The term “Columbian Exchange” refers to the massive transfer of life between the Afro-Eurasian and American hemispheres that was precipitated by Columbus’ voyage to the New World. The Columbian Exchange is widely appreciated by historians, social scientists and economists as a major turning point that had profound and lasting effects on the trajectory of human history and development.I propose that the Columbian Exchange should also be appreciated by biologists for its role in the creation of novel human genomes that have been shaped by rapid adaptive evolution. Specifically, I hypothesize that the process of human genome evolution stimulated by the Columbian Exchange was based in part on selective sweeps of introgressed haplotypes from ancestral populations, many of which possessed pre-evolved adaptive utility based on regional-specific fitness and health effects.Testing of this hypothesis will require comparative analysis of genome sequences from putative ancestral source populations, with genomes from modern admixed populations, in order to identify ancestry-specific introgressed haplotypes that exist at higher frequencies in admixed populations than can be expected by chance alone. Investigation of such ancestry-enriched genomic regions can be used to provide clues as to the functional roles of the genes therein and the selective forces that have acted to increase their frequency in the population.Critical interrogation of this hypothesis could serve to underscore the important role of introgression as a source of adaptive alleles and as a driver of evolutionary change, and it would highlight the role of admixture in facilitating rapid human evolution.This article was reviewed by Frank Eisenhaber, Lakshminarayan Iyer and Igor B. Rogozin The historian Alfred Crosby coined the term “Columbian Exchange” to describe the extensive transfer of life between the Afro-Eurasian (Old World) and American (New World) hemispheres following Christopher Columbus’ voyage of 1492 [The introduction of calorically rich and nutritional New World crop species – potatoes, maize and cassava in particular – facilitated agricultural developments that allowed for sustained population growth in the Old World . The demi.e., combinations of linked alleles) that are specific to individual populations; the process of admixture during the Columbian Exchange then led to the repeated introgression of these population-specific haplotypes onto distinct genomic backgrounds. In other words, population mixing during the Columbian Exchange generated novel human genome sequences with combinations of haplotypes that had never previously co-existed in the same genome. I am interested in exploring the implications of the rapid creation of such novel, admixed American genome sequences for adaptive human evolution, fitness and health.From a population genomic perspective, the Columbian Exchange can be considered to have facilitated genetic admixture among three human population groups – African, European and Native American – that had previously evolved separately for many thousands of years –10. Duride novo mutation.As mutation is the ultimate source of novel adaptive alleles, it can be considered to be a critical rate limiting step for adaptive evolution. Human germline mutation rates are low , and accIf genetic admixture between previously isolated populations is extensive, it can provide introgressed haplotypes at intermediate to high frequencies to the resulting admixed population. Introgression could thereby increase the rate of adaptive evolution by elevating the frequency of potentially beneficial alleles available in the population. In this sense, introgression of adaptive alleles can be considered to provide an opportunity for so-called ‘soft’ selective sweeps, which are defined as causing rapid molecular evolution via the simultaneous increase in frequency of multiple adaptive alleles in the population . Soft seI hypothesize that genetic admixture and introgression among the human population groups brought together via the Columbian Exchange provided the opportunity for rapid adaptive evolution based on the existence of numerous pre-adapted haplotypes. In other words, introgression during the Columbian Exchange provided extensive standing genetic variation to New World populations, much of it with potential adaptive significance, which could have provided the raw material for numerous  selective sweeps.The three ancestral human population groups – African, European and Native American – that were brought together over the last 500 years during the course of the Columbian Exchange began to diverge ~60–100,000 years ago (ya) as modern humans emerged from Africa and spread around the world . Europe The New World environment that served as the selective crucible for the introgressed haplotypes would have consisted of both the external physical environment as well as the internal microbial environment, which was shaped by the mixture of microbes endemic to the ancestral source populations. The novel microbial environment in particular is likely to have exerted strong selective pressure on New World populations, based on the need to respond to the challenge of infectious pathogens, suggesting that immune system genes would be particularly prone to introgression-accelerated adaptive evolution –30.It should be noted that the ~500 years that have elapsed during the era of the Columbian Exchange is an extremely short amount of time with respect to human evolution; indeed, it would correspond to a mere 20–25 generations assuming a generation time of 20–25 years. Irrespective of the role of introgression in providing standing adaptive genetic variation to an admixed population, this would not likely be enough time to allow for the complete fixation of adaptive alleles. Thus, the kind of introgression-facilitated adaptive evolution proposed here would amount to partial (or ongoing) selective sweeps . Neverthi.e. evolution over ecological time scales, has received substantial attention over the last few years [Analysis of human population genetic change over such a relatively short time period provides an opportunity to consider the possibility of rapid human evolution in the context of recent studies related to the convergence of evolutionary and ecological time. The question of extremely rapid evolution, ew years . Traditiew years . Howeveri.e., SNPs) when possible. Having characterized the overall ancestry contribution proportions for an admixed American population, one can then search for specific genomic regions (loci) or SNPs that significantly deviate from the expected patterns  in human genome sequences are based on complex statistical models of sequence substitution and may lack power to unambiguously distinguish among different models of selection – e.g., hard versus soft selective sweeps, the role of de novo versus standing genetic variation and the prevalence of polygenic selection [Analysis of admixed American populations using this conceptual framework has the potential to reveal human evolution in action. Current methods for detecting the signatures of adaptive evolution  of the domestic animals that serve as sources of zoonotic infections [, e.g., cows are the natural reservoir for measles, smallpox and tuberculosis, and pigs can harbor whooping cough and flu. The low burden of infectious disease in the Americas, and the related lack of genetic resistance mechanisms, left these populations extremely vulnerable to Old World pathogens and was the major cause of the population crash that occurred during the conquest of the Americas.Author’s response: fections , e.g., cOne possible exception to this pattern is syphilis [. There is evidence to suggest that syphilis was brought to Europe by Columbus’ returning crew members, and the first recorded epidemic was from Naples, Italy in 1495. Shortly thereafter, syphilis quickly spread throughout Europe with devastating effects. Thus, it may in fact be the case that the burden posed to naïve European and African populations by syphilis in the New World also served as a selection pressure for adaptive introgression of resistant Native American haplotypes.syphilis . There iThe process of admixture as evolutionary process to gain fitness might be contrasted with the consequences of the process of (self-)isolation of populations as it is known, for example, for certain religious groups and heavily stratified societies  doi: 10.1073/pnas.1513197113). It would be interesting to study whether at all or to which extent increasingly inbred mating might have detrimental effects on the populations’ fitness.This is another interesting point, which is entirely feasible although there is less direct evidence in support of it at this time. It is certainly possible that Native American populations had lower innate (genetic) resistance to infectious disease owing to lower genetic variability associated with bottleneck event(s) that accompanied their initial migration to the Americas from Asia [. High genetic diversity, among African populations for example, has been associated with increased resistance to infectious disease [, and low diversity could accordingly place populations at risk. Consistent with the idea raised by the reviewer, our own analysis of Native American populations from the Human Genome Diversity project suggests that they have far lower overall genetic diversity than other populations from this same set of samples and/or from the 1000 Genomes Project. However, this could also be a result of a bottleneck that occurred later as a result of contact with European populations [.Author’s response: rom Asia . High ge disease , and lowulations .The article must be published. It is an original idea, untested and likely to provide new insights.This is a timely, well articulated and well defined hypothesis to test for the presence of partial selective sweeps of haplotypes derived from ancestral populations during the “Columbian Exchange”. It is only in the past 5 years or so that we are even beginning to understand the impact of admixtured populations across various regions and chronological times, leading to fascinating insights on the origins of modern populations, the presence of selective sweeps of alleles and even the impact of these on languages and cultural memes . In this backdrop of development, King’s proposal to test a very specific aspect on the presence of adaptive allele sweeps, from at least 3 or more ancestral populations that contributed to the admixtured populations of the modern Americas, is timely. The idea of using an introgression-based framework is interesting and perhaps quite agnostic as pointed out. Given that both the European and African populations that entered the Americas themselves are derived from multiple ancestral populations, studying the above might further throw light on the evolutionary dynamics of those alleles or loci known to have been selected for in these populations prior to the “Columbian exchange” (e.g. 