STRUCTURE to estimate ancestry proportions and tested the sensitivity of these estimates to the choice of the number of clusters, K. We used partial correlation analyses to examine the relationship between gene diversity and geographic distance from Beringia, controlling for non-Native American ancestry , and taking into account alternative paths of migration. Principal component analysis and multidimensional scaling were used to investigate the relationships between Andean and non-Andean populations and to explore gene-language correspondence. We found that 1) European and East Asian ancestry estimates decline as K increases, especially in Native Canadian populations, 2) a north-south decline in gene diversity is driven by low diversity in Amazonian and Paraguayan populations, not serial founder effects from Beringia, 3) controlling for non-Native American ancestry, populations in the Andes and Mesoamerica have higher gene diversity than populations in other regions, and 4) patterns of genetic and linguistic diversity are poorly correlated. We conclude that patterns of diversity previously attributed to pre-Columbian processes may in part reflect post-Columbian admixture and the choice of K in STRUCTURE analyses. Accounting for admixture, the pattern of diversity is inconsistent with a north-south founder effect process, though the genetic similarities between Mesoamerican and Andean populations are consistent with rapid dispersal along the western coast of the Americas. Further, even setting aside the disruptive effects of European contact, gene-language congruence is unlikely to have ever existed at the geographic scale analyzed here.Our current understanding of pre-Columbian history in the Americas rests in part on several trends identified in recent genetic studies. The goal of this study is to reexamine these trends in light of the impact of post-Columbian admixture and the methods used to study admixture. The previously-published data consist of 645 autosomal microsatellite genotypes from 1046 individuals in 63 populations. We used  Our current understanding of pre-Columbian history in the Americas rests in part on three trends identified in recent genetic studies. The first trend is a negative correlation between population-level genetic diversity and geographic distance from the Bering Strait, which Wang et al.  attributIn a subsequent study of the same data, Hunley and Healy 2011) found that the level of European ancestry in the 29 Native American populations was also negatively correlated with geographic distance from Beringia. Moreover, after controlling for European ancestry in partial correlation analyses, they demonstrated that the magnitude of the correlation fell dramatically and lost statistical significance. This finding potentially undermines the role of serial founder effects in shaping patterns of Native American diversity. However, Hunley and Healy failed to consider the possibility that more than three ancestral groups contributed to extant admixed Native American populations . In particular, they failed to consider potential contributions from East Asian populations that may have occurred subsequent to initial peopling 1 found t–6. To thThe second trend is that Western South American populations have higher diversity than populations in eastern portions of the continent ,7–9. In The third trend is a correspondence between patterns of genetic and linguistic variation ,13–17. MThe goal of this study is to reexamine these trends in the pattern of Native American diversity in light of the limitations imposed by the admixture process itself and by the data and methods used to measure the contribution of ancestral sources to admixed populations. We concentrate on four questions that are commonly addressed in genetic studies of Native American prehistory:What is the level of African, European, and East Asian ancestry in Native American populations?What routes did people take during their initial dispersal across the Americas?How are native Andean populations related to populations from other regions of the Americas?Are patterns of population genetic and linguistic variation correlated?The genetic data come from published sources. They consist of 645 autosomal microsatellite genotypes from 1046 individuals in 63 populations . Microsatellite data are well-suited for this study because they are relatively free of ascertainment bias and therefore provide accurate estimates of gene diversity. The data were compiled by Pemberton et al.  from RosFor most analyses, we labeled the Native American populations as belonging to one of nine geographic regions, initially defined based on geographic location and subsequently refined based on results of our analyses. The regions are: Canada (n = 3), northern Mexico , Mesoamerica excluding Costa Rica (n = 5), Costa Rica and Panama (n = 2), coastal Colombia (n = 6), inland Colombia (n = 2), the Andes (n = 3), Amazonia and Paraguay (n = 5), and Southern Brazil (n = 2).STRUCTURE [K. The approach uses a statistic (ΔK) related to the second order rate of change in the posterior probability of the data given K  [K and related statistics.We used the model-based clustering approach implemented in TRUCTURE  to estimTRUCTURE  to assisen K see . Based oy plots) .We used y plots)  to calcuK on ancestry estimates, we averaged the African, European, East Asian, and Native American ancestry estimates in each population for each value of K (across 15+ runs), and