Lilium cernuum, and compared levels of allozyme-based genetic diversity of five populations from NE China with five populations from South Korea (home of its hypothesized refuge areas). As a complementary tool, we used the maximum entropy algorithm implemented in MaxEnt to infer the species’ potential distribution for the present time, which was projected to different past climate scenarios for the Last Glacial Maximum (LGM). Permutation tests revealed that Korean populations harbored significantly higher levels of within-population genetic variation than those from NE China . Our results suggest that the lowered levels of genetic diversity in NE Chinese populations might be due to founder effects associated with post-glacial migration from southern regions. Congruent with genetic data, past distribution models showed higher probability of occurrence in southern ranges than in northern ones during the LGM. In addition, a positive correlation was detected between the expected heterozygosity and environmental LGM suitability. From a conservation perspective, our results further suggest that the southern populations in South Korea may be particularly worthy of protection.The so-called “Baekdudaegan” (BDDG), a mountain range that stretches along the Korean Peninsula, has been recently proposed as a major “southern” glacial refugium for boreal or temperate plant species based on palaeoecological and, especially, genetic data. Genetic studies comparing genetic variation between population occurring on the BDDG and more northern ones (i.e. in NE China and/or in Russian Far East) are, however, still too few to draw firm conclusions on the role of the BDDG as a refugium and a source for possible northward post-glacial recolonizations. In order to fill this gap, we selected a boreal/temperate herb,  Unglaciated regions and/or regions that provided relatively stable environmental conditions during the Last Glacial Maximum  are generally regarded as candidates for glacial refugia for plant species, at least in the Northern Hemisphere . TherefoIn agreement with these expectations, a large body of studies—especially conducted in Europe—have revealed a common pattern of latitudinal decrease of genetic variation, which is often referred to as “southern richness vs. northern purity” ,6,7,8,9.On the Korean Peninsula, the so-called “Baekdudaegan” (hereafter the “BDDG”) is undeniably one of the most important mountain systems in East Asia in terms of length, ecological value, and sacredness ,12. Morein situ in northern areas, we may expect similar levels of genetic diversity in northern and southern populations.Broadly, two scenarios could be proposed in relation to the levels of within-population genetic variation of boreal/temperate plant species that are native to the BDDG but that also occur in more northern latitudes. If a given species vanished from northern areas during the LGM and then recolonized the area through post-glacial migration from its putative refugia in the BDDG (see below), we may expect diminished levels of genetic diversity in populations located at more northerly latitudes, due to founder effects. Alternatively, if the species would have also endured the LGM Lilium cernuum Kom. (Liliaceae). We believe that this is a good study system because of the following reasons. First, the species is distributed from Primorsky Krai in Russian Far East, Jilin and Liaoning provinces in NE China to the south of the Korean Peninsula ;;Lilium cninsula  are solitary or arranged in racemes of 2–7, nodding, and fragrant, and open in July. Fruits (capsules) are globose to obovoid, 1.5 cm long. The breeding system and pollinators are unknown  but, to our knowledge, it is not protected because it was not included in the laws of 1984  and 1999 . In South Korea, L. cernuum was delisted as an endangered species in 2012. Now it has been listed as “LC” (“Least Concern”), i.e., not considered threatened.n = 207) and five populations from South Korea (n = 217), and these were sampled from areas of similar size to prevent area effects (n = 43 vs. 34). In addition, the resolved allozymes are not the same .We selected five populations from NE China , mean number of alleles per locus (A), allelic richness (AR) using a rarefaction method that compensates uneven population sample sizes  values reached a plateau )nce; see ) by meanL. cernuum  were polymorphic across 10 populations, resulting in high levels of genetic variation in pooled samples . Regarding the allelic composition, we identified 22 alleles in the northern populations and 28 alleles in the southern ones; six alleles were exclusive to the Korean populations and none of them to the Chinese ones . Deviation from H–W expectations due to allele frequency differences between populations were, despite low, significantly different from zero for the two regions . However, we found no statistically significant differences between the two groups of populations (P = 0.571). Across 10 populations, the value of FST was 0.133 (P = 0.001). Consistent with these results, the AMOVA indicated that most of the variation resided within populations (84%), with variation attributable to differences between regions (South Korea vs. NE China) and among populations within regions being only 7% and 9%, respectively (We found a significant deficiency of heterozygotes (at the 0.05 level) relative to H–W expectations in all but LC-4 and LC-7 populations . These rectively .K = 2, according to both the ln Pr(X|K) and the ΔK statistic; The UPGMA phenogram  revealedFST/ and logarithm of pairwise linear geographic distances , large parts of the Korea Strait, and southern Japan (L. cernuum north of 40°N (The AUC scores averaged across 20 runs were high (> 0.93) for the four models built , which supported their predictive power. According to the MaxEnt jackknife tests of variable importance, the most informative variables for all models were bio11, bio14, and bio8 (in this order). The present day distributional predictions for rn Japan . Indeed, of 40°N .He and habitat suitability for the studied populations of L. cernuum, for both the present time and the LGM  harbor greater genetic variation than northern ones (NE China)  would not have harbored climatically suitable areas for L. cernuum during the Pleistocene glacial periods. Given the positive relationship between He and habitat suitability in L. cernuum, ENM might constitute a simple and cost-effective surrogate of genetic diversity studies. Although not explicitly tested, some studies with boreal/temperate plant species that are also native to our study region (Korea plus NE China and Russian Far East) also show an apparent congruence between genetic diversity levels and habitat suitability, identifying the mountains of South Korea as a putative glacial refugium [Quercus mongolica [L. cernuum usually occurs.Levels of within-population genetic diversity found in °N or so . Most of°N or so  is inclu°N or so . Thereforefugium ,73. Of tL. cernuum also indicate that the vegetation there adapted to much colder conditions than today. For instance, in a couple of sites at latitude ca. 43°N, in the southernmost tip of Primorsky Krai, boreal forests dominated by Betula  would have occurred [Betula and Picea with the presence of Abies and Pinus) would have occurred [L. cernuum would have not been present in situ in areas such as NE China or Russian Far East during the LGM.The results from the present study are in agreement with a recently published review of the region’s Pleistocene biogeography , in whicoccurred . Similaroccurred . Taken aMore favorable habitats for this lily, instead, would have occurred further south. According to the tentative LGM vegetation map for the Korean Peninsula provided by , non-arbL. cernuum (neither populations from Liaoning Province nor Russian Far East have been studied), there is a clear pattern of higher genetic diversity for the Korean populations compared to the Chinese ones; such results are fully compatible with the genetic studies conducted for species endemic or native to the region, which have been recently compiled by [Hep = 0.159; [Hep for the Korean populations of L. cernuum (0.173) is close to the mean value reported by [Hep = 0.095). Second, the still small but growing body of studies on genetic diversity and phylogeography at regional level including populations located in southern refugia on the Korean Peninsula and populations located further north, are mostly consistent with the “southern richness vs. northern purity” pattern. That is, Korean populations harbor more genetic diversity, ancestral haplotypes, and/or significant amounts of unique haplotypes/alleles  by means of allozyme and RAPD markers. The authors found that levels of within-population genetic variation significantly decreased with increasing latitude. More recently, [Acer mono; both cpDNA and microsatellites suggested that the BDDG was the origin for the recolonization of NE China, a scenario also recovered from ENM [Although the results must be interpreted with caution because of the limited sampling in the northern range of piled by . First, = 0.159; ,78). Theles . However, other populations, such as LC-7 (Mt. Deokhang), would enjoy large effective population sizes (Ne) [FST values both at local , as alsble 1 in . Therefozes (Ne) . The lowf <1 km; ), countrf <1 km; ) and regf <1 km; .Ne) and rates of gene flow (m) are higher at the range center compared to range margins [Even being relatively isolated geographically at the southern tip of the species’ distribution, the Korean population LC-10, that occurs at a high-elevation mountain within the BDDG , has the lowest amount of genetic diversity among the five southern populations (but still higher than the northern populations). On the other hand, the populations LC-7 (Mt. Deokhang) and LC-8 (Yongyeon Cave), located approximately at the center of the current range for the species within South Korea, maintain the highest levels of genetic diversity among the studied populations. As mentioned above, populations around LC-7 maintain large effective population sizes on the order of hundreds . These f margins . Histori margins , fit wel margins . Accordi margins ) and, thL. cernuum), stressing the importance of empirical genetic studies in plant species [L. cernuum should not be regarded as genetically extremely depauperate. Reference values for plants in general .In contrast to some populations distributed along the ridge of the BDDG, populations from NE China and Russian Far East that are close to the border with North Korea are relatively large and continuously distributed . Given present-day population sizes, population genetics theory predicts that genetic diversity should be higher in the populations from NE China compared to populations from South Korea. However, our results show the opposite pattern , plants= 0.118; ), and sh= 0.096; ) are onlFIS and t (outcrossing rates) or s (selfing rates) (FIS = s/(2 − s) or FIS = (1 − t)/(1 + t); [t of 0.688 for L. cernuum, suggesting a mixed mating system in the species. The occurrence of selfing, however, might not be the only factor explaining the observed heterozygote deficiency at most populations, as other factors, including non-random mating with relatives  and Wahlund effect (population subdivision), may account. Because NE Chinese populations of L. cernuum have been observed to be large and continuous , the higher levels of inbreeding for NE Chinese (FIS = 0.280) compared to South Korean (FIS = 0.136) populations are probably indicating different reproductive behavior between the two groups instead of population subdivision. Detailed field work would be necessary to see whether there is reduced pollinator service in the northern populations compared to the southern ones. The significantly larger FIS estimates found in the NE Chinese populations compared to those in the southern ones may have conservation implications, as inbreeding may lead to reduced fitness [If we use the equilibrium equation between (1 + t); ,85), we  fitness .The loss or reduction of genetic diversity is of particular concern for conservationists and managers because it may lead directly to the decrease of biological fitness of populations. Thus, maintenance of genetic diversity is often viewed as a prerequisite for adaptation to environmental changes  and, thuL. cernuum vanished from the Chinese provinces of Jilin and Liaoning, and Primorsky Krai in Russian Far East during the LGM, and that the species was only able to persist in southern glacial refugia, presumably located along the BDDG in South Korea. Our results are in concordance with previous studies showing that a pattern of survival in southern refugia followed by post-glacial recolonizations would have been common for the boreal and temperate flora of NE Asia [L. cernuum in South Korea are particularly worthy of protection.Our results from genetic and ENM analyses support the hypothesis that  NE Asia ,12. Our S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 TableP values of sign and Wilcoxon sign-rank tests conducted using the program BOTTLENECK.Numbers reported are (DOCX)Click here for additional data file.