26595274). I agree that such a study is likely to result in some really interesting insights on the impact of introgression between at least three distinct populations separated by many tens of thousand years.I appreciate the positive feedback from the reviewer. I agree that it will be important to take into account the fact that the Europeans and Africans who came to the Americas during the course of the Columbian exchange were themselves derived from multiple (and/or distinct) populations. It should be possible to tease apart the sub-continental admixture contributions from such ancestral populations, and our own group is currently doing so for Afro-Colombian populations in an effort to more precisely locate their origins within the African continent [.Author’s response: ontinent , 52.The reviewers’ point also underscores the importance of choosing the most appropriate ancestral populations to perform the kind of ancestry enrichment analysis described here. For example, one may expect that European ancestry for Latino populations would be better modeled using Spanish ancestral populations  as opposed to the more widely used CEU sample, which corresponds to broadly distributed Northern European ancestry. Our own preliminary results, suggest that this is in fact the case, and New World admixed populations can be distinguished by virtue of different European ancestry profiles. I have added a paragraph to the “Testing the hypothesis” section of the manuscript to address the need for analysis of appropriate ancestral populations.The author hypothesized that the process of human genome evolution stimulated by the Columbian Exchange was based on partial selective sweeps of introgressed haplotypes from ancestral populations, many of which possessed pre-evolved adaptive utility based on regional-specific fitness and health effects. I think that this is a promising hypothesis. The author suggested that in order to test this hypothesis, one would need to compare genome sequences between putative ancestral populations with sequences characterized from admixed American populations.It is known that natural selection is not the only force that changes allele frequencies, other evolutionary forces include genetic drift, migration, mutation, and biased gene conversion. One potential problem is how to define “putative ancestral populations”, I think that only small  samples from putative ancestral populations contributed to the Columbian Exchange (the founder effect). The founder effect might confound the signal of adaptive evolution. However this may not have a substantial impact on the Columbian Exchange, this needs to be evaluated using sequence data although the author may add a brief discussion of this problem to the paper.The reviewer raises important points about defining ancestral populations for enrichment analysis and the need to distinguish between the effects of natural selection and demographic processes, both of which can change allele frequencies over time. The issue of the appropriate choice of ancestral populations was also raised by Reviewer #2. Fortunately, there are more and more genomic data sets available from potential ancestral populations, and these data can be compared to genomic data from admixed American populations in order to determine the most appropriate ancestral population for analysis. Nevertheless, the issue of founder effects from ancestral populations, which is entirely reasonable given the history of the conquest and colonization of the Americas, could still confound attempts to quantify changes in allele frequencies by comparing modern populations. This fact, along with the need to distinguish selection from drift present both challenges and opportunities for the development and application of population genetic approaches to this question, in addition to the more straightforward genome sequence analysis approaches described here. I have added a paragraph to the “Testing the hypothesis” section of the manuscript to address the points raised by the reviewer.Author’s response:


Styela plicata, a solitary ascidian introduced in harbours and marinas of tropical and temperate waters, across spatial and temporal scales. A fragment of the mitochondrial gene Cytochrome Oxidase subunit I (COI) was sequenced from 395 individuals collected at 9 harbours along the NW Mediterranean coast and adjacent Atlantic waters  at two time points 5 years apart (2009 and 2014). The levels of gene diversity were relatively low for all 9 locations in both years. Analyses of genetic differentiation and distribution of molecular variance revealed strong genetic structure, with significant differences among many populations, but no significant differences among years. A weak and marginally significant correlation between geographic distance and gene differentiation was found. Our results revealed spatial structure and temporal genetic homogeneity in S. plicata, suggesting a limited role of recurrent, vessel-mediated transport of organisms among small to medium-size harbours. Our study area is representative of many highly urbanized coasts with dense harbours. In these environments, the episodic chance arrival of colonisers appears to determine the genetic structure of harbour populations and the genetic composition of these early colonising individuals persists in the respective harbours, at least over moderate time frames (five years) that encompass ca. 20 generations of S. plicata.Spatio-temporal changes in genetic structure among populations provide crucial information on the dynamics of secondary spread for introduced marine species. However, temporal components have rarely been taken into consideration when studying the population genetics of non-indigenous species. This study analysed the genetic structure of  The increase in maritime traffic and aquaculture activities in recent decades has fostered the spread of non-indigenous marine species (NIS) across the globe . At the International traffic among large commercial ports is a common pathway for the spread of NIS into new and often distant areas  and adjacent Atlantic waters. Contrary to other introduced ascidians in this area  were sampled: seven on the Mediterranean side and two on the Atlantic shores, near the Strait of Gibraltar . These lCOI) gene was amplified using the universal primers LCO1490 and HCO2198 . A fragment of the mitochondrial Cytochrome Oxidase I ( HCO2198 . Amplifi HCO2198 .Nh), haplotype diversity (Hd), and nucleotide diversity (π) were computed with DnaSP v.5 , and a correction for multiple comparisons was applied following the Benjamini-Yekutieli false discovery rate (FDR)  with a FDR-corrected probability. A value of Dest was deemed significant when the confidence interval around its mean did not contain 0.Number of alleles  . For DesIn order to complement these differentiation methods based on allelic frequencies, we used two approaches that explicitly consider the spatial distribution of alleles across samples. First, we performed a spatial analysis of shared alleles SAShA, , which tSTF and Dest) were computed for each population between sampling years. A correlation between these values among pairs of populations at both years was also calculated. Finally, an analysis of the molecular variance (AMOVA) was performed using haplotype frequencies with Arlequin, grouping the populations per sampled year, and its significance was tested by running 10,000 permutations of the data.For temporal comparisons, differentiation values (STF and with the R package ade4 (function mantel-rtest) .Mantel tests were also computed to correlate genetic and geographic distances separately at each year. The shortest distances by sea between points were calculated using Google Earth (Google Inc.). Mantel tests were performed with Arlequin for l-rtest)  for DestStyela gibsii as an outgroup (GenBank accession number HQ916447) with Mega v6.06 .Twenty to twenty-six individuals were sequenced per locality in 2009 and 2014 , resultiHd = 0) was found in La Herradura (HER) in 2014 and in Torre Ladrones (TOR) and Chipiona (CHI) at both years. No clear geographic or temporal trend was apparent for haplotype diversity, with some populations showing higher values in 2009 and others in 2014  . The hig in 2014 .Haplotype 2 was the most abundant and widespread (only absent in Xàbia in 2009), followed by haplotype 1, which was dominant in Blanes (BLA), Arenys de Mar (ARE), and Xàbia (XAB). Haplotype 5 was the third most frequent and the dominant haplotype in Carboneras (CAR). The remaining haplotypes were found only in 6 or less individuals . The hapThe haplotype network revealed that haplotype 5 was most divergent, separated by 16 mutational steps from the remaining haplotypes, which were grouped more closely together, albeit with some divergent sequences . A majority of pairwise comparisons were significant . Overall, Vilanova i la Geltrú (VIL) was the locality that showed fewer significantly different comparisons with other populations, while Carboneras (CAR) showed significant differentiation in all cases. In contrast, the values of differentiation between years for the nine localities were extremely low and non-significant in all cases   .STF and Dest values for both years , combining information of both years, showed a significantly restricted spatial distribution of the alleles in our samples  . These rSTF-2009: r = 0.287, p = 0.065; STF-2014: r = 0.252, p = 0.086; Dest-2009: r = 0.273 p = 0.066; Dest-2014: r = 0.252, p = 0.084), indicating an overall relationship between genetic and geographic distances, albeit with the geographic component explaining little variance in genetic structure.The results of the AMOVA grouping populations by sampling year showed that most genetic variability occurred between populations in a given year  and within populations  . In contStyela clava in New Zealand, Our setting is representative of many highly urbanized coasts along the Mediterranean Sea and elsewhere, with a dense network of harbours and marinas of different sizes. We focused specifically on small to medium-size harbours, which mainly host short-range fishing fleets and recreational boats. These are typically sites of secondary dispersal for introduced species  were generally low in the sampled populations and lower than reported for the same marker in other introduced ascidians in harbours  and gene diversity values  and the rare haplotypes (right of break). Asterisks indicate haplotypes that were private  at the corresponding year.Click here for additional data file.10.7717/peerj.2158/supp-2Supplemental Information 2p-values indicate significant differences obtained through permutation of data.Frequency distributions of observed (triangles) and expected (circles) distances between co-occurrences of the alleles present in more than one locality, both sampling dates pooled. Click here for additional data file.10.7717/peerj.2158/supp-3Supplemental Information 3Click here for additional data file.