displayed the results using line graphs.To assess the effects of We constructed scatter plots of gene diversity  vs. geogTo assess and control for the effects of non-Native American admixture , we performed partial correlation analyses between gene diversity and geographic distance controlling for combined African, European, and East Asian ancestry, referred to here as “non-Native American ancestry.” We report the partial correlation coefficients and display the results of the partial correlation analyses using a scatter plot of the residuals of linear models of non-Native American ancestry and gene diversity vs. non-Native American ancestry and geographic distance. We also used a jackknife approach to identify the contribution of each population and region to the magnitude of the correlation and partial correlation between gene diversity and distance from Beringia. For these analyses, we dropped each population (and region) from the analysis, one at a time, and then re-computed the correlation.We used principal component analysis (PCA) to summarize the major axes of genetic variation in the full 63-population dataset. We extracted the first 25 PCA factors, first scaling the allele frequencies using the scaleGen function from the adegenet package in R , and theWe used the Ethnologue , in conj2 [Nei-(δμ)2 = 0.945 for the Native American populations. The results and interpretations that we report below were unaffected by the choice of the distance measure. In the plot of the first two principal coordinates (PCos) for the Native American-only sample, we color-coded the points by language family and scaled the point size according to the level of non-Native American ancestry.We replotted the PCA factors from the Native American-only analyses described above, color-coding the populations by language family affiliation. We also constructed multidimensional scaling (MDS) plots from Nei’s minimum genetic distances  for the 2 , which tAnalyses and plotting were carried out using the base R package and the packages ade4, ppcor, fields, vegan, vioplot, and RcolorBrewer ,38–41.STRUCTURE analyses are consistent with those from previous studies in showing the existence of geographically-patterned genetic structure at all values of K [K increased steadily from K = 5 to K = 9, after which it decayed, with the exception of a spike at K = 15  and the Andes (mean = 9.9%) and lowest in Amazonia (mean = 0.02%). The mean for the largest Native American-specific ancestry component, which was shared by all Native American populations, shown in purple, was 67.5%. We hereafter refer to this general Native American-specific ancestry component as “pan-Native American” ancestryAt K = 15, the mean African ancestry across the populations was essentially unchanged at 1.0%, but the mean East Asian ancestry fell to 0.8%, the mean European ancestry fell to 4.9%, and the mean pan-Native American ancestry fell to 50.3%. These estimates continued to fall at even higher values of K. These results indicate that ancestry estimates in Native American populations are potentially sensitive to the choice of K.At K, we plotted the mean ancestry for each population for each value of K in K. In contrast, European ancestry decreased in all populations as K increased. The decrease leveled off in some cases, but in others cases, it continued through K = 21 . The most notable decrease occurred in the three Canadian populations and the Colombian Arhuaco population. East Asian ancestry also decreased steadily in the Canadian populations from K = 5 to K = 9, at which point it stabilized at a mean of about 1%. The pan-Native American ancestry component also decreased as K increased; these changes reflect the continual formation of Native American region-specific ancestry clusters.To further explore the sensitivity of ancestry estimates to the choice of STRUCTURE analyses of the 29 Native American populations alone .The violin plots in r = -0.699, p < 0.000). Second, when populations with high or low non-Native American ancestry are removed from the analysis, the magnitude of the correlation changes. In cases where admixed populations are located far from Beringia , their removal strengthens the correlation (makes r more negative). Conversely, in cases where admixed populations are located close to Beringia , their removal reduces the magnitude of the correlation (makes r closer to zero). Third, the Amazonian and Paraguayan populations have particularly low diversity, and they have essentially zero non-Native American ancestry. The regional jackknife results in There are three salient features of the pattern of diversity. First, the correlation between gene diversity and geographic distance from Beringia is high and statistically significant  decreased only slightly from the initial correlation, but the pattern of lack of fit is different in that the highest level of diversity is now in Mesoamerica and the Andes, not in Canada. This change is due to the drop in ancestry-corrected diversity in the Canadian Ojibwa and Cree. Another important feature of the plot is that Amazonian and Paraguayan populations are still driving the correlation. When they are removed from the analysis, the partial correlation drops substantially and becomes non-significant . With one exception, the PCs show little variation in Europe and East Asia. That exception is PC3, which separates the Tundra Nentsi from the other East Asian populations and groups them with the Native