Lygus pratensis (L.) is an important cotton pest in China, especially in the northwest region. Nymphs and adults cause serious quality and yield losses. However, the genetic structure and geographic distribution of L. pratensis is not well known. We analyzed genetic diversity, geographical structure, gene flow, and population dynamics of L. pratensis in northwest China using mitochondrial and nuclear sequence datasets to study phylogeographical patterns and demographic history. L. pratensis (n = 286) were collected at sites across an area spanning 2,180,000 km2, including the Xinjiang and Gansu-Ningxia regions. Populations in the two regions could be distinguished based on mitochondrial criteria but the overall genetic structure was weak. The nuclear dataset revealed a lack of diagnostic genetic structure across sample areas. Phylogenetic analysis indicated a lack of population level monophyly that may have been caused by incomplete lineage sorting. The Mantel test showed a significant correlation between genetic and geographic distances among the populations based on the mtDNA data. However the nuclear dataset did not show significant correlation. A high level of gene flow among populations was indicated by migration analysis; human activities may have also facilitated insect movement. The availability of irrigation water and ample cotton hosts makes the Xinjiang region well suited for L. pratensis reproduction. Bayesian skyline plot analysis, star-shaped network, and neutrality tests all indicated that L. pratensis has experienced recent population expansion. Climatic changes and extensive areas occupied by host plants have led to population expansion of L. pratensis. In conclusion, the present distribution and phylogeographic pattern of L. pratensis was influenced by climate, human activities, and availability of plant hosts. Cytoplasmic and nuclear data combined with coalescent theory are commonly used for phylogeographic studies. They are powerful tools for evaluating the possible influence of climate changes, geological events, environmental changes and hosts on extant population structure and tracing the possible evolutionary history of species . MoleculBactrocera dorsalis  from 286 adults individuals, originating from nine populations covering the main distribution region of this species in China. Our aims were to (i) determine the phylogeographic structure and possible influential factors; (ii) study the demographic history and gene flow among populations; and (iii) identify human activities and ecological conditions responsible for the current population structure and distribution patterns.In this study, we sequenced five DNA fragments , except for the ITS2, and the genes of 5.8S and 28S, which were amplified using primer pairs P1 [COI, KX447219–KX447261 for COII, KX447262–KX447288 for Cytb, KX447289–KX447360 for ITS2 plus 5.8S and 28S, KX447361–KX447403 for ND5.Data for five mitochondrial gene sequences and three ribosomal DNA sequences were obtained by polymerase chain reaction (PCR) amplification. All primers were specifically designed for pairs P1  and 28Z pairs P1 . All seqCOI+COII+ND5+Cytb+16S rRNA and three combined nuclear markers 5.8S+ITS2+28S. For nuclear sequences, allele determination is a major obstacle to phylogeographic usage [S), haplotype diversity (Hd), nucleotide diversity (π), average number of nucleotide differences (K), and number of haplotypes (Ht) were calculated using DnaSP 5.0 [The sequence polymorphisms were assessed by calculating statistical parameters for five combined mitochondrial genes ic usage . Therefoic usage  to inferic usage . This pric usage , and refic usage , Haplotyic usage , HaploReic usage , Arlequiic usage , GCHap [ic usage , Gerbil ic usage  and HAPIic usage . ConsensnaSP 5.0  or ArleqnaSP 5.0  for the K values (number of groups) were set from 2 to 8 and each independent simulated annealing processes was performed with 100 replicates. For each pre-specified K value, the method searches for the best clustering option which was defined as the highest FCT value (the level of genetic differentiation among groups). When SAMOVA analysis did not adequately delimit populations, the population structure was defined according to zoogeographic divisions of China (two geographic units: Xinjiang and Gansu-Ningxia region) for subsequent analysis.The genetic structures of populations were delimited using the SAMOVA 1.0 program  based onFST matrix was obtained using Arlequin software, and then converted into a matrix of (FST)/(1- FST) to obtain linearized estimates. The linear geographic distance (km) was obtained from Google Earth. The Isolation-by-distance (IBD) model was tested using the genetic distance (FST)/(1- FST) matrix and the linear geographic distance (km) across sampling areas, with the Mantel test employing 1,000 randomizations in program IBDWS 3.2 [The genetic distance BDWS 3.2 . The meaHidden genetic barriers associated with genetic discontinuities across the sample areas were further analyzed using the maximum difference Monmonier’s algorithm in the Barrier 2.2 software . This prNem: Ne is the effective population size; m is the immigration rate; i.e. ΘM: Θ is mutation-scaled population size; M is mutation-scaled migration rate). Also the gene flow between groups (in the Xinjiang and Gansu-Ningxia region) was assessed based on genetic data. A Bayesian search strategy was used to calculate parameters Θ and M. The parameters were set as follows: long-chains = 1, long-inc = 20, long-sample = 1,000,000, burnin = 100,000, heating = YES: 1: {1.0, 1.5, 3.0, 6.0}, heated-swap = YES and replicate = YES: 5. In order to obtain consistent results four runs of MIGRATE analysis were performed. In the first run, Θ and M were estimated from FST values, in the subsequent three runs, the parameters of the previous run were used as starting values for the next run until a convergent result was obtained. The datasets of Θ, M and ΘM present here were from the final run. In addition, gene flows among populations were also tested by the Arlequin program.To verify whether there is asymmetrical gene flow between populations, MIGRATE 3.6  softwareL. pratensis, we constructed haplotype and allele networks with mtDNA and rDNA datasets in Network 4.6 [In order to study the haplotype and allele phylogeny of work 4.6  based onL. pratensis was studied using parameters of Tajima’s D, Fu’s FS, demographic expansion time (τ1) and spatial expansion times (τ2), which were calculated in Arlequin 3.5 based on two datasets. Tajima’s D and Fu’s FS were used to detect departure from the population equilibrium. For a species, departures from the null model might be caused by many factors. One factor is fluctuation in population size (e.g. population expansion) which results in a large amount of low frequency variants and negative D values. Conversely, processes such as recent population bottlenecks, could result in an excess of intermediate frequency variants and positive D values [FS values indicated that the population may have experienced a recent range expansion [F*, Fu and Li’s D* were determined using DnaSP 5.0.Demographic history of D values , 49. Larxpansion . The parCOI; GTR for COII, Cytb, and 16S rRNA; GTR+I for ND5; and GTR+I+G for the three nuclear fragments, respectively. Trace 1.4 [S were greater than 200, with a burn-in of 10%. A relaxed uncorrelated lognormal molecular clock was applied, with a pairwise sequence divergence rate of 0.0115 per site per million years for the COI gene [7 generations with sampling every 1,000 generations, and the Yule process model was used.We used the Bayesian skyline plot (BSPs) method implemented in BEAST 1.8  to estimrace 1.4  was usedCOI gene  was usedCOI: 856 bp, COII: 740 bp, ND5: 570 bp, Cytb: 910 bp and 16S rRNA: 970 bp) from 286 individuals. No insertions or deletions were detected among the above five fragments. The nuclear dataset included 1942 bp , sequences from 220 individuals that were successfully sequenced. Five sequences were filtered out by CVhaplot 2.0 due to poor quality and 215 sequences were used for analyses.The alignment of the combined mitochondrial dataset included 4,046 bp  were identified. Xinjiang had 49 unique haplotypes, Gansu-Ningxia had 15 unique haplotypes; three haplotypes were shared by populations from both regions  when K = 2   , which cFST)/(1−FST) and geographical distance among populations based on the mtDNA dataset . Similar results were found between Xinjiang and Gansu-Ningxia groups . The relatively low pairwise genetic differentiation and only reached a low level of differentiation was also found between populations  for all populations . Overall, genetic differentiation among populations was low and both defined groups showed weak genetic structure. The mean genetic distance was 0.001 among populations based on the MEGA program.For the mtDNA dataset, the genetic differentiation among all populations was low but significant (ulations  based onNem) were observed between populations, and for the Akesu and Suoche populations, the levels were relatively high (Table F in Nem < 2); the remaining four populations  had high numbers of immigrants (Nem: 5.79–15.86). When the geographical groups (Xinjiang and Gansu-Ningxia) were analysed, the effective population number from Xinjiang (θ = 0.006) was relatively higher than Gansu-Ningxia (θ = 0.002).BI-based analysis using MIGRATE 3.6 showed that all populations are well connected in both directions, with high levels of gene flow based on both datasets . ContrarNem among populations were high except for the Akesu and Kuerle populations. Asymmetrical effective migrants per generation (Nem) were observed among Akesu and the other seven populations, with more individuals emigrating from Akesu to the other regions. Similar asymmetrical migration occurred between Kuerle and the other seven populations , and between Kuerle and each of these three populations   were similar to the above two datasets, high level of gene flow were found among populations (Table H in Nm > 1 (Nm (gene flow) > 1, a population will be no significant genetic differentiation and it is replaced by immigrants [Nm ranged from 1.17 to 288.59 based on the rDNA dataset. The mtDNA dataset showed Nm values ranging from 1.70 to 948.97A high level of gene flow was also demonstrated using the Arlequin program analysis based on the mtDNA and rDNA datasets. A large amount of gene flow values Nm > 1 , indicatmigrants . The Nm Network analysis for the mitochondrial dataset revealed that H2, H3, H5 and H6 haplotypes were the four most frequent haplotypes, which included 12%, 15%, 42% and 9% individuals, respectively . H3, H5 For the nuclear dataset, rH1, rH2, rH3, and rH7 were major alleles, which accounted for 23%, 13%, 15%, and 8% all individuals, respectively. The four main alleles were all shared by the Xinjiang and Gansu-Ningxia populations. All of these were in the star center . This isD, Fu’s Fs, Fu and Li’s D* and Fu and Li’s F* were highly significant whether the nine populations were considered as one unit or they were delimited into the Xinjiang and Gansu-Ningxia groups , which lasted until recently . Time toL. pratensis on different host plants in northwest China, showed that this species mainly feeds on cotton and alfalfa. However, population size is obviously reduced in groups feeding on native host plants . Therefore, we did not examine molecular markers on samples from native host plants of L. pratensis. We only collected samples from cultivated cotton and alfalfa. Native host plants might play a role in the population genetics of L. pratensis, but this possibility was not discussed here because no insect samples from these hosts were available.Studies on the relationship between population genetic structure and host plants of agricultural insect pests are difficult and complicated, especially for species that have diverse host plants and high dispersal capacity. Our previous study of the population dynamics of L. pratensis has relatively high genetic diversity based on the mtDNA and rDNA datasets. The mtDNA dataset indicated relatively high genetic diversity in the Xinjiang region but low genetic diversity in Gansu-Ningxia populations. However, the rDNA dataset indicated similar genetic diversity in both groups. Discrepancies between rDNA and mtDNA genetic markers could be due to the maternal inheritance of mitochondrial DNA; and the ITS rDNA regions evolving more rapidly than the coding regions. These regions would therefore show more polymorphisms which would be useful for phylogeographical studies. We suggest that the relatively high genetic diversity in the Xinjiang region based on the mtDNA dataset is related to the host plants. The abundance of cotton hosts provides ample food for L. pratensis, resulting in successful reproduction, high rates of population growth, and large populations. Neutrality tests also indicated that the Xinjiang populations have experienced recent expansion. These results demonstrate that larger populations tend to possess high genetic diversity compared to smaller populations [Compared to other mirid species , 5, L. pulations .