (EG). To determine which among several scenarios of the history of divergence of populations from these regions was the most probable, we used the ABC procedure in DIYABC (ver. 2.0.4) software  was significantly smaller than NST .Seven perfect chloroplast microsatellite loci were used to assay 222 Siberian apricot individuals, resulting in 18 different alleles. In total, 15 unique cpSSR haplotypes were produced by combinations of the 18 different alleles. Details of the haplotype frequency and population genetic diversity are provided in Supplementary Tables atpB-rbcL and trnQ-rps16) was 1,400 base pairs in length. Six nucleotide substitutions and five indels were found in the combined fragment which revealed eight cpDNA haplotypes  was not significant higher than GST (0.862). Five populations were polymorphic while the remaining 17 were fixed for a single haplotype ; ; ; (P13); (P17); and (P22). Meanwhile, AMOVA of the six groups revealed that 91.70% of the variance was distributed among groups  were divided into two clades, one associated with the EG and the other with the WG , based on chloroplast variation across all populations, was much smaller than the total genetic diversity, HT (0.809). A permutation test showed that NST (0.871) was significantly higher than GST .In combination, the mixed cpDNA sequences identified 21 chlorotypes , , and . SAMOVA of chloroplast data variation separated all populations into two large groups and one small one. The two large groups had a distinct boundary, which was the longitude lines of P16 , and the second largest ΔK was at K = 2 (ΔK = 9.30). However, the absolute values of ΔK were very small  showed an upward trend until 0 Figure . The vall Figure . The CLUK Figure . The bigmWGEG →  was 344.75, and that of 4 NmEGWG →  was 322.86. Linear regression analysis showed that the annual precipitation had a significant correlation with mean ancestry value (P = 0.00103 < 0.01) whereas annual mean temperature had a significant correlation with mean ancestry value at P = 0.05 level . The genetic diversity of cpDNA (HT = 0.652) showed a relatively lower level of genetic diversity than those in other deciduous tree species, such as, Prunus spinosa  than that of 170 plant species  was significantly higher than that of GST (0.758) using mixed cpDNA sequence data, indicating the presence of a phylogeographic structure. The grouping of populations produced by SAMOVA of mix-sequences variations strikingly similar to that of STRUCTURE, based on nSSRs  during the LGM  originated from a single glacial refugium, which was located in the Changbai Mountains  were likely the refugia for the WG and EG, respectively. Moreover, an isolated microrefugium (P22), near the Changbai Mountains, was suggested within the EG. Overall, apart from geographic barriers, our findings highlight the importance of climate in the present-day distribution of temperate deciduous trees in northern China.KY000823-KY000832.All cpDNA sequences were deposited in GenBank under the accession numbers ZW, RW, and XP planned and designed the research. ZW, ZZ, JT, and SS performed experiments and analyzed data, and ZW, YZ, and XP wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.