American populations. PCs 4–6 account for an additional 1.6% of the variation. They capture substructure in the African populations, separating the San, Mbuti and Biaka from other African populations. The Native American populations are largely invariant for these three PCs. These results provide additional evidence of the pervasive influence of African and European admixture on patterns of Native American genetic diversity.To explore these patterns of diversity in more detail, we used principal components analysis (PCA) to summarize the major axes of genetic variation in the full 63-population sample. PCs 1–3, shown using box plots in r = 0.92), and therefore with European ancestry. Subsequent PCs capture substructure within the Americas, associated mainly with the Amazonian and Paraguayan populations. Mesoamerican and Andean populations have similar scores on PCs 2–10, only separating from one another for PC 11 (not shown), and only then because the Mixe and Mixtec populations separate from the other Mesoamerican populations.We repeated the PCA after removing African, European and East Asian populations. Boxplots of the first six PCs from this analysis are shown in In the 29 Native American populations, there are 16 well-attested language families, with an average of 1.8 languages per family. Only two of the families have more than two population representatives: Chibchan (n = 4), and Tupi-Guaraní (n = 4).The PCs also provide information about the relationship between patterns of genetic and linguistic diversity in the Americas. r = -0.649). PCo2 distinguishes the Ache and Suruí from the other Native American populations. Consistent with the PCA analyses described above, the plot shows that Andean populations (in red) have relatively short genetic distances to Mesoamerican populations (in light pink).We also constructed MDS plots from Nei’s minimum genetic distances. K in the STRUCTURE analyses. In many studies, the value of K is chosen based on the posterior probability of the data for a given K, P(X|K). In our analyses of the 63-population dataset, K = 9 had the highest posterior probability. Evanno et al. [X|K) plateaued or continued to increase beyond the true value of K, and 2) the variance in this probability across runs  increased beyond the true value. Based on these observations, the authors determined that a statistic related to the second order rate of change in the estimate of the posterior probability, termed ΔK, peaked at the true value of K. This approach performed well in simulations of several models of evolution, including a hierarchical island model consisting of sub-populations nested inside of larger demes, even when one deme was excluded from the analysis. This model is a reasonable representation of the structure of human diversity [Our inferences about Native American prehistory were based in part on the choice of o et al.  demonstriversity .K = 9. At this and higher values of K, estimates of European and East Asian ancestry were much lower than at values of K from 5 to 8. Independent of P(X|K) and ΔK, sensible geographically-based genetic substructure continued to form at values of K well above 9. These results imply that some previous studies of admixture in Native Americans may have overestimated European and East Asian ancestry. In supervised runs of STRUCTURE at K = 5, for example, Wang et al. [K = 9 and higher, East Asian ancestry was low across the Americas, and it was uncorrelated with geographic distance from Beringia.In our analyses of the 63-population dataset, ΔK also peaked at g et al.  identifiIn a related vein, a recent cluster analysis of autosomal SNP diversity by Riech et al.  found thSTRUCTURE runs of the same data, Hunley and Healy [K increased . Further, in our partial correlation analysis, the strong negative correlation between gene diversity and geographic distance from Beringia persisted when total non-Native American ancestry was controlled. These results demonstrate that studies of evolution in admixed populations are sensitive to the choice of the value of K.In unsupervised nd Healy  identifiWhile is tempting to view the strong correlations between gene diversity and distance from Beringia as evidence for a north-south serial founder effect process, our partial correlation analyses revealed that 1) gene diversity is highest in the Mesoamerican and Andean populations, not the Canadian populations, and 2) the correlation between gene diversity and geographic distance from Beringia loses statistical significance when the Amazonian and Paraguayan populations are removed from the analysis . We emphThat said, it is important to note that we cannot reject the hypothesis that a north-south serial founder effect process ever occurred in the Americas. We have attempted to control for the effects of European contact on patterns of Native American diversity, but many other complex evolutionary processes occurred subsequent to initial peopling, including continued movements and interactions between peoples in the Americas and East Asia ,42, possIn our analyses, we also found that patterns of diversity point to close affinity between Andean and Mesoamerican populations, consistent with findings of previous studies ,8,9. As It is difficult to assess the extent of gene-language congruence in the Americas prior to European contact because that contact resulted in the deaths of millions of people and the extinction of hundreds of languages ,48. EvenTo the extent that the current distribution of Native South American languages is in any way representative of the pre-contact period, the only language family that was genetically cohesive in the current study was Chibchan, which has a limited geographic distribution compared to other South American languages . However