Turkey is a crossroads of major population movements throughout history and has been a hotspot of cultural interactions. Several studies have investigated the complex population history of Turkey through a limited set of genetic markers. However, to date, there have been no studies to assess the genetic variation at the whole genome level using whole genome sequencing. Here, we present whole genome sequences of 16 Turkish individuals resequenced at high coverage (32 × -48×).MAPT locus) and found increased allele frequency of 31.25% for H1/H2 inversion polymorphism when compared to European populations that show about 25% of allele frequency.We show that the genetic variation of the contemporary Turkish population clusters with South European populations, as expected, but also shows signatures of relatively recent contribution from ancestral East Asian populations. In addition, we document a significant enrichment of non-synonymous private alleles, consistent with recent observations in European populations. A number of variants associated with skin color and total cholesterol levels show frequency differentiation between the Turkish populations and European populations. Furthermore, we have analyzed the 17q21.31 inversion polymorphism region  contains supplementary material, which is available to authorized users. High throughput sequencing technologies have prompted sequencing of human genomes at the population level. For instance, the 1000 Genomes Project has reported genome resequencing data from 14 populations and aims to analyze the genomes of a total of 27 populations .th Century A.D., which occurred with disputed intensity and frequency, but with undoubtedly profound linguistic impact [Alu[Western Asia has been the main corridor through which initial out-of-Africa migrations have populated Eurasia . Moreovec impact . In addic impact . Locus sc impact , 10, mtDc impact , Alu[12act [Alu in the gIn addition to providing insights into the demographic history of the population, resequencing studies are gaining prominence for identifying variants that are associated with complex, sometimes population specific diseases . Such asIn this paper, we present, for the first time, high coverage (32X to 48X) whole genome re-sequencing data from 16 individuals from Turkey, covering at least 99.1% of the genome at 1X, and at least 98.79% at 5X  sequence data at high coverage ranging between 32 to 48X, using the Illumina platform  of our SNP calls, we analyzed the same samples using Affymetrix 6.0 SNP arrays (Methods) to genotype for common SNPs. We then compared the microarray results with the SNP calls we generated from WGS. Assuming the SNP arrays as the gold standard, we estimated the FDR to be at 0.174% and FNR as 0.209%.Next we studied the 16 genomes with respect to major patterns of population diversity, structure, and migration, comparing these profiles with those in the 1000 Genomes Project . We founTo obtain an overview of population relationships, we conducted principal component analyses using 16 individuals randomly selected from each one of the 14 populations within the 1000 Genomes Project, combined with the profiles from Turkey (Methods). The first two principal components of the combined dataset replicated the global genetic structure identified in earlier studies (e.g. ). WithinWe then estimated the maximum likelihood population tree with migration using the Treemix software  , to the Turkey-Tuscan clade, was only 0.048. In comparison, the weight for the migration event predicted to originate from the branch ancestral to East Asia  into current-day Turkey was 0.217. Although this implies a major population event from the East to West Asia, we note that these weights are not direct estimates of the migration rates. First, the original contributing populations to the ancestral population in Turkey are not known. For instance, we do not know the exact genetic relationship between current-day East Asian populations and the Turkic speakers from Central Asia who migrated into Anatolia about 1,000 years before present. In fact, Hodoglugil and Mahley, using HGDP genotyping data, predict that South Asian contribution to Turkey's population was significantly higher than East/Central Asian contributions , suggestFinally, we investigated possible population structure within Turkey. Principal component analyses did not reveal any subclustering among the 16 individuals, and we found no evidence for longitudinal or latitudinal divergence Figure C. SupporCCDC82, was shown to evolve under positive selection in humans, and diverged from Neandertals [The assessment of whole genome and exome sequencing data at the population level has shown that due to recent population growth, human populations carry an excess of rare genetic variants , many ofndertals . Indeed,We then explored potentially functional variants at higher or lower frequency in the Turkish population relative to the closely related European populations Figure . For thiAmong these 7 SNPs, 2 exist at lower frequencies in Turkey compared to Europe. We found that both are related to pigmentation , and haiTo our surprise, 2 of the 5 SNPs that show higher frequency in Turkish population as compared to Europeans are associated with lower total cholesterol counts. This is particularly interesting, given that the Turkish population has indeed been shown to have lower cholesterol than Western European populations, including total cholesterol, and high and low density lipoprotein cholesterol measures, while carrying relatively high triglyceride levels . Althougin silico validate our discovery callset (Methods). In summary, we identified 1,751 deletions per individual, with a non-redundant total of 3,292 deletions. To assess novelty, we compared against the 1000 Genomes Project datasets (Methods), and found 199 novel deletions per sample on average . We did not find any whole-gene deletions, but 21/494 deletions were predicted to delete coding exons where 10/21 were predicted in more than one chromosome . We collected blood samples from 16 volunteers after receiving signed informed consent forms from each of the individuals. The individuals that participated in this study were selected to represent different geographical regions of Turkey.Approximately 10 ml peripheral blood was collected from each subject into a tube containing K3EDTA to prevent coagulation. Each blood sample was then transferred into a 50 ml centrifuge tube and 30 ml of ice cold red blood cell (RBC) lysis buffer was added. The contents were mixed thoroughly and the mixture was kept at 4°C for at least 20 minutes to lyse the cell membranes. Centrifugation was performed at 5000 rpm at 4°C for 10 minutes. The supernatant containing the RBC debris was removed and the pellet containing the leukocyte nuclei was washed with 3 ml RBC lysis buffer to remove the cell debris. The pellet was suspended in 10 ml of cold RBC lysis buffer by vortexing. After centrifugation at 5000 rpm at 4°C for 10 minutes, the supernatant was discarded, the pellet was cleaned with 3 ml RBC lysis buffer and centrifuged again. 3 ml nuclei lysis buffer was added and the pellet was dissolved by vortexing. In order to digest nuclear proteins, 30 ul Proteinase K (20 mg/ml) and 50 ul of 10% SDS were added, mixed gently and incubated at 37°C overnight or at 56°C for 3 hours. After the incubation step, 10 ml 2.5 M NaCl solution was added to the mixture, shaken thoroughly and centrifuged at 5000 rpm at 20°C for 30 minutes. The supernatant was transferred into a new 50 ml centrifuge tube and approximately 30 ml absolute ethanol was added. The tube was inverted gently until the DNA threads become visible. DNA was fished out with the aid of a micropipette and was left to dry for 2 hours. It was dissolved in 300 ul TE and stored at -20°C. The purity and quality of the extracted DNA were measured at A260/A280, and were found to be within the acceptable range (1.8 - 2.0).Whole genome shotgun sequence data were generated using the Illumina HiSeq2000 platform (paired-end 101 bp reads clipped to 90 bp) at BGI-Shenzhen. The sequencing reads that contained Illumina adapter sequences or high number of low-quality bases were removed. We finally obtained sequence data coverage between 32- to 48-fold per sample.sampe). For easier comparison and mitochondrial DNA correspondence, we used the GRCh37 version that is used by the 1000 Genomes Project. Next we used the GATK tool [We aligned the reads to the human reference genome (NCBI GRCh37) using the BWA aligner  with defATK tool  to realiWe hybridized the DNA extracted from 15 of the samples with the Affymetrix 6.0 SNP arrays to test for sensitivity and specificity of the call sets. We compared the Affymetrix 6.0 SNP array results with SNPs we obtained from WGS. Assuming that microarray results provide the gold standard, we calculated false discovery rate to be 0.174% on average and false negative rate to be 0.209%.in silico validation, and we filtered out any calls that were not successfully genotyped using split-read or read-depth information. We reasoned that any true variation discovered with VariationHunter using read-pair information will be supported by read-depth and split-read information used by GenomeSTRiP to genotype the variant in the genomes of multiple individuals. Although it is still possible that GenomeSTRiP may have false negatives in genotyping, therefore incorrectly invalidating a true variant detected by VariationHunter, this effect is acceptable to obtain a more reliable (lower FDR) call set.Using the BAM files generated in the previous step, we filtered read pairs that map concordantly to the reference genome within 4 standard deviations around the mean span size. In order to discover all possible mapping locations of the remaining discordant read pairs and unmapped reads in GRCh37, we used the mrFAST aligner  in pairein silico validation strategy, we counted the number of heterozygous SNPs detected by GATK within the novel deletion intervals. We applied this validation to only the novel deletion calls when compared to the 1000 Genomes Project. We reasoned that, if a deletion is real, then it couldn’t contain heterozygous SNPs within, as at least one of the copies is deleted. The only exception to this rule is if the deletion is found within duplication. However, such deletions are too difficult to detect to start with, and filtered out by both VariationHunter and GenomeSTRiP. We found that >85% of our novel deletion calls contain no heterozygous SNPs; where only 10% contains exactly one heterozygous SNP.As a second We determined novelty in our call set by filtering those deletions that overlap  with any of the 1000 Genomes Project releases , or callGenotypes of each individual were confirmed by PCR by using the primer specific for Tau deletion region using primers 5’GGAAGACGTTCTCACTGATCTG, and 3’AAGAGTCTGGCTTCAGTCTCTC and subsequent capillary based sequence analysis , 50.We used ANNOVAR version 2012Oct23) to annotate the detected variants. For gene based and filter based annotation, we used the April 2012 version of the annotation database , and the dbSNP version 132. For comparison against the 1000 Genomes Project, we used the data released by the consortium as Phase I of the project [012Oct23 Treemix, that allows constructing maximum likelihood phylogenetic trees while allowing migration from edges to nodes [To compare Turkish populations with worldwide populations, we used the phase 1 version of the 1000 Genomes Project dataset . Averageto nodes , providito nodes  and simiTo compare the frequencies of SNPs associated with phenotypes and disease between Turkish population and other world populations, we calculated the frequencies of SNPs listed in the GWAS Catalog  among wohttp://turkiyegenomprojesi.boun.edu.tr/tgp_vcf/TGP.integrated_callset.vcf.gz.Sequence reads are deposited to the SRA read archive (SRP021510). The VCF file that lists all genomic variation characterized in this study is available at: Additional file 1: Table S1: Geographic locations of the samples used in the project and their NCBI accession numbers. (PDF 81 KB)Additional file 2: Figure S1: Average nucleotide diversity (π) across the genome calculated for the 1000 Genomes dataset populations and the 16 Turkish genomes. Positions with missing data were removed. Hardy-Weinberg filtering was not applied. The populations included are as follows: Turkey (TUR); Utah residents with Northern and Western European ancestry (CEU); Toscani in Italia (TSI); British from England and Scotland (GBR); Finnish from Finland (FIN); Iberian populations in Spain (IBS); Han Chinese in Beijing, China (CHB); Japanese in Tokyo, Japan (JPT); Han Chinese South (CHS); Yoruba in Ibadan, Nigeria (YRI); Luhya in Webuye, Kenya (LWK); African Ancestry in Southwest US (ASW); Mexican Ancestry in Los Angeles, CA (MXL); Puerto Rican in Puerto Rico (PUR); Colombian in Medellin, Colombia (CLM). (PDF 454 KB)Additional file 3: Figure S2: Neighbor joining tree of Tuscan individuals from Italy from the 1000 Genomes Project. Individuals are indexed according to their order in the 1000 Genomes dataset. Note the star-like topology of the tree. (PDF 78 KB)Additional file 4: Table S2: Characterization of the 7 private SNPs. (PDF 97 KB)Additional file 5: Table S3: Novel deletions discovered in the TGP dataset. (PDF 2 MB)A) Read alignments for three individuals within MAPT deletion region (238 bp) [B) Genotypes of each individual are also confirmed by RT-PCR and subsequent Sanger-based sequencing PCR analysis are performed to confirm the genotype of H1 and H2 individuals using a diagnostic indel [Additional file 6: Figure S3: To validate our prediction for common inversion polymorphism, we have selected three individuals that are shown to be polymorphic for H1/H1 (38I220611), H1/H2 (33 M140611) and H2/H2 (32A140611) haplotypes. (238 bp) , 50 are Additional file 7: Figure S4: Pairwise comparison of GWAS SNP allele frequencies. Note the expected high correlation of allele frequencies between Turkish and European populations. (PDF 266 KB)