Θ value) and recent population expansion  it was divided. The time to the most recent common ancestor of both groups occurred in MIS3 periods, and the amounts of shared haplotype/alleles among current populations all support this conclusion. In addition, the significant IBD patterns were found based on the mtDNA dataset. This pattern usually results from limited gene flow and increased genetic differentiation among geographical habitats. However, high levels of gene flow and low genetic differentiation were found based on the mtDNA dataset in L. pratensis from our sample regions.The geographical division between Xinjiang and Gansu-Ningxia was studied using mtDNA analyses. The overall genetic structure was weak; and nuclear DNA did not show the expected genetic pattern. Instead, nuclear DNA showed a lack of distinct genetic structure across the entire sample region. orphisms . We specL. pratensis populations is related to host plant introduction events. For example, cotton and alfalfa were introduced into China relatively recently. Asiatic cotton from Southeast Asia was introduced to China in 221 B.C. (during the Qin and Han dynasties) and cotton was intensively cultivated from 1840 to 1921 (i.e. the end of the Qing Dynasty) [L. pratensis, we found that cotton is one of the most important hosts in the Xinjiang region. However, in the Gansu-Ningxia region, alfalfa is the most important host. We suggest that the short time period of differentiation on alternate hosts might contribute to the weak genetic structure. The genetic data support this hypothesis; the most common haplotype H5 was predominant over other haplotypes (S3 Table), and this occurred in all populations. In contrast, haplotype H2 was exclusive to the Xinjiang populations and a large number of unique haplotypes (30) were found in these populations. The biased distribution of haplotypes among populations and reduced divergence indicated lineage sorting of the five mitochondrial loci in populations. In addition, introgression may disturb the reciprocal monophyly of populations and should occur when there is a high level of gene flow.Our original hypothesis was that the recent divergence among Dynasty) ; AlfalfaDynasty) . During L. pratensis may result from geography exerting different selective pressures within each region. The complex landscape of Xinjiang, including the Tian Shan Mountains, Dzungarian Basin (with the Gurbantunggut Desert), and Tibetan Basin (with the Taklamakan Desert), present diverse habitat climate types for L. pratensis. The Tian Shan Mountains may have had an important effect on the current species distribution and this has been demonstrated in previous studies [L. pratensis was not prevented by the Tian Shan Mountains. Therefore, we suggest that the homogeneity of populations was more likely associated with the high dispersal ability of L. pratensis and the effects of human activities.Secondly, the genetic structure of  studies . HoweverNesidiocoris tenuis [Grapholita molesta [Dermacentor variabilis [L. pratensis. In ancient China , the Hexi Corridor was an important corridor for traffic between inland China and the Western Region; it is now among the most centralized areas of the economy related to transportation, communication, education, and information [L. pratensis among populations in western China and increased the level of gene flow.Long distance dispersal of insects by human transport or host dissemination has been documented in s tenuis , Graphol molesta , and Derriabilis . The Hexormation , 66; as ormation . AlfalfaL. pratensis. However, the major factors affecting the population expansion of L. pratensis remain unclear. We propose that humidity, precipitation, and host plant availability might be the main factors limiting population expansion. Du (1996) found that air temperature has increased in winter and precipitation has increased in the summers since 1977 in the western arid region of China [L. pratensis. L. pratensis has a period of rapid population growth from June through September and increasing rainfall in the summer can benefit population growth. In conclusion, increased precipitation and availability of numerous host plants might have resulted in L. pratensis population expansion.The Neutrality test showed significant negative parameters. A star-shaped network and BSPs analysis indicted a period of rapid population growth. These results indicate recent population expansion in of China . In addiof China . These sS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe genetic barriers are shown in red line ‘a’.(TIF)Click here for additional data file.S4 FigThe genetic barriers are shown by red line ‘a’.(TIF)Click here for additional data file.S1 FileLygus pratensis from each site (Table A). Primer sequences used for amplification in this study (Table B). Haplotype frequency by population based on the mtDNA dataset of Lygus pratensis (Table C). Pairwise FST values of nine populations of Lygus pratensis based on the rDNA dataset (Table D). Migration parameter  estimates for nine populations of Lygus pratensis based on the rDNA dataset (Table E). Number of effective migrants per generation (Nem) for nine populations of Lygus pratensis based on the mtDNA dataset (Table F). Number of effective migrants per generation (Nem) for nine populations of Lygus pratensis based on the rDNA dataset (Table G). Migration parameter  estimates for nine populations of Lygus pratensis based on the combined mtDNA and rDNA datasets (Table H). Number of effective migrants per generation (Nem) for nine populations of Lygus pratensis based on the combined mtDNA and rDNA datasets (Table I).Collection information of (DOCX)Click here for additional data file.


Gadus morhua), inversions of several megabases have been identified that dominate genomic differentiation between migratory and nonmigratory ecotypes in the Northeast Atlantic. Here, we show that the same genomic regions display elevated divergence and contribute to ecotype divergence in the Northwest Atlantic as well. The occurrence of these inversions on both sides of the Atlantic Ocean reveals a common evolutionary origin, predating the >100 000-year-old trans-Atlantic separation of Atlantic cod. The long-term persistence of these inversions indicates that they are maintained by selection, possibly facilitated by coevolution of genes underlying complex traits. Our data suggest that migratory behaviour is derived from more stationary, ancestral ecotypes. Overall, we identify several large genomic regions—each containing hundreds of genes—likely involved in the maintenance of genomic divergence in Atlantic cod on both sides of the Atlantic Ocean.Chromosomal rearrangements such as inversions can play a crucial role in maintaining polymorphism underlying complex traits and contribute to the process of speciation. In Atlantic cod ( Genomic islands of divergence  but can Drosophila (Helianthus sunflowers (Anopheles mosquitoes (Agrodiaetus butterflies (Gadus morhua L.)  . The facrhua L.)  suggestsHere, we investigate Atlantic cod populations from both sides of the Atlantic Ocean  that havWe sampled 316 Atlantic cod , consist−1. All samples were individually genotyped using a 12K Illumina SNP chip for which 8165 SNPs were polymorphic in this data set, had a call rate of >95% and showed Mendelian inheritance in a separate set of individuals with a pedigree. Out of these SNPs, 602 were close to selected candidate genes, 1470 were nonsynonymous SNPs and the remaining 5857 SNPs were randomly distributed throughout the 23 different linkage groups (LGs). Genotype clustering was performed in Genome Studio 2011.1 . The nomenclature of the LGs follows www.ncbi.nlm.nih.gov/snp).DNA was extracted from muscle tissue using the E.Z.N.A Tissue DNA kit  and normalized to 100 ng μlHo and He) within each population were calculated in ARLEQUIN 3.5.1.3  and the median q-value. As outlier tests may have a high rate of false positives because of the effects of population demography and bottlenecks  were selected using PLINK v1.07  within all identified groups, and nucleotide divergence between these groups (DXY), using a sliding windows approach with a 50-SNP window and 10 SNPs per iteration in DnaSP 5.10  on the different data sets. Delta K and the best K-value for each data set in STRUCTURE was identified with CLUMPAK , using all 8165 SNPs were performed, using the ADEGENET . The corr2 estimate using PLINK, was evaluated in all populations separately and within the identified groups , in any of the populations  were generally orders of magnitude larger than those based on the neutral SNPs in any pairwise comparison and only the Can-S_BB comparisons with Can-S_SB and Can-S_GM were not significantly different from zero. Elevated FST values predominantly occurred within distinct regions in LGs 2, 7 and 12  in the Northwest Atlantic populations, primarily in LGs 1, 2 and 7 in the Northeast Atlantic populations, and in a distinct region in LG23 and in a few SNPs in LG11 between the two continents (DXY) patterns observed  between Northwest and Northeast Atlantic populations, using both the neutral and the outlier data sets (K=3) revealed that the NCC population is distinctly different from the other Northeast Atlantic populations. Further neutral population structuring within both the Northwest and Northeast Atlantic was evident from the network analyses  in the Northwest Atlantic populations, 361 SNPs (4.4%) in the Northeast Atlantic, and 518 SNPs (6.3%) in all pairwise population comparisons  as candidates for divergent selection  (Oncorhynchus mykiss) (Solenopsis invicta) (Gasterosteus aculeatus) (Anopheles mosquitoes (Clupea harengus) across the Atlantic Ocean  known to be expressed in early-stage embryos of Danio rerio  have been described as reflecting a differentiation similar to that reported between NEAC and NCC populations  between Frontal and Coastal ecotypes, whereas significant nonneutral divergence  are observed (FST=0.0547). Both the Frontal and the Coastal ecotypes are inshore cod that spawn at the same spawning grounds and at the same time. The observed pattern with no neutral divergence, and significant nonneutral divergence, is consistent with a divergent selection hypothesis where individuals potentially interbreed at the spawning grounds followed by de novo selection, discriminating the two ecotypes.Even though a distinction between Coastal and Frontal ecotypes in Icelandic waters has been investigated recently , but also to some extent by differences in the LG1 inversion. This is consistent with results reported by FST values