Mytilus galloprovincialis has established invasive populations across the globe and in some regions, have completely displaced native mussels through competitive exclusion. The objective of this study was to elucidate global connectivity patterns of M. galloprovincialis strictly using archived cytochrome c oxidase 1 sequence data obtained from public databases. Through exhaustive mining and the development of a systematic workflow, we compiled the most comprehensive global CO1 dataset for M. galloprovincialis thus far, consisting of 209 sequences representing 14 populations. Haplotype networks were constructed and genetic differentiation was assessed using pairwise analysis of molecular variance.The invasive mussel, M. galloprovincialis as was found in previous studies. These results suggest a complex dispersal pattern for M. galloprovincialis driven by several contributors including both natural and anthropogenic dispersal mechanisms along with the possibility of potential hybridization and ancient vicariance events.There was significant genetic structuring across populations with significant geographic patterning of haplotypes. In particular, South Korea, South China, Turkey and Australasia appear to be the most genetically isolated populations. However, we were unable to recover a northern and southern hemisphere grouping for The online version of this article (10.1186/s13104-018-3328-3) contains supplementary material, which is available to authorized users. Mytilus galloprovincialis Lamarck, 1819 by repurposing archived cytochrome c oxidase 1 (CO1) sequence data from public databanks.Quantifying dispersal in marine environments has been a long standing challenge due to the difficulty in tracking large numbers of microscopic larvae within oceanic basins . As a coMytilus galloprovincialis is a relatively small marine bivalve (5–8 cm) that is native to the Mediterranean but has aggressively extended its range to the Americas, Asia, southern Africa and Australasia [M. galloprovincialis along with its palatability have resulted in the transplantation of stock populations for aquaculture purposes in different regions of the world [M. galloprovincialis across multiple global localities. We hypothesized that M. galloprovincialis would exhibit marked genetic differentiation between northern and southern hemisphere mussels but overall very low levels of genetic differentiation within hemispheres due to repeated introductions.tralasia –9. The ptralasia . In addihe world . The suche world , broad the world  and resihe world . The objM. galloprovincialis DNA sequences. These databases included GenBank, the European Nucleotide Archive, DNA Database of Japan and the Barcode of Life Database (BoLD). An automated search was preferentially chosen over a manual search due to the speed of sequence acquisition, and its highly discriminative nature . The mitochondrial DNA marker, cytochrome c oxidase 1 (CO1) was chosen due to its overrepresentation in population genetic studies for this species relative to other markers. The following qualifiers were incorporated into the coding script: ‘Mytilus’, ‘galloprovincialis’ ‘mitochondrial’, ‘CO1’. After scanning 2480 mitochondrial gene sequences, 322 CO1 fragments were recovered . For verification, each database entry was manually checked and discarded if: (i) the sequence could not be linked to published research  and or (ii) the sequence could not be traced to a specific geographic locality. In a few cases the mining program recovered ‘CO1-like sequences’ which were discarded. To avoid compiling duplicated sequences, database entries were cross referenced and any duplications were also discarded. To confirm collection dates, sequences were cross-referenced to their corresponding publications and in cases where no collection date was specified, authors were contacted directly for confirmation. Based on these aforementioned filters, a finally tally of 209 sequences representing 14 distinct populations were tagged as ‘useable’ for this study and they were all accessible from the GenBank database  with 88% of the genetic variation nested within individual hemispheres. Pairwise comparisons showed generally high ɸST values indicating strong genetic differentiation across all localities. Tasmania, South China and South Korea were the most genetically isolated populations, with all three generating significant ɸST values of 0.80–0.93 when compared with other populations (Table ST = 0.07–0.26) and the Chilean and North Atlantic/Mediterranean populations (ɸST = 0.07–0.13). There was also low and non-significant genetic differentiation between British Columbia and Chile, China (NP), Portugal and western Australia (0.07–0.18).The ɸM. galloprovincialis [M. galloprovincialis is used as evidence for the support of the ‘northern migration’ hypothesis which states that M. galloprovincialis diverged from an ancestral M. edulis in the Mediterranean Sea more than 1.5 mya and then migrated to the southern hemisphere during the Pleistocene via the Atlantic Ocean. The lack of distinct northern and southern hemisphere groupings could be due to cryptic dispersal where frequent anthropogenic