Alopias pelagicus. Though an earlier study using mtDNA control region data, showed evidence for differences between eastern and western Pacific populations, the study was hampered by low sample size and sparse geographic coverage, particularly a lack of samples from the central Pacific. Here, we present the population structure of Alopias pelagicus analyzing 351 samples from six different locations across the Pacific Ocean. Using data from mitochondrial DNA COI sequences and seven microsatellite loci we found evidence of strong population differentiation between western and eastern Pacific populations and evidence for reciprocally monophyly for organelle haplotypes and significant divergence of allele frequencies at nuclear loci, suggesting the existence of two Evolutionarily Significant Units (ESU) in the Pacific Ocean. Interestingly, the population in Hawaii appears to be composed of both ESUs in what seems to be clear sympatry with reproductive isolation. These results may indicate the existence of a new cryptic species in the Pacific Ocean. The presence of these distinct ESUs highlights the need for revised management plans for this highly exploited shark throughout its range.There has been an increasing concern about shark overexploitation in the last decade, especially for open ocean shark species, where there is a paucity of data about their life histories and population dynamics. Little is known regarding the population structure of the pelagic thresher shark,  Sharks of the open ocean are wide-ranging, highly migratory species that routinely cross national borders. Such characteristics may lead to the misconception that these marine resources are unlimited and resistant to localized depletion. When population subdivision exists, these misconceptions lead to an unsustainable use of the resource, ending in the depletion of the population under pressure. If severe enough, these population depletions may result in reduced genetic diversity and a concomitant reduction in the ability of a population to adapt to environmental or disease stressors Isurus oxyrinchusCetorhinus maximusRhincodon typusPrionace glaucaHighly migratory and broadly distributed open ocean shark species are expected to show little to no population heterogeneity. As expected, previous population genetic studies on epipelagic sharks, such as the shortfin mako shark Alopias pelagicus) is a large (up to 330 cm TL) aplacental viviparous epipelagic shark with a distribution restricted to the Indian and Pacific Oceans. Fecundity is very low with litters of only 1–2 pups The pelagic thresher shark  Are the patterns observed by Trejo similar when bi-parental nuclear data are included? 2.) Given the strong vicariance between Eastern and Western Pacific locations observed by Trejo Though a strong biogeographic signal was detected for A total of 351 samples were collected between 1997 and 2011 . The majet al.Samples were identified by experts in the field and for the samples for which identification was uncertain, we implemented the molecular identification protocol described by Caballero 5′ TCWACCAACCACAAAGAYATYGGCAC) and FishCoxI R (TARACTTCWGGGTGRCCRAAGAATCA) modified from Ward et al.A portion of the mitochondrial COI gene (655 bp) was amplified using the universal primers FishCoxI F (http://www.geneious.com/) and aligned using Seaview 4.4.1 software All forward and reverse sequences were checked and edited manually using Geneious Pro v. 3.6.1  or an ABI 3730 Genetic Analyzer at the NOAA SWFSC  using the internal ROX 500 size standard. Electropherograms were visualized and allele size calling was done using ABI PRISM GeneMapper Software v4.1 (Life Technologies). A reference set of samples were used to cross-calibrate scores between analysis platforms and labs. All authors scored all samples for all loci individually in order to minimize scoring errors across readers.ad hoc statistic  with most populations being significantly different from each other (ST (ΦST 0.0768–0.4971) with significant differentiation for all comparisons involving Baja  and non-significant differences in pairwise comparisons for both FST and ΦST  and 39 (AV-H8) across all sampled populations.E) and observed heterozygosity (HO), were obtained for seven loci in all populations units, along with deviations from H-W equilibrium  and RST (−0.032–0.135) comparisons were significant for most comparisons with Taiwan and between Hawaii and Colombia and Ecuador (ST (−0.003–0.109) and RST (−0.032–0.338) comparisons remained large and significant between clade A and B groups while comparisons within clade B group were low and not significant . In all analyses K = 2 had the highest likelihood . Among tCT = 0.018 p = NS), but significant within group variance remained (ΦSC = 0.018 p<0.05). Samples grouped by mtDNA clade (i.e. clade A and B) explained 4.9% of the variance (ΦCT = 0.049 p<0.05), while within group variance was fairly low (ΦSC = 0.008 p<0.05) but still significant.AMOVA comparisons of microsatellite data were performed to compare hypothesized groups . GroupinA. pelagicus in the Pacific Ocean using both mitochondrial and nuclear molecular markers. Though an earlier study using mtDNA control region data This study presents the first extensive analyses of the molecular ecology of A core task of this study was to evaluate and extend earlier findings by Eitner These results support the existence of two groups on separate evolutionarily trajectories, conforming to the concept of Evolutionarily Significant Units (ESUs). ESUs have been described based on the presence of reciprocal monophyly for organelle haplotypes  and significant divergence of allele frequencies at nuclear loci  showing the true value of K after testing K = 1–10 with 20 repetitions each.Plot of the second order rate of change of the likelihood (Δ(TIF)Click here for additional data file.Table S1Name, PCR profile number, annealing temperature, reference or Genbank accession numbers for primers used in this study.(DOC)Click here for additional data file.Table S2Structure Likelihood values for multiple Ks with No Location Prior, Geographic Location Prior and mtDNA Clade Prior.(DOC)Click here for additional data file.


Podarcis muralis, on Jersey  and in the Chausey archipelago. We sequenced a fragment of the mitochondrial cytochrome b gene in 200 individuals of P. muralis to infer the phylogeography of the island populations using Bayesian approaches. We also genotyped 484 individuals from 21 populations at 10 polymorphic microsatellite loci to evaluate the genetic structure and diversity of island and mainland (Western France) populations. We detected four unique haplotypes in the island populations that formed a sub-clade within the Western France clade. There was a significant reduction in genetic diversity  of the island populations in relation to the mainland. The small fragmented island populations at the northern range margin of the common wall lizard distribution are most likely native, with genetic differentiation reflecting isolation following sea level increase approximately 7000 BP. Genetic diversity is lower on islands than in marginal populations on the mainland, potentially as a result of early founder effects or long-term isolation. The combination of restriction to specific localities and an inability to expand their range into adjacent suitable locations might make the island populations more vulnerable to extinction.Populations at range limits are often characterized by lower genetic diversity, increased genetic isolation and differentiation relative to populations at the core of geographical ranges. Furthermore, it is increasingly recognized that populations situated at range limits might be the result of human introductions rather than natural dispersal. It is therefore important to document the origin and genetic diversity of marginal populations to establish conservation priorities. In this study, we investigate the phylogeography and genetic structure of peripheral populations of the common European wall lizard,  There is a growing interest in the patterns and processes associated with geographical variation in population genetic structure across species’ ranges since these often shift, expand and contract over time –4. HistoPelophylax lessonae) in Britain. Initially considered to be present solely as a result of human introductions the native status of pool frogs was confirmed just in time to witness its extinction  and Hpod [3′-GGTGGAATGGGATTTTGTCTG-5′] ,26–28. AGeneious 6.1.7 [cyt-b region to amino acid sequences, to verify that no premature stop codons disrupted the reading frame. Unique sequences were submitted to GenBank under the accession numbers KP118978-KP118990.Sequencing reactions were carried out with BIGDye Terminator v3.1 Ready Reaction kit  in both directions. Products were precipitated in isopropanol and analysed on an ABI 3130 automated capillary sequencer . Mitochondrial DNA sequences from both directions were corrected by eye and aligned to obtain a consensus sequence. Accepted sequences were then aligned using MAFFT  implemenet al. [et al.[2O. PCR conditions were as follows: 15min of initialization step at 95oC, 26 cycles of 30sec at 94 oC, 90sec at 57 oC (for multiplex 1 and 2) or 55 oC (for multiplex 3) and 1min at 72oC and a final extension step of 20min at 60oC. The 5’-end of each forward primer was labeled with a fluorescent dye either 6-FAM, HEX or NED. PCR products were run with an internal ladder (red ROX-500), on an ABI 3130 genetic analyser (Applied Biosystems Inc.) We scored alleles in Geneious 6.1.7 and any ambiguous peaks were repeated to confirm genotype.To infer the genetic structure and diversity of our populations we genotyped 484 individuals at 10 polymorphic microsatellite loci; four described by Richard et al.  and six . [et al. (Table AP. siculus (AY185095) [P. liolepis (JQ403296) [P. melisellensis (AY185097) [MrBayes [Mega 5.2 . The BI analysis was run with four chains of 1,000,000 generations and sampling every 100 trees. We discarded (burn-in-length) the first 10% of the trees after checking for convergence of the chains and the posterior probability branch support was estimated from the 50% majority-rule consensus tree.We used the phylogenetic tree approach to assign haplotypes to known lineages by combining our sequences with 68 sequences (of varying lengths), obtained from GenBank, across the native distribution of the species (see Table B in Y185095) , P. liolQ403296)  and P. mY185097)  were use[MrBayes  under thMega 5.2 40. ThTo investigate evolutionary relationships of our sequences, we constructed a parsimonious phylogenetic network using a median—joining algorithm in Network v.4.6.12 . The meticrochecker V.2.2.3 [O, HE) were calculated with Genalex 6.5 [R) with Fstat v.2.9.3 [IS) and deviations from Hardy-Weinberg equilibrium were also evaluated at the 0.05 nominal level for multiple tests using sequential Bonferroni corrections in Fstat v.2.9.3 [O, HE, AR in island versus mainland populations with a Welch Two Sample t-test and evaluated the correlation between expected heterozygosity and latitude with a Spearman’s rank correlation test in R [We checked the microsatellite data in M V.2.2.3  for nullalex 6.5  and alle v.2.9.3 ,45. Inbr v.2.9.3 ,45. We cest in R .Structure v.2.3.4 [6 iterations from K = 1 through 5. Runs for each K were replicated 10 times and the true K was determined according to the method described by Evanno et al [Structure Harvester v.0.6.93 [Arlequin 3.5.1.3 [ST values and visualized with a Principle Coordinate Analysis (PCoA) in Genalex 6.5 [To infer population structure, we implemented a Bayesian analysis in  v.2.3.4  using th v.2.3.4 . The simno et al  in the ov.0.6.93 . We testv.0.6.93 ) in Arle 3.5.1.3 . Populatalex 6.5 .Analysis of mtDNA sequences of 192 individuals revealed 13 unique haplotypes all nested within the Western France Clade . The mosp<0.05) in the island populations of Jersey and Chausey than in mainland France populations  between latitude and expected heterozygosity  and multiplex three (3) was developed by Richard et al. (2012). Table B, List of sequence data used in the phylogenetic analysis. Information on sampling location, GenBank accession numbers and the reference study. Table C, Historical information on the island populations of the wall lizard. Table D, Table of null alleles per population per locus. Bold values indicated significant deviation from Hardy-Weinberg equilibrium (p<0.05). Table E, Matrix of pairwise FST values. Figure A, Plot of Delta K (ΔΚ). Calculated as in Evanno et al. (2005) from K = 2 to K = 4. Highest Delta K for K = 3. Figure B, Plots of genetic diversity indexes between island (group 1) and mainland populations (group 2). Genetic diversity is expressed as HO, HE and AR. Differences in the mean numbers were compared with a Welch Two Sample t-test.Multiplexes one (1) and two (2) were developed by Heathcote (DOCX)Click here for additional data file.