was observed between spring and winter spawning populations within the LG2 region , relative to the neutral trans-Atlantic divergence (FST=0.081). This suggests local selection pressures acting differently on both of the variant on each side of the Atlantic, but less so for the I/I variant that is associated with migratory behaviour in the Northeast Atlantic. The fact that we detect a similar trans-Atlantic pattern at the LG1 inversion is consistent with The inversion in LG1 is involved in ecotype divergence of Northeast Atlantic populations  and NCC populations that may seem inconsistent with the proposed association with migratory behaviour. However, this might reflect the fact that the Ice_C ecotype presumably has a NEAC origin that consists predominantly of the I/I variant. Hence, the standing genomic divergence may not contain sufficient variation-needed for adaptation. Alternatively, the selection pressure on this genomic region might not be strong enough to cause significant differentiation within the selection timeframe.SNPs within the presumed inversions in LGs 2 and 7 have previously been linked to temperature  is high relative to the I/I variant (FST=0.076) and the neutral trans-Atlantic divergence (FST=0.081) that may indicate local selection pressure acting differently on the NI variant in Northeast and Northwest Atlantic.The presumably inverted genomic region in LG12 has recently been used to discriminate between two Atlantic cod stocks inhabiting the Norwegian Skagerrak coast , the I/I variant is almost fixed in LGs 2 and 7 (frequency: 0.98 and 0.94). As a result, almost all variation within the putative LG 2 and 7 inversions will be collinear, allowing for normal recombination to take place. As such, the genomic divergence between Coastal and Frontal ecotypes within these regions may provide valuable insight into the actual targets of selection, because the normal limitations associated with highly linked genomic regions here are omitted. The highest FST values between Coastal and Frontal ecotypes are found in a SNP close to Synaptotagmin (SYT3) in LG2 (FST=0.1152) and in two SNPs (FST=0.1240 and 0.1222) associated with Tyrosinase (TYR), one of which is nonsynonymous in LG7. SYT3 is known to show different expression patterns in resident ‘sneaker’ individuals of Atlantic Salmon  relative to normal migratory individuals —a gene known to mediate dim light vision—is strongly divergent between the two Icelandic ecotypes, indicating an involvement of visual systems in local adaptation of Atlantic cod  all of the inversion events occurred before the split between Northeast and Northwest Atlantic cod populations, ∼100 000 years ago; (2) the nonmigratory/Can-S group is always dominated by the ancestral collinear inversion genotype (NI/NI) containing the highest nucleotide diversity; (3) nonmigratory behaviour in the Northeast Atlantic appears to be ancestral to migratory behaviour; and (4) inshore Icelandic ecotypes have a presumed NEAC origin. As such, we provide fundamental insight into the evolution of distinct morphs and ecotypes of Atlantic cod with different life-history strategies across the trans-Atlantic barrier. Overall, the data indicate a central role for a few distinct large genomic regions, presumably inversions. The genomic content of these regions are targets of selection, likely to be involved in generating and maintaining adaptive divergence and population differentiation among Atlantic cod throughout its distribution range.www.ncbi.nlm.nih.gov/SNP/. All individual genotype data are available from the Dryad data repository (http://dx.doi.org/10.5061/dryad.b20ps).All SNPs are referred to by their ss# or rs# available in dbSNP at


DNA to better understand the current status of those goat genetic diversity and the effects of natural landscape in fashion of domestic animal genetic diversity. The genetic variability of 16 goat populations in the littoral zone of the Yangtze River was estimated using 21 autosomal microsatellites, which revealed high diversity and genetic population clustering with a dispersed geographical distribution. A phylogenetic analysis of the mitochondrial D‐loop region (482 bp) was conducted in 494 goats from the Yangtze River region. In total, 117 SNPs were reconstructed, and 173 haplotypes were identified, 94.5% of which belonged to lineages A and B. Lineages C, D, and G had lower frequencies (5.2%), and lineage F haplotypes were undetected. Several high‐frequency haplotypes were shared by different ecogeographically distributed populations, and the close phylogenetic relationships among certain low‐frequency haplotypes indicated the historical exchange of genetic material among these populations. In particular, the lineage G haplotype suggests that some west Asian goat genetic material may have been transferred to China via Muslim migration.The objective of this study was to assess the genetic diversity and population structure of goats in the Yangtze River region using microsatellite and mt The goat is also one of the major landmarks in the evolution of human modes of subsistence  and the Animal Protection Law of China. In total, 426 individual blood samples from goats in 16 populations in the littoral zone of the Yangtze River Table  was usedAll goats were genotyped using twenty‐one microsatellite markers Table  as recomNA), observed (Ho) heterozygosity, expected heterozygosity (HE), polymorphism information content (PIC), and inbreeding coefficient (FIS) were estimated using FSTAT 2.9.3.2   with a total reaction volume of 50 μl, containing 150 ng of DNA, 25 μl of 2× PCR‐Mix , 20 pmol/L each of the forward and reverse primer, and ddH2O to bring the volume up to 50 μl. The PCR protocol was as follows: an initial denaturation step at 95°C for 3 min, followed by 28 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min. The final cycle was followed by a 72°C extension for 10 min. Tanyibiotech  performed the sequencing using CAP‐F.The mtDNA D‐loop variants of 326 related goats are shown in Table AF533441 as the reference sequence. DnaSP5.10 , lineage B (EF617965 and DQ121578), lineage C (AY155708 and DQ188892), lineage D (AY155952 and DQ188893), lineage F (DQ241349 and DQ241351), and lineage G (EF618084)) from a previous study .The sequence alignments of the D‐loop region in all goats were constructed using Clustal X software (1.83)  to 0.698 (MAF065) and 0.640 (0.399 (ILSTS029) to 0.768 (SRCRSP5)), respectively. The average PIC across the markers was 0.590, which ranged from 0.364 (ILSTS029) to 0.721 (SRCRSP5)  and was above 0.20 in six populations . Most of the populations (12/16) had a significant p‐value for FIS.The In total, 125 private alleles were distributed across 16 populations and 21 microsatellite markers. The frequency of certain private alleles was high in certain populations. For example, the frequency of the 208‐bp allele SRCRSP15 was 30.43% in the YW population, and the frequencies of the 193 and 194 bp alleles INRA023 were 8.82% and 13.24% in the MG populations, respectively. The 156‐bp allele SPS113 had a frequency of 17.24% in the QH population, and the 217‐bp allele CSRD247 had a frequency of 22.92% in the WG , and the smallest divergence was observed between WG and BJ , had a significantly lower divergence compared with that in populations from geographically distant populations  and separated into Cluster II; the DZ and HW populations, which were collected in Chongqing and separated into Cluster III; the BJ (from Chongqing) and WG (from Yunnan) populations, which were separated into Cluster IV; the UW and NB populations, which were separated into Cluster V; the JY, CN and MG populations, which were sampled in Sichuan and separated into Cluster VI; and the EB and YW populations, which were sampled in Hubei and separated into Cluster VII, Figure b.K ≤ 16 was estimated using the STRUCTURE software by setting the optimal K value to 2 and 8 (Appendice S2). At K = 2, the populations were separated into two different groups; the first group comprised YW, EB, MG, CN, and JY, and the other group comprised the remaining populations. At K = 8, the population structure was separated into seven group, which was similar to the groups separated using the neighbor‐joining tree. The population structure analysis using both STRUCTURE and the phylogenetic neighbor‐joining network revealed a population structure without a unanimous ecogeographical distribution  were used for further analyses.To analyze the mtDNA D‐loops of domestic goats in the littoral zone of the Yangtze river, samples from 326 goats from 13 populations were obtained, and the sequences were 582 bp in length as published in the GenBank database (Table We found 117 single‐nucleotide polymorphism (SNP) sites across the 452‐bp alignment sequence, and no insertions/deletions were identified , PIC, and NA were observed in all microsatellite markers across 16 goat populations, indicating that these markers were adequate for estimating diversity within each population. In addition, several markers showed a deviation from HWE and allele privatization, which could have been due to the small sample size or the possible occurrence of recent population genetic events.In this study, high heterozygosity levels , suggesting that these populations could have inbreeding. The positive association between number of markers that deviated from HWE and FIS within population  explains the deficiency of heterozygotes in populations that deviate from HWE. Of course, some other reasons could explain the deviation, including nonrandom mating, selection, genetic drift, and small population size. In this study, the populations came from different areas and all were kept in conservation farm as large populations with systematic nonrandom mating. In addition, it is well known that inbreeding occurs within populations when some individuals in large populations separate into smaller flocks or are improperly managed by humans. Thus, nonrandom mating should be the major reason for the deviation from HWE and high FIS.Using 21 markers in sixteen goat populations, a high diversity was observed within the populations according to the In addition, the sample size, as one of reason above, in this study could not completely represent the real diversity of each population or other types of population genetic events that occurred within the goat populations. Moreover, certain high‐frequency private alleles within populations indicated that these populations could have independently undergone long‐term domestication history  in this study, the distribution of FST showed low genetic divergence (not significant) among populations in general. The EB population, which is an indigenous breed of goats that live on the Enshi Grand Canyon in Hubei, was highly divergent from most populations. Thus, we inferred that this population had a lower gene flow than the other ecogeographic populations due to the specific geographical structure of their habitat. In addition, three populations  in Sichuan were less genetically diverge than the other populations, which could be because Sichuan is an important economic cultural center and a hub in West China, which could easily promote the gene fowl between domestic animals  and the Hubei populations (EB and YC), which grouped together into Cluster II and Cluster VII, respectively. These results were consistent with many previous reports regarding the different population relationships among goats , in the CM population was clustered into lineage G in this study. Lineage G has previously only been observed in goats in West Asia (Iran), the Eurasian continental shelf (Turkey), and the Asian‐African continental shelf (Egypt). This finding supports forward an inference and hypothesis that the local Qinghai goat population has experienced gene flow from abroad. This gene flow could be explained by the large‐scale