Cyclura cychlura inornata) is endemic to the Allen Cays, a tiny cluster of islands in the Bahamas. Naturally occurring populations exist on only two cays (<4 ha each). However, populations of unknown origin were recently discovered on four additional cays. To investigate patterns of genetic variation among these populations, we analyzed nuclear and mitochondrial markers for 268 individuals. Analysis of three mitochondrial gene regions  and data for eight nuclear microsatellite loci indicated low genetic diversity overall. Estimates of effective population sizes based on multilocus genotypes were also extremely low. Despite low diversity, significant population structuring and variation in genetic diversity measures were detected among cays. Genetic data confirm the source population for an experimentally translocated population while raising concerns regarding other, unauthorized, translocations. Reduced heterozygosity is consistent with a documented historical population decline due to overharvest. This study provides the first range-wide genetic analysis of this subspecies. We suggest strategies to maximize genetic diversity during ongoing recovery including additional translocations to establish assurance populations and additional protective measures for the two remaining natural populations.The Endangered Allen Cays Rock Iguana ( Invasive species, anthropogenically mediated sea level rise and stochastic natural events such as hurricanes disproportionately affect island populations . InsularCyclura inhabit two main island groups in the West Indies: the Greater Antilles and the Bahaman archipelago. Although taxonomic classifications remain controversial, of the 10 species and 15 subspecies identified , currently classified as Endangered by the IUCN  and the Exuma Island Rock Iguana (C. c. figginsi). Cyclura c. inornata was believed extinct due to human harvest when it was originally described  have increased steadily over the last fifty years, with current combined populations of approximately 1000 individuals, with each population likely approaching carrying capacity (Mus musculus). Strong indicators suggest that the presence of rodents negatively affects iguana populations  in less than three years (Procyon sp.) was linked to a 35–67% decrease in the only existing population of White Cay Iguana (Cyclura rileyi cristata) in the Bahamas within a year , and altered blood chemistry, in the Allen Cays Iguana  to better inform conservation management planning for the northern Exumas. Specifically, we (1) assess genetic relatedness among populations; (2) identify the likely origins of recently discovered translocated populations; and (3) evaluate the potential for natural dispersal, and translocation to aid in population persistence and resilience in the face of increasing anthropogenic impacts. The current study investigated twelve DNA microsatellite loci and three mitochondrial DNA regions to examine genetic diversity in C. c. inornata across the known  distribution of the subspecies in the Exuma Islands  in the Exumas that collectively comprise the entire known range of the Allen Cays Rock Iguana . Tissue We extracted genomic DNA from blood and tissue using Qiagen DNeasy Blood and Tissue Kits  and an Applied Biosystems 6,100 Nucleic Acid PrepStation  following manufacturer’s instructions. Final elutions of DNA were stored at −20 °C.We sequenced 95 of the 285 individuals sampled for three mitochondrial loci . These 95 individuals included representatives from all sampling sites  to assesPCR products were cleaned using AMPure magnetic beads , and cycle-sequenced using BigDye Terminator v 3.1 Kit . Sequencing products were purified using CleanSeq  and run on an ABI 3730xl DNA Analyzer . We aligned and edited sequences using Sequencher 5.0 .Cyclura . Thermal cycling conditions were as follows: 5 min initial denaturation at 96 °C, 35 cycles of 94 °C for 30 s, Ta °C (primer specific) for 30 s 72 °C for 30 s, and a final extension at 72 °C for 10 min.We attempted to genotype all 285 individuals at 12 microsatellite loci  previousCyclura . These 1cychlura . PCR amp® size standard. Alleles were identified using GeneMarker® .Amplified products were visualized on a 1% agarose gel using GelRed™  and microsatellite genotyping was performed using an ABI 3100 DNA Analyzer with GeneScan™ 500 LIZAll mtDNA sequences were aligned against published sequences available from Genbank  using Clα), Hardy-Weinberg Equilibrium (HWE), tests of linkage disequilibrium, F-statistics based on HE), and observed heterozygosity (HO), were calculated using the program GENEPOP v. 4.1 (α) corrected for unequal sample sizes was calculated using the rarefaction method , as well as between island size and genetic diversity measures  (Frankham 1996) were computed using IBM SPSS Statistics . One-tailed tests for significance were determined at α = 0.05 (Frankham 1996). The relationship between geographic distance and genetic distance (FST) was tested for significance with a Mantel test using the Isolation by Distance Web Service  resulted in a single haplotype . Phylogenetic analysis of our data with published Cyclura sequences resulted in a combined C. c. inornata and C. c. figginsi subclade as in Extractions for 15 of 285 individuals (5%) did not produce high enough quality DNA for sequencing and genotyping and were excluded from further analyses. Removal of individuals of unknown locality and poor quality DNA left 268 individuals available for analysis. Our alignment of concatenated mitochondrial sequences for NADH-4, cytochrome b and the control region for the subsample of p = 0.009) in a subset of populations. However, as the pattern was not consistent across all populations, we chose to include both loci in our final analyses.Of the 12 microsatellite loci screened for polymorphisms, eight  amplified and were variable, one locus (D111) amplified but was monomorphic, and three primer pairs  did not produce consistent PCR products and were therefore not included in further study . Two locα) ranged from 1.76 to 2.09  Cay. Among islands, expected heterozygosity (HE) ranged from 0.13 to 0.46, and observed heterozygosity (HO) ranged from 0.09 to 0.58. The population from SEFRR Cay exhibited both the lowest HE and HO. When this site was excluded, the ranges of HE and HO were 0.35–0.46 and 0.38–0.58, respectively. Effective population sizes were low relative to estimated census sizes. In the two presumed natural populations, parametric estimates ranged from 16.4 to 105, and 95% confidence intervals had variable if not infinite ranges  were not significant . While not significant, the relationships between island area with α(p = 0.06) and HE(p = 0.093) were both positive. According to the Mantel test (p = 0.33). Prior to the test, Alligator Cay was removed from this analysis due to its history as a confirmed human-introduced population.The correlations between population size and genetic diversity measures  that exhibited a per cluster allelic richness ranging from 3.04 to 3.84  in the southern region of the same island chain  revealed allelic richness values of 5.55 and 4.78 for two separate island populations (Sphenodon sp.) exhibited 84 unique alleles across six microsatellite loci and allelic richness ranged from 2.2 to 5.6 in 14 island populations (Cyclodina whitakeri) from insular New Zealand also exhibited relatively high diversity with 114 unique alleles across twelve microsatellite loci , excluding the small all-female population on Southeast Flat Rock Reef Cay, are comparable to those for Anegada Iguanas . This suggests that these two populations have been isolated since rising sea-levels associated with the current interglacial partially inundated the Great Bahama Bank, a finding consistent with observations made regarding C. c. figginsi . However, our data suggest that unauthorized translocations of iguanas among some islands are highly likely (FST = 0.016), yet they are separated by the greatest geographic distance, excluding Alligator Cay which is known to result from translocation.Leaf and U Cays host the only two populations of figginsi . If therFST excluding the Flat Rock Reef Cay population illustrated a positive trend and implied a stronger correlation, but was not significant . Long-term capture records identifying the presence of individuals on Flat Rock Reef Cay that were previously marked on U Cay (most) or Leaf Cay  are suggestive of such unauthorized translocations.A Mantel test for geographic distance and C. c. inornata was relatively low compared to other studied Cyclura species, genetic structure was still evident among populations. Overall, Bayesian STRUCTURE analyses revealed that two unique genotypic clusters exist within the subspecies and mirrored patterns of genetic diversity described above  clearly reflects the recent (1988–1990) translocation event.Although the genetic diversity of ed above . The majed above . MoreoveNE), defined as the actual number of breeding individuals in a population, is predicted to influence the rate at which this loss is expected to happen . Factors likely responsible for the high N:NE in C. c. inornata include largely overlapping generations, and highly variable reproductive success. Further, the abundance of juveniles in the population likely influences N:NE because the age of maturity in C. c. inornata is 12 years, the longest known for a lizard , which also is endemic to islands once joined as a single landmass during the last glacial maximum . Our results suggest a positive relationship between these parameters, although the correlations are not statistically significant. Low levels of genetic diversity, low variation in island size, and the confounding effects of historic and ongoing anthropogenic factors including translocations likely hinder our ability to detect such correlations . In addi species . During  species . Combine maximum .inornata, which once doubtless existed on several islands about Allen’s Harbor, is now beyond doubt extinct. Since these creatures are excellent for food, they are constantly hunted…often with dogs trained for the purpose.”The low genetic diversity in the Allen Cays Rock Iguana could reflect geologic history, recent anthropogenic effects, or a combination, and these factors warrant additional investigation. In particular, the most dramatic impact on populations of the Allen Cays Rock Iguana may be sourced to the extreme overharvesting of the species at the beginning of the twentieth century. Rapid decreases in population size may result in rapid loss of genetic diversity, which subsequently predisposes populations to genetic drift and inbreeding . As prevC. c inornata revealed low levels of genetic diversity. However, small samples sizes for some populations and historical influences make interpretation of these results difficult. Biogeography and historical and current anthropogenic factors are all suspected to have contributed to observed patterns.Molecular data can augment data from ecological and behavioral research to benefit those managing wild populations. Our preliminary analyses of nuclear and mitochondrial markers in C. cychlura between the current ranges of inornata and figginsi in the Exumas that have gone extinct (For future planning, it is important to recognize that high levels of natural inbreeding in this insular species in combination with anthropogenic and environmental stressors could send the species into an extinction vortex . Consist extinct .C. c. inornata. Our evidence for genetically distinct natural populations on U Cay and Leaf Cay supports their treatment as separate management units. However, it appears that animals from Leaf Cay have already been translocated without authorization to U Cay, potentially compromising any genetic distinction of the latter population. Hence, additional translocations to historically occupied sites may still be warranted in order to maximize the preservation of remaining genetic diversity. In addition, these sites could provide beneficial assurance populations in the event of stochastic extirpations.The maintenance of genetic diversity is an important component of conservation planning. Expected increases in hurricane activity and sea level rise under projected climate change scenarios pose significant threats to the persistence of 10.7717/peerj.1793/supp-1Supplemental Information 1Click here for additional data file.


Nesidiocoris tenuis (Hemiptera: Miridae), is one of the most thermophilous dicyphines in agroecosystems and is widely distributed in China. Little is known regarding the genetic structure of N. tenuis and the effect of historical climatic fluctuations on N. tenuis populations. We analyzed partial sequences of three mitochondrial protein-coding genes  and nuclear genes  for 516 specimens collected from 37 localities across China. Analyses of the combined mitochondrial dataset indicated that the Southwestern China group (SWC) was significantly differentiated from the remaining populations, other Chinese group (OC). Asymmetric migration and high level of gene flow across a long distance within the OC group was detected. The long-distance dispersal of N. tenuis might be affected by air currents and human interference. Both the neutrality tests and mismatch distributions revealed the occurrence of historical population expansion. Bayesian skyline plot analyses with two different substitution rates indicated that N. tenuis might follow the post-LGM  expansion pattern for temperate species. Pleistocene climatic fluctuation, complicated topography and anthropogenic factors, along with other ecological factors (e.g. temperature and air current) might have accounted for the current population structure of N. tenuis.The plant bug,  Nesidiocoris tenuis is considered as a major natural enemy and has shown its beneficial role in control of several pests such as whiteflies, leafminers, aphids, thrips and spider mites1N. tenuis populations are well established, pest infestation is less severeN. tenuis is currently widely commercialized and mass supplied in southern and warm production areas1N. tenuis covers various climate types, from temperate to subtropical and tropical regionN. tenuis, due to, wide range of climate types and the presence of several refugia.The plant bug N. tenuis is a model organism of biological control potential, and knowledge of its population genetics and associated factors is fundamentally crucial for species management and conservation strategies. Multiple factors can influence population dynamics, genetic diversity and population structure7N. tenuis, and its efficacy against pests346N. tenuis in China remained unknown.N. tenuis related to current factors ; and (2) investigate demographic history of N. tenuis affected by Pleistocene climate fluctuation in China.Herein we investigated its genetic diversity, population structure and demographic history using sequences of mitochondrial and nuclear data. The objectives of this study were to (1) reveal the genetic distribution of COI, 65 haplotypes for ND2, and 30 haplotypes for CytB were identified, respectively. A total of 130 haplotypes were detected in a combined mitochondrial dataset, which included 2, 226 bp of protein-coding regions . Among these identified haplotypes, 102 were unique haplotypes and six haplotypes  were most widely shared. The H1 haplotype was shared by 120 individuals and was detected in most populations. The H4, H6, H12, H13, H17 haplotypes accounted for 9.50%, 7.75%, 10.47%, 5.04%, and 6.40%, respectively. In addition, we observed 145 polymorphic sites, which were composed of 54 parsimony-informative sites (2.29%) and 91 singleton-variable sites (4.09%). High haplotype diversity (Hd) and low nucleotide diversity (Pi) were shown in all 37 populations . Compared to the results of the combined mitochondrial dataset, fewer haplotypes (HN = 30) and a lower level of genetic diversity (Hd = 0.183) were observed in the nuclear data. In addition, we observed 34 polymorphic sites, which were composed of one parsimony-informative site (0.13%) and 33 singleton-variable sites (4.38%).For the nuclear data, 509 sequences were successfully obtained with the length of 753 bp  exhibited the most significant genetic differentiation ; and the OC group included the remaining 34 populations in China .N. tenuis, based on analyses of Mantel tests for both the combined mitochondrial dataset  effect was detected among populations of ST = 0.01809, P < 0.05) was observed. AMOVA analysis showed that 1.81% of the variation was partitioned among populations and the other 98.19% was within populations. SAMOVA analysis revealed that the FCT value reached the peak at K = 2 and decreased subsequently  also showed the significant genetic differentiation between two defined groups (SWC and OC). Moreover, the three-level AMOVA analysis indicated extremely significant structure between two groups , with 12.97% genetic variation.For the combined mitochondrial dataset, significant genetic structure among all populations  were consistently low and ranged from 0.00096 for Southern District (SD) to 0.04566 for Central District (CD)  was found for samples from Southwestern District (SWD) to CD, while the lowest M value (686.5) was for samples from the Northern District (ND) to SWD. Significant asymmetrical migration rates among long-distance district pairs, e.g. SWD-CD pair, were discovered by non-overlapping 95% confidence intervals of each estimate.When four geographical districts were analyzed, estimates of effective population size (ict (CD) . Estimatθ values and M values were translated into effective migrants per generation (Nem = θM), most migrants into CD (290.39) and ND (257.42) were obtained , which indicated that xpansion .Rg values but non-significant SSD values also indicated sudden population expansion, which was in line with the results of neutrality tests  was detected in the HNA population as well ] and geographic distance (ln Km) were determined using Mantel tests based on both molecular datasets, indicating IBD effect was non-significant among populations of N. tenuis in China  between CHS population and TS population (geographic distance = 1385.467 Km), and a non-significant FST value (0.007) was detected between the two furthest populations . We therefore speculated the homogeneity of populations was likely related to long-distance dispersal by human interference. It might be possible that biological companies introduced N. tenuis into different locations as a biological control agent and long-distance dispersal might be carried out due to the distribution chain operational in many countries11Grapholita molestaLiposcelis bostrychophila was capable of long-distance dispersal  due to human-transportTribolium castaneum widely colonized grain storages primarily through anthropogenic dispersalin China . Gene flet al. had shown limited dispersal abilities of N. tenuis from autumn to spring in certain temperate regions, while some dispersal would be possible in the summerN. tenuis was consistent with that of the Asian subtropical monsoon, which occurs from southern and southwestern to northern direction in spring and summerFurthermore, our gene flow analyses confirmed long-distance dispersal, which demonstrated asymmetrical migration and high migration rates . High miN. tenuis. Mitochondrial and nuclear network construction showed multiple star-like shapes, which implied that N. tenuis experienced population expansion events more than onceN. tenuis experienced recent demographic expansion events. The results were in line with the estimates using Bayesian skyline plots with different rates . The interspecific substitution rate used in the previous COI analysis was much lower than mutation rate within species due to the delayed effects of purifying selectionN. tenuis populations were estimated to start expanding nearly 15 Kya, which was consistent with the result of population expansion after the LGM. Although the results from the substitution rates based on COI gene and the combined mitochondrial dataset were different, the same post-LGM expansion pattern was obtained.A variety of analyses indicated recent demographic expansion events for nt rates . The estN. tenuis after the LGM was related to climate changes, because temperate species may present contracted distribution during glacial periods and experience range expansions during interglacial periodsN. tenuis was proved to be the most thermophilous of all dicyphines in the Mediterranean region3Stomoxys calcitransLocusta migratoriaUnlike the expansion during the LIG to LGM transition, which has been observed in other organisms in Asia34N. Tenuis.In summary, a combination of historical factors , ecological factors (specific topography and comfortable climate) and anthropogenic factors  might have shaped the current population structure pattern and dispersal routes of N. tenuis individuals were collected from 37 locations, covering all representative distributions in ChinaIn this study, a total of 516 adult COI, a fragment of ND2 and partial CytB gene) and nuclear genes  were used as molecular markers. Primers for three mitochondrial genes were specifically designed based on the complete mitochondrial genome of N. tenuisCOI, KT587084 - KT587148 for ND2, KT587054 - KT587083 for CytB, and KT587149 - KT587178 for ITS2 plus partial 5.8S and 28S.Considering the genetic variability of different genes, three mitochondrial protein-coding genes  were inferred from the amino acid alignment. The number of polymorphic sites (S), the number of haplotypes (HN), haplotype diversity (Hd), nucleotide diversity (Pi) and average number of nucleotide differences (K) for each location were calculated using Arlequin version 3.5 Sequences of mitochondrial and nuclear markers were aligned independently using Clustal W implemented in MEGA version 5 N. tenuis. The spatially explicit BAPS model for clustering of individuals, implemented in BAPS version 6.0FST analysis with 10,000 permutations was also calculated using Arlequin version 3.5 Several approaches were used in order to understand the population genetic structure of Trigonotylus caelestialium, was chosen as the out-groupND2, GTR+G for COI and GTR+G for CytB, respectively. The analysis was performed with two runs and four chains for 15 million generations, and the chains were sampled every 1000 generations. The first 25% of samples were discarded as burn-in.To reveal the relationships among mitochondrial haplotypes, the splits network was constructed using SplitsTree version 4.13.1 FST/(1 − FST)] vs. linear geographic distance (ln Km) were investigated using Mantel tests implemented in the software Arlequin version 3.5To detect the effect of geographical isolation, Mantel testsFST values between defined groups and hierarchical analyses of molecular variance (AMOVA) with 10,000 permutations were executed using Arlequin version 3.5COI sequence (DNA barcoding marker) were performed using MEGA version 5The spatial analysis of molecular variance (SAMOVA) was performed using SAMOVA version 1.0 Four districts were defined based on the zoogeographic regions of China52θM), the mutation-scaled population size  and the mutation-scaled migration rate  were estimated using Bayesian inference implemented in Migrate version 3.6.4FST values, and three subsequent runs were started with θ and M from the previous run to confirm the consistency of results. For each run, one long chain with five independent replicates was contained, other parameters were as follows: long-inc = 20, longsample = 1,000,000, burn-in = 100,000. To increase the efficiency of the MCMC, four heating chains were used with approximately exponential increasing temperatures at 1.0, 1.5, 3.0, and 1,000,000. The final estimates of parameters along with 95% confidence intervals were reported here.To estimate effective number of migrants entering and leaving each region per generation  index of the observed distribution and the sum of square deviations (SSD) between the observed and the expected mismatch were also calculated using Arlequin version 3.5 rg index indicated that populations had undergone recent demographic expansions, and a significant value of SSD was an indication of population stabilityMultiple approaches were explored to investigate the demographic history of COI gene, a substitution rate of 1.77% per million yearsTo estimating population expansion through time, Bayesian skyline plots were implemented in BEAST version 1.6.1 How to cite this article: Xun, H. et al. Population genetic structure and post-LGM expansion of the plant bug Nesidiocoris tenuis (Hemiptera: Miridae) in China. Sci. Rep.6, 26755; doi: 10.1038/srep26755 (2016).