migration of Muslim people into mainland China from West Asia, which resulted in the exchange of genetic material and caused the presence of lineage G in the Qinghai goat population , particularly during the Han Dynasty, and Zhang Qian built the Silk Road in the western region, which made an extraordinary contribution to the development of the economy and cultural exchange in Asia, Europe, and Africa , there was a dispersed geographical clustering, indicating that a high frequency of genetic material exchange and gene flow among these populations of goats occurred. In particular, we discovered lineage C mtDNA in the southwestern goat populations and lineage G mtDNA in the Qinghai goat population, which was inconsistent with previous studies. Based on our results, we infer that high anthropic movement and migration related to the acclimation process of domestic animals occurred. Therefore, the results of this study are helpful for the understanding of the domestication process of domestic animals in South China and can be used to support new, planned strategies for their conservation.None declared.Yong‐Fu Huang, Guang‐Xin E, and Li‐Peng Chen participated in the experimental design and wrote the manuscript; Yong‐Ju Zhao, Ming‐Xing Chu, Yue‐Hui Ma, Jia‐Hua Zhang, Hui‐Jiang Gao, Yuan‐Zhi Sun, and Mei‐Lan Jin performed the laboratory experiment and carried out the bioinformatic data analysis; Gao‐Fu Wang, Hang‐Xing Ren, Peng Zhou, Ji‐Jun Guo, Huai‐Zhi Jiang, Lan Zhu, and Yan‐Guo Han conducted sample collection; Cao‐De Jiang, Qiong‐Hua Hong, Xiang‐Long Li, and Lan‐Hui Li have read and approved the final manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Dingoes play a strong role in Australia’s ecological framework as the apex predator but are under threat from hybridization and agricultural control programs. Government legislation lists the conservation of the dingo as an important aim, yet little is known about the biogeography of this enigmatic canine, making conservation difficult. Mitochondrial and Y chromosome DNA studies show evidence of population structure within the dingo. Here, we present the data from Illumina HD canine chip genotyping for 23 dingoes from five regional populations, and five New Guinea Singing Dogs to further explore patterns of biogeography using genome-wide data. Whole genome single nucleotide polymorphism (SNP) data supported the presence of three distinct dingo populations (or ESUs) subject to geographical subdivision: southeastern (SE), Fraser Island (FI) and northwestern (NW). These ESUs should be managed discretely. The FI dingoes are a known reservoir of pure, genetically distinct dingoes. Elevated inbreeding coefficients identified here suggest this population may be genetically compromised and in need of rescue; current lethal management strategies that do not consider genetic information should be suspended until further data can be gathered. D statistics identify evidence of historical admixture or ancestry sharing between southeastern dingoes and South East Asian village dogs. Conservation efforts on mainland Australia should focus on the SE dingo population that is under pressure from domestic dog hybridization and high levels of lethal control. Further data concerning the genetic health, demographics and prevalence of hybridization in the SE and FI dingo populations is urgently needed to develop evidence based conservation and management strategies. Dingoes are controversial in Australia; like many other top-order carnivores, dingoes pose a risk to livestock and are thus extensively managed in the livestock grazing regions of southeastern Australia . There iAs the mainland top-level predator, dingoes play a strong role in shaping the ecosystems of Australia , 9–11. TIn many parts of Australia dingoes are subject to lethal control in an effort to mitigate risks to agricultural activities , 27, 28.Knowledge concerning the genetic health of natural populations, particularly isolated or threatened populations, is important for the development of effective conservation and management programs. Severe inbreeding is a particular risk to conservation efforts because it may lead to inbreeding depression, leaving a population more vulnerable to environmental or demographic fluctuations, and possibly resulting in extinction –43. GeneCurrent genetic phylogenies based upon mitochondrial, Y chromosome and whole genome single nucleotide polymorphism (SNP) data suggest that dogs, wolves and dingoes form a monophyletic group –54. DogsPrevious genetic studies have focused largely upon uniparental haplotypes, using mitochondrial and Y chromosome markers. Mitochondrial control region studies asserted that modern dingo populations were likely the result of a single very homogeneous founder population, possibly even a single pregnant female . HoweverAll applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This research was approved by the Animal Care and Ethics Committee of the University of NSW (Permit Number: 12/36B).To investigate patterns of biogeography, we sampled 25 wild dingoes from five geographical regions: The Kimberley , The Gibson Desert , The Simpson Desert (Northern Territory), Fraser Island (Queensland) and the Australian Alpine region  ; Fig 1. Canis lupus) were included [Samples were genotyped on the 170K Illumina HD Canine SNP array  at the Cornell Genomics Core Facility . Genotypes were called using GenomeStudio  and quality control filtering was conducted in PLINK v1.7 . Specifiincluded , 74. To included . AustralFIS), examining differences in the observed and expected homozygosity levels for each individual were calculated in PLINK v1.7 [IS is a relative comparison between a sample and the reference populations expected and observed homozygosity. Individual inbreeding coefficients were then averaged across geographical populations. Individual inbreeding coefficients were also calculated for only dingoes versus only NGSDs, although it is important to note that NGSDs are known to be inbred based on pedigree.Using ‘Dataset A’, a sex check analysis was also performed in PLINK to confirm gender assignments. As dingoes and NGSD are closely related , inbreedINK v1.7 . FIS is FST values between the four population clusters were calculated in ADMIXTURE v1.23 [ADMIXTURE v1.23  was usedRE v1.23 . ClusterA principal components analysis (PCA) was performed on ‘Dataset A’ in PLINK v1.9 , 80. Thep <10−4 were considered to be strongly associated with population structure in the dingo and NGSD.The phylogenetic relationships between dingoes and NGSDs were investigated using SNPhylo  a pipeliConcatenation is a method of combining sequences from multiple genetic loci and is particularly useful for intraspecific datasets where divergences may be recent . HoweverAdditionally, SNPhylo  was usedW population) incorporated samples from four geographical wolf populations: European, Chinese, Middle Eastern, and Russian. Data from dog populations (the X populations) in Borneo, Vietnam, Portugal and from the Australian cattle dog breed were included in the analysis [Y population and the non-alpine dingo populations were the Z population. Standard error and Z-statistics were also calculated.To investigate the extent of modern domestic dog introgression into dingoes D-statistics were calculated using ADMIXTOOLS . This ananalysis . In the 10.5061/dryad.sq8d0) had a genotyping rate before filtering of 0.974, and 0.990 after filtering. A total of 58,512 autosomal SNPs remained after filtering. A sex check performed in PLINK confirmed the gender identity of samples.Of the 25 dingoes genotyped, two were excluded from the analyses due to >10% missingness. The remaining 23 dingoes from 5 geographical populations and 5 NGSDs (FIS) calculated based on 58,512 autosomal SNP loci suggested that some dingo populations were inbred .Individual inbreeding coefficients , 15.1%  and 9.8%  of the genetic variance respectively and indicate the presence of four population clusters: SE, FI, NW and NGSD. PC1 differentiates NGSDs from dingoes, PC2 separates out SE dingoes and PC3 distinguishes FI dingoes from the other populations . The thrWhen PC analyses were repeated with ‘Dataset B’, we see the same four population clusters: SE, FI, NW and NGSD as well as three new clusters representing the Vietnam and Borneo village dogs, Portuguese dogs and Australian cattle dogs. The top three PC vectors accounted for 37.8% , 9.1%  and 5.5%  of the genetic variance respectively. PC1 differentiates dingoes and NGSD from both Asian village dogs and European dogs while PC2 and PC3 distinguish NGSD from dingoes and Portuguese village dogs from ACD . It is ip<10−4) indicating a strong association between these SNPs and geographical population. These SNPs may be useful for developing a SNP assay to investigate population structure in a larger geographic survey of dingoes. The unrooted ML tree constructed in DNAml [For ‘Dataset A’, SNPhylo identified 4913 SNPs that were variable and ancestry informative. Of these a total of 460 SNPs were identified has having significant Wald test values (in DNAml  identifiCanis lupus) samples as outgroup taxa. The rooted ML analysis in raxmlGUI [To further explore the phylogenetic relationship between dingoes and NGSD a second rooted analysis was completed using ‘Dataset C’, this incorporated 12 wolf  if it is: (1) geographically separated; (2) is genetically differentiated due to reduced gene flow or; 3) carries locally adapted phenotypic traits , 91. FSTivergent . An alte carries ivergent . This maWe used Bayesian clustering, PCA and D-statistics to investigate hypotheses concerning the evolutionary history of dingoes and the presence of domestic dog introgression Hybridization between dingoes and dogs is considered to be widespread in southeastern Australia . There aClustering analyses did not find evidence of admixture within northwestern or Fraser Island dingoes from Asian village dogs or European domestic dogs . This isFIS = 0.04 [FIS = 0.06–0.08 [Inbreeding coefficients can be used to assess the genetic health of a population or species , 39. HigS = 0.04  and wolv.06–0.08 , 109. An.06–0.08 . The SE .06–0.08 .FIS = 0.647–0.732 , mountain pygmy possums (Burramys parvus) and Rocky Mountain bighorn sheep (Ovis canadensis) [Genetic rescue has been used to safeguard inbred populations of threatened species around the world, including Florida panthers (adensis) , 117–120adensis) .Whole genome SNP data indicate that there are at least three divergent dingo populations forming discrete conservation units (ESUs). This builds upon previously published mitochondrial and Y-chromosome studies , 67. FI This study highlights the need to conserve dingoes from all geographical regions of Australia to preserve the full range of genetic diversity and identity of the dingo. However, dingoes in southeastern Australia are in urgent need of protection due to the increased pressures of lethal control and hybridization in the region. Concerningly, FI dingo populations and the captive NGSD population appear to be under threat of inbreeding. Management strategies may need to be adapted to preserve and improve the genetic health of these important canid populations.S1 FigCross validation errors for each K value were averaged across ten independent runs in ADMIXTURE v1.23. Error bars represent standard error calculated across the ten runs. Cross validation error was lowest for K = 4.(PDF)Click here for additional data file.S2 FigCross validation errors for each K value were averaged across ten independent runs in ADMIXTURE v1.23. Error bars represent standard error calculated across the ten runs. Cross validation error was lowest for K = 5.(PDF)Click here for additional data file.S1 TableIdentifier, Geographical Locale, Latitude, Longitude and Genetic Identity for genotyped samples.(PDF)Click here for additional data file.


Michelia yunnanensis Franch., is a traditional ornamental, aromatic, and medicinal shrub that endemic to Yunnan Province in southwest China. Although the species has a large distribution pattern and is abundant in Yunnan Province, the populations are dramatically declining because of overexploitation and habitat destruction. Studies on the genetic variation and demography of endemic species are necessary to develop effective conservation and management strategies. To generate such knowledge, we used 3 pairs of universal cpDNA markers and 10 pairs of microsatellite markers to assess the genetic diversity, genetic structure, and demographic history of 7 M. yunnanensis populations. We calculated a total of 88 alleles for 10 polymorphic loci and 10 haplotypes for a combined 2,089 bp of cpDNA. M. yunnanensis populations showed high genetic diversity (Ho = 0.551 for nuclear markers and Hd = 0.471 for cpDNA markers) and low genetic differentiation (FST = 0.058). Geographical structure was not found among M. yunnanensis populations. Genetic distance and geographic distance were not correlated (P > 0.05), which indicated that geographic isolation is not the primary cause of the low genetic differentiation of M. yunnanensis. Additionally, M. yunnanensis populations contracted ~20,000–30,000 years ago, and no recent expansion occurred in current populations. Results indicated that the high genetic diversity of the species and within its populations holds promise for effective genetic resource management and sustainable utilization. Thus, we suggest that the conservation and management of M. yunnanensis should address exotic overexploitation and habitat destruction. Investigating population genetics, which includes genetic diversity, population differentiation, and degree of gene flow, is crucial in the conservation and sustainable utilization of a species  in Yunnan Province Table  to studyIntact genomic DNA was extracted from dried leaves with the modified CTAB method Doyle, . Extract2 (25 mM), 2.0 μL dNTPs (10 mM), 0.5 μL of each primer, 0.4 μL Taq DNA polymerase , and 16.6 μL double-distilled water. PCR amplifications were performed in accordance with the following conditions: 3 min of initial denaturation at 94°C; 35 cycles of 1 min at 94°C; 1 min of annealing at 50°, 53°, and 54°C for trnL-trnF, psbA-trnH, and matK, respectively; 1.5 min of extension at 72°C; and 10 min of final extension at 72°C. PCR reactions of 10 microsatellite markers were referenced and modified into a total reaction volume of 20 μL that contained 20 ng DNA, 2.5 μL 10 × PCR buffer, 1.25 μL MgCl2 (25 mM), 2.25 μL dNTPs (10 mM), 0.5 μL of each primer, 0.4 μL Taq DNA polymerase , and 11.1 μL double-distilled water. For each reaction, we used the following conditions: initial 5 min of denaturation at 94°C, followed by 20 cycles of 50 s at 94°C, 50 s of annealing at Tm under different primers, 1.5 min of extension at 72°C, and a final extension for 7 min at 72°C.cpDNA PCR reactions were performed in a total reaction volume of 25 μL that contained 15 ng DNA, 2.5 μL 10 × PCR buffer, 1.5 μL MgClThe fluorescent dye  was labeled with the forward SSR primers and visualized on an ABI 3730xl Capillary DNA Analyzer by a professional laboratory . Fragment sizes were assessed using GeneMapper version 4.0. Meanwhile, all purified PCR products by cpDNA markers were bidirectionally sequenced by Sangon in accordance with standard sequencing protocols.* 4.0b10  under a congruency test. The combined cpDNA sequences were used in the following analyses.Sequences were edited and assembled by SeqMan II  and haplotype diversity (Hd) indices. Using ArcGis version 10.2 (http://desktop.arcgis.com), the haplotypes of combined cpDNA sequences were plotted on the map of the region from which the samples were collected. Within-population gene diversity (HS) and gene diversity in total populations (HT) were calculated using Permut version 1.0 (http://www6.bordeaux-aquitaine.inra.fr/biogeco/Production-scientifique/Logiciels/Contrib-Permut/Permut). Meanwhile, the level of population differentiation (GST) and phylogenetically ordered alleles (NST) were also calculated by this software to test for the presence of phylogeographic structures  was used to construct a genealogical haplotype network to estimate the degree of relatedness per haplotype. Indels were treated as single mutational events. Phylogenetic relationships of cpDNA haplotypes in M. yunnanensis populations were inferred without an outgroup by Bayesian methods in MrBayes version 3.1.2  was determined by Modeltest in Mega version 6.06 .Evolutionary rates estimated for D and Fu's FS, Tajima, P-values were also calculated by this program.To further investigate population expansion and signatures of demographic change in populations, we used DnaSP version 5.0 , effective number of alleles (NE), number of private alleles (NP), expected heterozygosity (He), observed heterozygosity (HO), information index (I), percentage of polymorphic loci (PPB), and fixation index (FIS = 1-Ho/He) across all microsatellite loci. We mutually corrected statistics with FSTAT version 2.9.3 , and coefficient of gene differentiation (GST) were estimated using FSTAT version 2.9.3  was also calculated by this program according to Leberg /4FST  − 1) − 2 (1/FST(M) − 1)]/(1/FST(M) − 1), in which FST(B) and FST(M) were F statistics for nSSRs and cpDNA markers calculating by Arlequin respectively.Dataset editing and formatting were performed in GenAlEx version 6.3  with geographic distance for all population pairs using GenAlEx version 6.3 , was used as an additional estimator of the likeliest number of genetic clusters  was tested by analyzing the correlation of .8 Earl, .Mc value of 0.68, which indicated that the populations decreased in population size or vice versa . A total of 17 variable sites were identified from the cpDNA dataset, containing 13 SNPs, 3 indels, and 14 segregating sites. Finally, 10 haplotypes (Hap1-10) were obtained from 7 populations of M. yunnanensis (Table Hd) and nucleotide diversity per site (Pi) in total populations were 0.471 and 1.06, respectively  occurred in population YL, followed by TJ , and SM ; whereas no diversity was found in the 3 remaining populations . Total genetic diversity (HT = 0.522) was higher than the average intrapopulation diversity (Hs = 0.243). The permutation test revealed a higher value for NST (0.728) than GST (0.534) in M. yunnanensis populations. The difference between the two statistics, however, was not significant (P > 0.05). This result indicated the absence of phylogenetic structure among populations. Moreover, the AMOVA result indicated significant genetic differentiation among all populations  with most chloroplast diversity occurring among populations  compared with only 36.44% partitioned within populations  and a HRag of 0.204  loci, almost all the remaining loci conform to the equilibrium state in HWE test (P > 0.05). Five of the 45 locus pairwise showed LD (P < 0.05) after Bonferroni correction, but not significant. Diversity estimates from 10 microsatellites varied among populations (Table NT) of each population varied from 38 (SM) to 46 (QZ); rarefied allelic richness (Ra) varied from 3.539 (YL) to 4.175 (BY); number of private alleles (NP) varied from 2 (SM) to 9 (QZS); and percentage of polymorphic loci (PPB) varied from 90%  to 100% . The lowest values of observed heterozygosity (HO = 0.513) and expected heterozygosity (HE = 0.513) all occurred in population SY, whereas the maximum values of observed heterozygosity (HO = 0.605) and expected heterozygosity (HE = 0.581) were distributed in YL and TJ populations, respectively. The above parameters showed that M. yunnanensis populations have a relatively high genetic diversity.A total of 88 alleles were identified in 10 polymorphic loci, ranging from 3 (ssr22) to 22 (ssr27) among 7 FIS, Supplementary Table FST = 0.058). Estimates of gene flow between each pair of the 7 populations are shown in Table M. yunnanensis populations were separated into two clusters with high bootstrap values (100)  and indicated no significant effect of IBD  Figure . SimilarP > 0.05) indicated that all populations did not experience a recent bottleneck event  in M. yunnanensis were considerably higher than the values of “narrow” species (He = 0.420) and approached those of “widespread” species  deeply decreased the level of differentiation among populations. Therefore, we speculated that substantial gene flow likely caused the low genetic differentiation and indistinct phylogeographic structure of M. yunnanensis , although the evolutionary trees and genealogical haplotype network showed two reciprocally monophyletic lineages with robust support. However, based on a meta-analysis of isolation by distance, Jenkins et al. . Thus, we speculated that Quaternary glaciers  may have affected the genetic variations and structure of this long-lived species . These populations with a wider genetic base may be significant to adapt future scenarios and used as source material for germplasm banks and/or nurseries (Poudel et al., M. yunnanensis should focus on exotic overexploitation and habitat destruction. Little evidence of inbreeding and high gene flow was detected. Reduced gene flow may be expected, however, given that the species is undergoing dramatic population decline and isolation.Information on the genetic diversity, genetic structure, and demographic history of endemic species can aid the development of appropriate conservation and management strategies. In the present study, the genetic variation and demographic history of seven wild M. yunnanensis has undergone a past population contraction ~20,000–30,000 years ago. This event was likely influenced by Quaternary glaciations. Furthermore, no recent population expansion occurred in this species because of their limited seed dispersal over long distances and human interference. Therefore, we suggest establishing protection zones or plots in the distribution areas of M. yunnanensis, controlling seed collection and minimizing the habitat destruction by local people. Moreover, we advise relevant local departments to develop corresponding conservation actions and mitigation measures to utilize M. yunnanensis, as well as to raise conservation awareness and implement rational management in distribution areas.Through the comprehensive analysis of demographic history, we observed that SS, XZ, and YW initiated and designed the research. SS obtained funding for this study. XZ, FW, and SS collected the materials and performed the experiments, SS and XZ analyzed the data and wrote the paper. All authors read and approved the manuscript.This study was financially supported by grant 31560224 and 31360155 from the National Natural Science Foundation of China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