Hilsa. Although the control region analysis revealed broad structuring between the Arabian Sea and Bay of Bengal  it was unable to detect structure among riverine populations. These results suggest that the markers used must be able to distinguish populations and control region has led to an underestimation of genetic differentiation among populations of Hilsa.The mitochondrial control region has been the first choice for examining the population structure but hypervariability and homoplasy have reduced its suitability. We analysed eight populations using control region for examining the population structure of Hilsa shad, Tenualosa ilisha, is an anadromous fish with broad distribution ranging from foreshore areas, estuaries, brackish water lakes, and freshwater rivers Indo-West Pacific region from the Persian Gulf, along the coast of Pakistan, India, Bangladesh, and Burma to South Vietnam [ Hilsa fisheries in east and west coast of India over the years because of the introduction of efficient mechanized crafts such as moshari (mosquito net) seine net, behundi jal (set bag net), and char ghera jal (fence-like net operation around the char) and makeshift gears and fishers are tempted to exploit Hilsa stocks without caring for size and season. Overfishing may reduce population sizes to a level at which inbreeding and loss of genetic diversity occur and may result in extinction of local populations [ Hilsa population.The Vietnam . It asceulations . Proper  Hilsa [The rate of evolution of mitochondrial DNA is generally higher than that of nuclear genes due to the lack of a known repair mechanism for mutations that arise during replication . The con Hilsa . Hilsa was investigated by various researchers using morphomeristical, biochemical, and molecular approaches and also they differentiated three stocks of Hilsa belonging to Hooghly, Padma, and Ganga rivers using biometrical parameters. Based on tagging experiment, Pillay et al. [ Hilsa individuals come up the Hooghly River during subsequent seasons, that is, winter and monsoon. Ghosh et al. [ Hilsa into slender and broad morphotypes using morphometric data. Brahmane et al. [ Hilsa from India using RAPD markers and cytochrome b region. So, the baseline information on genetic stocks needs to be authenticated. However several morphometric and molecular studies  were conducted in Bangladesh and India but no study was done using mitochondrial D-loop to understand population genetic structure and patterns of gene flow of T. ilisha [The population structuring ofy et al.  concludeh et al.  differene et al. , 9 repor. ilisha . T. ilisha from the geographical distribution range in India, namely, rivers draining in Bay of Bengal  as well as from Diamond Harbour and Paradip Port and rivers draining to Arabian Sea . T. ilisha was identified and discriminated from T. toli and Hilsa kelee based on morphometric and meristic data following Talwar and Jhingran [ Hilsa body is oblong and compressed with 30–33 spines like scutes on abdomen. Difference between two major Hilsa species, that is, T. ilisha and T. toli, is very minute. In the former, dorsal and ventral profile of the body is equally convex, while in the later abdominal profile is more convex than that of dorsal. Further, about 150 to 200 straight to slightly curved gill rakers are present on the lower part of first arch in T. ilisha, while in T. toli gill rakers are curved and the number of gill rakers is 80 to 90 as discussed by Huda and Haque [The present study included 77 specimens ofJhingran  and FishJhingran . The Hilnd Haque . The fisμL volume contained 1x buffer, 100 μM dNTPs, 2 mM MgCl2, 10 picomoles of each primer, 3 U Taq DNA polymerase, and 100 ng template DNA. Amplifications were performed in Veriti 96 fast thermal cycler . The thermal regime for control region consisted of initial denaturation of 3 min at 94°C, followed by 35 cycles of denaturation at 94°C for 50 sec, annealing at 47°C for 30 sec, and extension at 72°C for 80 sec with final extension of 10 min at 72°C. PCR products were visualized on 1% agarose gels stained with ethidium bromide and documented using a gel documentation system . DNA sequencing was performed following the dideoxynucleotide chain termination method [Approximately 50 mg of caudal or anal fin or muscle tissue was used for DNA isolation following standard phenol : chloroform : isoamyl alcohol method . Precipin method  using anS), haplotype diversity (h), and nucleotide diversity (π).Complete control region sequence was generated from forward and reverse sequence reads using MEGA 5.1 . AmbiguiThe evolutionary history was inferred using the ML method. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analysed. The evolutionary distances were computed using the Kimura 2-parameter method  and are Minimum spanning network of haplotypes was prepared by Network 4.6 software . IntrapoFST statistics [ D and Fu's FS values were estimated based on pairwise differences between sequences. Tajima's D test [ FS test [The isolation-by-distance effects on population genetic structure were estimated by IBDWS 3.23 and pairwise atistics . To detes D test  calculat FS test  is basedN), number of haplotypes (NHap), haplotype diversity (HapD), and nucleotide diversity (π) for each population. A total of 77 individuals were sequenced for the mtDNA control region (873 bp).Among 77 samples, 94 polymorphic sites and 58 haplotypes were detected. These polymorphisms included 64 parsimony informative sites and 32 singleton sites. The nucleotide composition (%) was 31.7 (A), 27.9 (T), 23.7 (C), and 16.7 (G).h) of the analysed populations was rather high with observed values between 1.000 ± 0.0058 in Ganga and 0.756 ± 0.01678 in Tapti population. The nucleotide diversity (π) within each population was very low, ranging from 0.00935 ± 0.0000012 in Hooghly to 0.01835 ± 0.0000059 in Tapti population . Estimates of genetic differentiation between eight populations using F statistics are given in There is significant geographical structuring among populations only when all populations were grouped in Bay of Bengal and Arabian Sea. AMOVA revealed 4.42% variation among populations and 95.58% variation within population , which w D was nonsignificantly negative for all except Narmada and Tapti populations. Fu's FS test also showed nonsignificant negative values for all except Diamond Harbour and Tapti populations. Large differences were observed between θ0 (population before expansion) and θ1 (population after expansion). In addition, the SSD and Hri values were also nonsignificant except for Diamond Harbour and Tapti populations   . FS and Tajima's D test except for Tapti and Diamond Harbour indicative of recent population expansion after a genetic bottleneck or founder events [The significant positive correlation between geographical and genetic distances usually referred to as “isolation by distance” is typically suggestive of migration-drift equilibrium. Our result showed high haplotype diversity (high genetic variation) and low nucleotide diversity and significant positive Fu'sr events , 28.F statistics (FST), AMOVA, haplotype network, and phylogenetic analysis revealed genetic differentiation among the T. ilisha populations, suggesting that T. ilisha does not have a single panmictic population. Brahmane et al. [ Hilsa by using cytochrome b region in Ganga and Hooghly rivers and also felt the need of advanced genetic markers like ATPase 8/6, nicotinamide dehydrogenase subunit 2 (ND2), and microsatellite for confirmation. Our results also strengthen the presence of more than one stock of Hilsa in Indian subcontinent; however Ganga and Hooghly Feeder Canal and Hooghly populations shared the same genepool. Geographical divergence and gene flow among subpopulations can be assessed by phylogenetic analysis and haplotype network provided that gene lineages have accumulated sufficient polymorphisms over time and populations have been isolated completely to allow genetic drift to act. In present study ML tree and haplotype network of control region have clearly distinguished four major clades/haplogroups, namely, Lineage 1 , Lineage 2 , Lineage 3 (Tapti population), and Lineage 4 (Narmada population). Lineage 2 had mixed haplotypes mostly from Godavari and some from Hooghly Feeder Canal (Hap_1), Diamond Harbour (Hap_12), Ganga (Hap_14), and common haplotype (Hap_8); this can be explained by slightly mistaken migration routes taken by some individuals. However formation of four clades may be explained by the philopatry driven genetic differentiation that also corroborates previous tagging studies that demonstrated limited natal and breeding dispersal of Hilsa [ Hilsa populations was also strongly supported by pairwise significant FST values of Tapti and Narmada with Godavari, Paradip Port, Diamond Harbour, Hooghly Feeder Canal, and Ganga. Although phylogenetic and phylogeographic analysis (minimum spanning network) shows that genetic variation is not randomly distributed among Bay of Bengal rivers, a pattern of population structure and gene flow has been difficult to verify statistically. Analysis of pairwise population FST provides strong support for divergence between Bay of Bengal and Arabian Sea population; however biologically accepted level of statistical significance (p ≤ 0.05) is too stringent to reveal the subtle genetic differences within Bay of Bengal and Arabian Sea populations when the Bonferroni correction for multiple p value is applied [In this study, e et al.  separatee et al.  reportedof Hilsa . These r applied .F statistics (FST) and AMOVA could not clearly demarcate the population structure among populations from Bay of Bengal. Mazumder and Alam [The genetic marker most commonly used to elucidate population structure in fish has been control region and was considered sensitive enough to test for structure among most populations due to rapid accumulation of mutation in the control region as well as the simplicity of mtDNA inheritance through maternal line . Howeverand Alam  used RFLand Alam . Hilsa.The populations are supposed to be separated by the philopatric behaviour. Phylogeographical structure defined by AMOVA and region specific haplotypes distinguished populations up to sea level  but population structuring at basin level was not noticed using D-loop as a marker so we suggest the use of some moderately evolving markers such as ATPase 8/6, nicotinamide dehydrogenase subunit 2 (ND2), and microsatellites for elucidating population structure of


Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae) was introduced into China more than 60 years ago, the genetic diversity and structure of this exotic insect pest and virus vector have not been studied. To investigate the population genetic characteristics of this invasive species and to identify potential invasion routes, the genetic diversity and population structure of 17 collections of T. vaporariorum from nine provinces in China were analyzed using seven microsatellite loci. The results of the analyses indicated that the genetic diversity for the populations examined from the four provinces: Jilin, Ningxia, Guizhou and Qinghai, was lower than the genetic diversity of populations from the five provinces: Yunnan, Shandong, Shanxi, Liaoning, and Gansu. The T. vaporariorum populations analyzed in this study grouped as two distinct genetic clusters based on the analysis using STRUCTURE, whereas, 8 clusters were identified based on the BAPS analysis. Of the 136 genetic distance (Fst) values, 128 (94%) were associated with a significant exact test. There was a significant relationship between Fst and geographical distance. These results demonstrate that populations of T. vaporariorum in China exhibit significant genetic differentiation, indicating the likelihood that multiple introductions of T. vaporariorum into China have occurred. Also, the populations collected from the provinces of Jilin, Ningxia, Guizhou and Qinghai appear to represent secondary introductions originating from other Chinese provinces.Though the greenhouse whitefly,  Trialeurodes vaporariorum (Westwood), which has its origin in tropical or subtropical America , is an economically important pest of horticultural and ornamental crops, worldwide [The greenhouse whitefly, orldwide ,3,4,5,6.orldwide ,11,12, aorldwide .T. vaporariorum was first discovered in Beijing during the 1940s. During the early 1960s, it was found in Tianjin, and then in Inner Mongolia in the 1970s [In China, he 1970s . Since the 1970s , it has he 1970s . Though Frankliniella occidentalis (Pergande) has revealed the invasion route and potential source of a secondary introduction in China [Extensive studies using molecular markers have provided estimates of the potential invasion routes, and of genetic characteristics of a number of invasive species ,17,18. Fin China , which cin China ,21. Micrin China ,22,23,24T. vaporariorum within China. To accomplish this, the genetic structure of 17 populations collected from nine provinces in China was analyzed using seven microsatellite loci.The objective of this study was to determine the genetic structure and subsequent spread of the greenhouse whitefly, Na) ranged from 2.1429  to 5.000 , and the effective number of alleles (Ne) ranged from 1.4428  to 2.4447 . The expected heterozygosity (He) ranged from 0.2324  to 0.5061 , while the observed heterozygosity (Ho) ranged from 0.1068  to 0.4550 . The value of He in each population was higher than the value of Ho. Nei’s expected heterozygosity (Nei) ranged from 0.2276  to 0.4945 . The values of genetic diversity indexes of the Chinese populations are provided in He, at 0.5061, while the population of Changchun, Jilin (JL3) had the lowest value at 0.2324. The populations from Yunnan (YN1-YN3), Shandong (SD), Shanxi (SX), Liaoning (LN), and Gansu (GS1-GS2) exhibited a He value of greater than the mean value of He 0.3683. In contrast, the He values of the other 9 populations from four provinces  were lower.The population of Yuxi, Yunnan (YN3) had the highest value of Fis, was not significant, indicating that there was no sub-structure within all populations (p < 0.05) was detected in the six populations: QH1, QH2, GS1, YN1, YN2, and JL1  were associated with a significant test  to 0.5194 (QH3/JL3). Obvious variation in allelic diversity and heterozygosity was found among the different populations   .K method was two  were associated with a significant exact test. However, the significant correlation was observed between Fst and geographical distance, suggesting that the spread of the populations was most likely associated with natural dispersal. The Fst data and analyses support the hypothesis that the introduced populations represent several distinct genetic lineages. Three possible explanations can be considered to explain the genetic differentiation of T. vaporariorum populations in China. One possible explanation could be a low rate of gene flow among the populations, which is known to result in the rapid genetic differentiation. The second possibility is that whitefly experienced rapid evolution to facilitate adaptation to new environments following the introduction. The genetic drift and natural selection resulting from exposure to some new plant hosts, climate, and management practices, all features that differ among regions in China, may have resulted in the selection of different variants [Of the 136 variants .He values indicates that the populations from four provinces: Jilin, Ningxia, Guizhou, and Qinghai might represent a secondary introduction stemming from the Yunnan, Shandong, Shanxi, Liaoning, and Gansu populations. When the results are considered together with the STRUCTURE and BAPS analysis, Jilin populations appear to have come from Shandong, whereas, the Ningxia population is more closely associated with Qinghai. However, it is not clear which population represents the primary introduction. Further, an association between a single population pair (the LN population from Liaoning and YN population from Yunnan) was observed, which can be explained by long distance transport of infested plant materials.The genetic diversity analysis based on T. vaporariorum in China. This is based on the observation that only 6 of the 17 populations exhibited significant heterozygosity excess, the signature of a bottleneck (under the IAM model), further because heterozygosity excess was not predicted under either the TPM or SMM models.The significant deviation from Hardy-Weinberg equilibrium (HWE) could have arisen from inbreeding, subpopulation structure, and/or null alleles. Null alleles can be ruled out as an explanation in this study. Our study revealed that only two of the seven loci had low frequencies of the null alleles and the estimated null allele frequency ranged from 0.09 to 0.19 among all populations. Dakin and Avise  concludeOverall, the genetic diversity and the distinct genetic structure revealed by the cluster analysis suggest the likelihood of multiple introductions into China since the initial introduction of this exotic species 60 years ago.T. vaporariorum were collected from cucumber, eggplant, and kidney bean plants in nine provinces throughout China during 2011–2012 , and stored at −20 °C. The T. vaporariorum-specific primers  were used to amplify the mitochondrial cytochrome oxidase I gene fragment (752 bp in size) to determine the identity of each whitefly individual (patent number is CN201310192726.5), resulting in the confirmation of 413 individuals as the whitefly T. vaporariorum. The PCR reactions were performed in 20 μL buffer containing 2 μL 10× buffer, 1.5 mM MgCl2, 0.2 μM dNTPs, 1 unit Taq DNA polymerase, 2 μL template DNA and 0.2 μM of each primer. PCR amplification was carried out as follows: initial denaturation at 94 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 60 s at 72 °C, and a final elongation step at 72 °C for 30 min.Genomic DNA was extracted from each individual adult T. vaporariorum DNA as the template [2, 0.2 μM dNTPs, 1 unit Taq DNA polymerase, 2 μL template DNA and 0.2 μM of each primer. PCR amplification was carried out as follows: initial denaturation at 94 °C for 4 min, followed by 35 cycles of 30 s at 94 °C, 90 s at the primer-specific annealing temperature  were used to amplify the microsatellite loci using template . The priperature  and 60 sT. vaporariorum, the average number of alleles per locus (Na), the effective number of alleles (Ne), the observed heterozygosity (Ho), the expected heterozygosity (He), and Nei’s expected heterozygosity (Nei) were calculated using POPGENE v.1.31 [Ar) [For each of the 17 populations of E v.1.31 . The pro.31 [Ar) .Fis [The Weir and Cockerham’s Fis  within eFis . ConformFis .Deviation of the mutation-drift equilibrium in the populations was tested using the approach in BOTTLENECK software . The hetFst values. The Weir and Cockerham’s estimator of the fixation index Fst [Fst among 17 populations. The genetic clustering of samples was examined using STRUCTURE v.2.3.2 and BAPS v.4.14 [K value, ranging from K = 1 to 17, and ΔK was used to calculate the optimal number of genetic clusters (K) [The traditional population differentiation approach was based on ndex Fst  was calcndex Fst . The corndex Fst .The distndex Fst , and by S v.4.14 ,28,29. Pters (K) . When esT. vaporariorum in China exhibit significant genetic differentiation, indicating the likelihood that multiple introductions of T. vaporariorum into China have occurred. Also, the populations collected from the provinces of Jilin, Ningxia, Guizhou and Qinghai appear to represent secondary introductions originating from other Chinese provinces.These results demonstrate that populations of