C. pyrenaica populations genetically and examine results in terms of (i) demographic history, (ii) subspecific classification and (iii) the implications for the management of Iberian ibex.Genetic differentiation in historically connected populations could be the result of genetic drift or adaptation, two processes that imply a need for differing strategies in population management. The aim of our study was to use neutral genetic markers to characterize victoria and hispanica). Our molecular analyses detected recent genetic bottlenecks in all the studied populations, a finding that coincides with the documented demographic decline in C. pyrenaica in recent decades. Genetic divergence between the two C. pyrenaica subspecies (hispanica and victoriae) was substantial (FST between 0.39 and 0.47). Unexpectedly, we found similarly high genetic differentiation between two populations (Sierra Nevada and Maestrazgo) belonging to the subspecies hispanica. The genetic pattern identified in our study could be the result of strong genetic drift due to the severe genetic bottlenecks in the studied populations, caused in turn by the progressive destruction of natural habitat, disease epidemics and/or uncontrolled hunting.We used 30 neutral microsatellite markers from 333 Iberian ibex to explore genetic diversity in the three main Iberian ibex populations in Spain corresponding to the two persisting subspecies (Capra pyrenaica conservation decision-making was based on the clear distinction between the two subspecies (victoriae and hispanica); yet our paper raises questions about the usefulness for conservation plans of the distinction between these subspecies.Previous  In the past century many animal populations were greatly reduced in size or driven to extinction by human activities such as habitat destruction or hunting . ConsequCapra pyrenaica . T. TCapra eninsula , 14]  the demographic history of these populations, (ii) their subspecific classification and (iii) the implications for Iberian ibex management.The aim of our study was to use neutral genetic markers to characterize genetically C. p. hispanica from Maestrazgo Natural Park (north-eastern Spain), 238 C. p. hispanica from Sierra Nevada Natural Space (southern Spain), and 26 C. p. victoriae from Sierra de Gredos  (http://lpdaac.usgs.gov) [Capara pyrenaica is adapted from [Between 2003 and 2008, we collected 333 Iberian ibex samples from the three main Spanish populations: 69 l Spain) . Map wassgs.gov) . The spated from .Samples consisted of tissue  obtained from deceased legally hunted animals, or from animals culled by park rangers as part of wildlife management plans aimed especially at combating outbreaks of diseases. This study was approved by the Ministry of Agriculture, Fishery and Environment of the Andalusian government . Sampling procedures were sanctioned as part of the application for permits for the fieldwork, which did not affect any endangered or protected species.C. p. hispanica may have suffered from two consecutive demographic bottlenecks, one in the period after the Spanish Civil War (1940–1945) and the other in the 1960s, when the population fell to just 30 animals and almost became extinct [The Maestrazgo population of  extinct . Howeverivil War 40–1945 a extinct .C. p. hispanica . This pC. p. victoriae numbered around 50 individuals in 1895 and just 12 individuals in 1905 [C. pyrenaica population and the main population of subspecies C. p. victoriae  and observed (HO) heterozygosity, and deviations from the Hardy-Weinberg equilibrium (HWE) were calculated using Genepop . Fisher .p< 0.001) in all populations. Hence, both microsatellite markers were excluded from further analysis. Significant deviation from HWE after a Bonferroni correction [p = 0.001), BM4505 (p = 0.034), MILSTS76 (p = 0.005) and URB058 (p = 0.017) in Maestrazgo Natural Park, and in BM4208 (p = 0.005), SR-CRSP24 (p< 0.001), BM415 (p = 0.007), MAF36 (p = 0.015), CSSM47 (p = 0.016), HAUT27 (p< 0.001) and GLA10 (p = 0.029) in Sierra Nevada Natural Space. The pairwise comparisons revealed that 2.52% of all pairs of microsatellite loci were in linkage disequilibrium, after correction for multiple tests.Of the 30 microsatellite loci, OarFCB48 was monomorphic in all three Iberian ibex populations, while OarVH34 deviated from HWE  in C. p. hispanica in Maestrazgo Natural Park, and 32 (45%) in C. p. hispanica in Sierra Nevada Natural Space. The number of private alleles per locus ranged from 0 (BM1818) to six (MAF36). Interestingly, the Sierra Nevada Natural Space population of C. p. hispanica had the highest proportion of private alleles, even higher than the C. p. victoriae population in Sierra de Gredos.In all, 71 of the 140 alleles in all loci were private alleles in one of the populations: 19 (26.8%) in FST value between the two C. p. hispanica populations  was of similar magnitude as the FST values between C. p. victoriae and C. p. hispanica . With K = 2, clusters  & Fig 3.C. p. victoria) and Maestrazgo Natural Park (C. p. hispanica) populations than between the two populations of C. p. hispanica .The dendrogram based on Nei’s standard genetic distance  showed aFor the Maestrazgo Natural Park population, all three tests for a bottleneck  detected significant heterozygosity excess compared to expectations if the population were at mutation-drift equilibrium. For the Sierra de Gredos population, two tests showed a significant result for a bottleneck, while the third test was just non-significant . None of the tests indicated a bottleneck for the Sierra Nevada Natural Space population .Different loci deviated from HWE in different populations. These deviations are more likely to be due to the subtle substructure of the Sierra Nevada and Maestrazgo populations than to the inherent characteristics of these loci; the possible exception is SR-CRSP24, which deviated from HWE in two of the three study populations and also deviated from HWE in some populations of Alpine ibex  since thC. pyrenaica hispanica in Sierra Nevada has experienced less severe bottlenecks than the other two populations. Therefore, in the Sierra Nevada population genetic drift was likely to be less than in the Maestrazgo and Sierra de Gredos populations. Genetic drift has a great effect on the proportion of private alleles [C. pyrenaica populations. The Sierra Nevada population with the least severe bottleneck has the highest proportion of private alleles. The generally high amount of private alleles in all C. pyrenaica populations indicates a lack of gene flow within the three populations.Historically  alleles  and seemThe significant genetic signature of a bottleneck in at least two of the three tests for the Maestrazgo and Sierra de Gredos populations matches the known severe demographic bottlenecks in these two populations, which were reduced to just 30 and 12 ibex, respectively, in the 1960s and at the beginning of the twentieth century . The SieC. p. hispanica from Maestrazgo Natural Park and from Sierra Nevada, a finding that was not predicted because these populations belong to the same subspecies, C. p. hispanica. Furthermore, these two mountain ranges are connected, albeit over a total distance of around 800 km [C. p. hispanica populations and the high genetic differentiation (FST = 0.3841) can only arise if there is only very limited gene flow between the populations and if genetic drift is occurring. The history of these populations and the bottleneck tests indicate that genetic drift is operating in each population, but more so in the Maestrazgo population.Our results from 28 neutral microsatellite loci highlighted unexpectedly high genetic differentiation between the populations of d 800 km  Fig 1)..C. p. hiC. p. victoria) and Maestrazgo (C. p. hispanica) Iberian ibex populations as between the two populations of C. p. hispanica. The high FST values between these two subspecies confirm that they are genetically quite different. However, we detected as much genetic differentiation within the subspecies C. p. hispanica as between the two subspecies. This unexpected genetic structure of these three populations raises the question as to whether genetic differentiation between subspecies reflects adaptive differentiation or is, rather, the consequence of random genetic changes due to genetic drift. Research into the differences in adaptation between these subspecies as needed to clarify this issue.We found as much genetic divergence between the Sierra Gredos  that are able to infer the adaptive potential better than the microsatellites . The strC. p. hispanica in Maestrazgo and Sierra Nevada, and (re-)establishing gene flow between them should be a future management goal.Admixture between genetically differentiated populations is a management option that can both decrease inbreeding and increase genetic variation in genetically impoverished populations, thereby enhancing the fitness of the population . AdmixinCapra pyrenaica subspecies denomination and the current avoidance of admixtures of these subspecies in conservation plans [C. p. victoriae from Sierra de Guadarrama into the Pyrenees where the subspecies C. p. pyrenaica is today extinct [The results of this study question the usefulness of the on plans . For ins extinct .Capra pyrenaica populations (including the three populations used in this study) found that extant Capra pyrenaica populations are as equally genetically distant as the extinct subspecies. This has led Manceau et al. [Capra pyrenaica populations to establish a new population with high genetic diversity in which selection can begin to operate. The results of our study with nuclear markers point in the same direction: the founding of a new Pyrenees population with ibex from all the remaining Iberian ibex populations–regardless of their subspecies–would enhance their adaptive potential in the new environment and only entail a low risk of outbreeding depression.However, a study of the mitochondrial DNA of seven u et al.  to suggePuma concolor coryi), for instance, was genetically rescued by introducing individuals from a different subspecies [Translocation of individuals from a genetically different population to increase the fitness of a recipient population has been successfully accomplished in some genetically imperilled populations . The Flobspecies . Interesbspecies  and its bspecies . Despitebspecies .S1 Table(DOC)Click here for additional data file.


Mytella charruana   became established along the coast of the southeastern United States (SE-US). Using mitochondrial DNA sequencing (cytochrome c oxidase subunit I), we compared genetic variation throughout its native range in South America to its invasive range in the SE-US. Samples from the SE-US were collected in 2006 and 2010 enabling a temporal comparison to evaluate possible genetic changes of the invasive population. We addressed two questions. First, what are the potential source populations (or geographic regions) for the SE-US invasion? Second, how has genetic diversity changed between the two sampling periods within the SE-US? We identified a total of 72 haplotypes, 64 of which were isolated to geographic sites and only 8 were shared among sites. The highly structured native range provides insight into the origin of invasive populations where our results suggest that the introduced SE-US population originated from multiple source populations with the Panama region as the primary source. Additionally, our results indicate that genetic composition of the non-native populations was unchanged between the two sampling periods. Mytella charruana exhibit a significant pattern of genetic structure among natural populations, owing to biogeographic barriers that limit natural dispersal, and an ability to persist in novel habitats, owing to a suite of life-history characters that favor survival under variable conditions. Overall, this study explains why M. charruana may become an increasing threat to locations founded by anthropogenic transportation.In 2004,  Human activities pose a significant threat to biodiversity and natural ecosystems due to the potential introduction of non-native species , 2. AlthIn particular, genetic characterization of invasive populations has proven to be important for several reasons, including species identification , 13, disDespite the detrimental conditions that may follow an invasion, initial colonizers do not always pose a threat when they first settle in an area due to low density and the likelihood that the invasion will fail to flourish . Indeed,Mytella charruana , a 3-5cm mussel that lives in muddy areas of bays and estuaries forming dense aggregations or covering mangroves roots and other hard substrates [Mytella charruana exhibit broad salinity and temperature tolerances, surviving in salinities ranging from 2–40 ppt [M. charruana was first discovered in Jacksonville, Florida, USA, attached to a seawater pipe of a power plant [M. charruana was considered extirpated from the Florida coast. However, in 2004 several individuals were discovered in the Mosquito Lagoon located 212 km south of the original discovery site [M. charruana has been found along the southeastern coast of the United States from Florida to South Carolina [M. charruana on native eastern oysters (C. virginica), confirming the invasiveness of the species [Studies seldom have the opportunity to characterize an invasion from its very beginning because of low densities and lack of ecological and economic impact of recent invaders. One exception to this is the invasive charru mussel bstrates . Mytella2–40 ppt  and temp2–40 ppt .This mus2–40 ppt –41. In 1er plant , 43. In ery site . Since 2Carolina  and seveCarolina –48. More species , 49.M. charruana along the Brazilian coast were genetically diverse, more structured than other related species  and resyanensis , 51. Samyanensis , 52. Add species ). PrevioM. charruana from 12 populations in 7 countries to investigate the relationship between native and nonnative populations of this invasive species. We used these data to address two primary questions: First, what were the possible source populations for the introduced populations? Second, did mtDNA haplotype composition change over time within the invaded region? Considering that a previous study [M. charruana in the SE-US was of mixed origin with likely sources from the southern Caribbean, we predicted that source populations would originate from the northern region of South America. Moreover, we used these data to investigate why invaded populations do not show marked founder effects (e.g. low genetic variability compared to source population areas) expected with typical new population founding [M. charruana after 2009/2010 extreme cold events throughout the invaded range. Finally, we discuss how spatial and temporal genetic structuring of populations can be used to better understand the initial phases of marine invasions.In this study, we analyzed the mtDNA cytochrome c oxidase subunit I (COI) gene of us study  suggestefounding , 54. Witfounding  found thM. charruana (™) for DNA preservation. Additionally, we collected samples from two separate time points for one population , Trinidad & Tobago (TRIN), Northeast Brazil (MACO), Southeast Brazil (VITO), South Brazil (PARA) and Uruguay (MONT) as well as previously published data representing the invaded region (SE-US ) with thharruana . The SE- fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene using polymerase chain reaction (PCR), with primers originally designed by Gillis et al. . Genomic primers ), 1 unitSequences were inspected and aligned in Sequencher 4.7 . Open reMytella guyanensis was used as an out-group in the phylogenetic reconstruction. Posterior probability values equal to or higher than 95% were considered statistically significant [To characterize phylogeographic patterns throughout native and introduced regions, we reconstructed the phylogenetic tree with unique COI haplotypes using the best-fit substitution model and partitioning schemes identified by PartitionFinder v1.1.0 . The phynificant . Becausenificant  to constnificant .M. charruana from throughout the native and invasive range, we estimated global and pairwise FST among all populations in Arlequin 3.5.2.2 [h) and nucleotide diversity (π) in Arlequin and employed a t-test in R statistical package version 3.2.3 [To investigate the genetic structure of  3.5.2.2 . Additioon 3.2.3  to evaluon 3.2.3 . Geograpon 3.2.3  by measuM. charruana, we used two approaches. First, we determined the number of shared haplotypes between native and invasive populations. Second, we conducted pairwise contrasts between native and invasive populations via analysis of molecular variance (AMOVA). Here, we ran a series of AMOVA analyses in which we grouped the introduced populations with populations from each native region. If these population pairings revealed groupings that were not significantly differentiated between native and introduced populations, then that native population was inferred to be a putative founder population of the invasion. All AMOVA analyses were based on pairwise difference tests with 1000 bootstrap simulations to build confidence intervals for estimations using Arlequin 3.5.2.2 [To infer which native population(s) were the most likely source(s) of the introduced SE-US  3.5.2.2 .FST value would indicate that genetic variation had changed between the two collection time points. Finally, we estimated gene diversity (h) and nucleotide diversity (π) in Arlequin for both time points of the invasive region to determine whether diversity indices had changed over time. Here, significance was determined if the 2010 estimates of genetic diversity were outside the 95% CI generated by the four SE-US populations in 2006.To address our second question, whether genetic diversity would change over time, we used AMOVA to compare the genetic composition of the 2006 SE-US introduced populations with that of the 2010 JAX population. Here, a significant M. charruana were analyzed using the mitochondrial COI gene. From those, 166 were new sequences and 154 were taken from literature [h = 0.9) while PARA exhibited the least genetic diversity  and was due to the occurrence of haplotypes from both female mtDNA lineages present in these populations (h = 0.702) in JAX2006. The nucleotide diversity ranged from π = 0.00738 (JAX2010) to π = 0.00904 (NSB). The entire data set is available in GenBank (Acc. No. MF074963–MF075128).Our sample set comprised eight populations ranging throughout the native distribution, from Central to South America, as well as five populations from the introduced range along the SE-US (four from 2006 and one from 2010). The average number of sequences per population was 23, with MACO and MONT having the fewest (n = 14) and COLO the greatest (n = 41). A total of 320 sequences of terature . All seqterature , and useterature . The numulations . Among tulations . The higZ = 761.9835, p = 0.765; r = - 0.1576; The phylogenetic relationship was reconstructed via a Bayesian consensus tree which identified two well defined clades, haplogroup A and B, that had been previously identified by Alves et al.  Fig 2)..Fig 2). FST = 0.633; FST values , and a large component of variation among localities (FST between native populations was between MONT and VITO (FST = 0.074), whereas the highest estimate of FST was between PARA and MONT (FST = 0.988). In contrast, introduced populations were not significantly differentiated from each other . Values contrasting genetic diversity between native and invasive populations were not significantly different for either gene diversity (p = 0.791) or nucleotide diversity (p = 0.6187)  .M. charruana, we ran independent AMOVA analyses to determine the degree of differentiation between native and non-native populations, as a means to identify if any native population could be a potential founder of the SE-US populations. We found that only a single native population exhibited a non-significant FST , then followed by the northern South American populations , showing a lack of change over the four-year period (h = 0.747–0.744), nucleotide diversity for JAX2010 was 0.0074 and fell slightly below the confidence interval found in 2006 SE-US populations (95% CI of π = 0.00881–0.00879).To determine whether there was a change in the genetic composition of populations over time in the invaded region, we ran an AMOVA contrasting the SE-US populations from 62006 with the JAX2010 population. This analysis revealed that the estimate of r period . A compaM. charruana populations. In contrast, variation among invasive populations along the southeastern coast of the United States exhibited little differentiation, even when contrasted with temporally displaced samples on either side of a major population die-off. Diversity within native populations exhibited an order of magnitude in variation among populations, but this level of variation was driven by populations either fixed for a single haplotype group or populations that exhibited mingling of haplotype groups. The genetic diversity of introduced SE-US population remained high considering all invasive populations were composed of a single haplotype group. Overall, compiling genetic information from throughout the native range provided the necessary data to determine that the introduced populations are likely closely related to Pacific Panamanian populations. Likewise, occurrence of shared haplotypes between northern South America and the SE-US provides further evidence that the introduced range included samples of mixed origin. Furthermore, admixture of haplotypes from Panama and southern Caribbean populations explains why within-population diversity is so high for the non-native populations.Our genetic assessment revealed a strong patterns of genetic structure throughout native Marine organisms have the potential to disperse throughout large areas where genetic isolation is generated with increasing distance , 71 and Gammarus zaddachi [Hydrobia ventrosa [Cnidoglanis macrocephalus [Evechinus chloroticus [Macrobrachium nipponense [M. charruana, M. guyanensis [M. charruana, as a typical estuarine mussel, exhibits a high degree of isolation throughout the native distribution, as demonstrated here.Connectivity among populations of estuarine taxa would be expected to be reduced compared to marine and coastal taxa , 87. In zaddachi ); gastroventrosa ); catfiscephalus ); echinooroticus ); shrimppponense ); as welyanensis . M. charMacrodon ancylodon, exhibit deep genetic divergence correlated with the northern flow of the Brazilian Current and the Malvinas Current, despite morphometric homogeneity [Perna perna, [M. charruana, we can see the impacts of currents driving differentiation between the populations of PANA and ECUA where the intervening habitat is ideal for M. charruana  exhibit high population genetic structuring between populations in the northern and southern Pacific coasts of Colombia owing to these currents [In addition to the estuarine environment leading to differentiation, ocean currents are another feature along the South American coast that also likely contributes to differentiation among populations , 94. Ocea perna, ). With rud flats ). These ud flats . Similarcurrents .M. charruana throughout the native range, what processes likely brought M. charruana to the SE-US? Anthropogenic translocation by ship transportation  from South America appears to be the most plausible vector for M. charruana. Many examples from other organisms support ballast water and ship hull fouling as efficient dispersal vectors for marine organisms [M. charruana SE-US invasion [M. charruana to arrive in Florida from Panama.Given these barriers to natural dispersal found for rganisms –101, esprganisms  and previnvasion . Given tM. charruana. A recent study investigating temperature and salinity tolerances of M. charruana sheds light on how abiotic factors limit spread throughout the invaded range [M. charruana survival was largely driven by temperature given that survivorship curves dropped off quickly at both low and high temperature extremes. With regard to biotic factors, M. charruana was found established on intertidal oyster reefs in the Indian River Lagoon, Florida [M. charruana has only been found on hard substrates, further spreading may be directly related to oyster density. However, a recent study investigating competitive interactions between M. charruana and oysters (Crassostrea virginica) found that oyster spat survival and growth was reduced by the presence of M. charruana [Callinectes sapidus) native to the SE-US equally prey upon M. charruana and the native ribbed mussel (Geukensia demissa) . Overall, there is a paucity of studies evaluating how biotic, abiotic, and the combination of these factors may impact the future spread of M. charruana throughout the SE-US. However, as climate change impacts North America, the range of invasive M. charruana, currently believed to be held in check by cold winter temperatures [Within the invasive range, there are both abiotic and biotic factors that can impact the spread of ed range . These r Florida . Given tharruana . Native harruana  found theratures , will liM. charruana. These results indicate that, even during periods when M. charruana were not observed, some individuals were protected in stronghold locations and persisted to re-establish the coast when favorable conditions returned.Furthermore, by comparing genetic diversity and differentiation over a temporal scale that spanned the 2009/2010  cold weaM. charruana, the differentiation identified among populations in the native range provided the necessary resolution to identify where the recent southeastern United States invasion originated. Moreover, this study identified an interesting contrast in dispersal patterns for M. charruana. Specifically, this species exhibits a pattern of minimal dispersal throughout the native range, yet is both persistent (this study) and spreading [M. charruana in both the native and invasive range, we suggest that although M. charruana will not likely be transported long distances on its own, anthropogenic transportation of this species can lead to foundation events in a variety of habitats that will likely be difficult to eradicate.In sum, this study illuminates how an understanding of genetic differentiation from throughout the native range can provide insights into the spread and persistence of an invasive species. With regard to preading  throughopreading , 108, hipreading , 36, andpreading ) cannot