transport could dilute phylogeographic signal and consequently decrease the value of standard population genetic parameters (e.g. FST) [Previous phylogenetic studies using RFLPS and 16S rRNA sequences recovered two distinct northern and southern hemisphere clades of incialis –21. In c.g. FST) .Mytilus mussels sampled in Australia are M. galloprovincialis [M. galloprovincialis is endemic and a secondary contact with M. edulis either before or after the founding population became established, could have resulted in the genetic isolation observed in the present study [While our study did not recover a northern and southern group, it did recover two Australasian haplogroups consisting of some individuals from east Australia and the entire western Australian, Tasmanian and New Zealand population. Approximately 99% of incialis . In partnt study .M. galloprovincialis populations also showed genetic isolation based on haplotype networks and pairwise AMOVA results. More surprising, however was that the Korean population was strongly differentiated from both the NP Chinese and South Chinese populations (ɸST = 0.88 and 0.91 respectively—P < 0.05) despite the fact that all three localities are located less than 2000 km away from each other in the Yellow Sea with ocean current direction conducive to fine scale connectivity. The high genetic structure observed in this region could be due to hybridization. In all three Asian localities, M. galloprovincialis’ range overlaps with that of M. edulis and M. trossulus. [M. galloprovincialis though further genetic analyses will be needed to definitively detect the presence of hybridization. Alternatively, past geological and climatic changes in this coastal system could have resulted in ancient vicariance events [Both South Korean and South Chinese ossulus. . Hybridiossulus. . When inossulus. . It is te events , leadingM. galloprovincialis using nuclear markers [M. galloprovincialis from the Asian Pacific region to British Columbia.There were also frequent instances of haplotype sharing along between NP Chinese populations, North Atlantic, Mediterranean and Pacific populations of Chile and British Columbia, which is congruent with past population studies of  markers –30. In a markers . TransocM. galloprovincialis has been studied extensively on the southern African coast where the mussel had rapidly colonized the western coast of the country upon in its introduction in the 1970s and has since displaced the native mussel Aulacomya ater in this region [M. galloprovincialis [The negative impacts of s region . Both haincialis .M. galloprovincialis and it likely involves a combination of natural and anthropogenic dispersal coupled with local adaptation and hybridization events. Most importantly, these findings were based on archived genetic data drawn from disparate studies. This study therefore shows that DNA sequence repositories possess valuable genetic data, from which informative results can be gleaned from post hoc analyses.In conclusion, our results are indicative of a complex dispersal pattern for Mytilus spp. complex of which the study species belongs to. However, we believe our workflow for repurposing sequence data was adequate in filtering bona fide M. galloprovincialis CO1 sequences. However, we did encounter some problems associated with sequence acquisition itself. For some of the DNA barcoding and phylogenetic studies, sampling localities were not included either as a source modifier in GenBank or in the published study, and in such cases authors had to be contacted directly for this information. In addition, GPS co-ordinates were also missing so we were unable to carry out some crucial tests commonly used in connectivity studies including isolation by distance (IBD) calculations and SAMOVA .Many researchers are very wary of using public databases to test new hypotheses especially in large scale population genetic studies. A significant issue is the taxonomic reliability of submitted sequences, especially with regards to closely related species like the Additional file 1: Table S1. GenBank accession data. List of GenBank accession numbers for all CO1 sequences used in the present study. In this file, sequences are separated by population and both sample size and the original purpose of the sequences are provided.


Quercus spinosa, Quercus aquifolioides, and Quercus rehderiana) using both cytoplasmic-nuclear markers and ecological niche models (ENMs), and elucidated the impacts of climate oscillations and environmental heterogeneity on their population demography. Our results indicate there were mixed genetic structure and asymmetric contemporary gene flow within them. The ENMs revealed a similar demographic history for the three species expanded their ranges from the last interglacial (LIG) to the last glacial maximum (LGM), which was consistent with effective population sizes changes. Effects of genetic drift and fragmentation of habitats were responsible for the high differentiation and the lack of phylogeographic structure. Our results support that geological and climatic factors since Miocene triggered the differentiation, evolutionary origin and range shifts of the three oak species in the studied area and also emphasize that a multidisciplinary approach combining molecular markers, ENMs and population genetics can yield deep insights into diversification and evolutionary dynamics of species.The East Himalaya-Hengduan Mountains (EH-HM) region has a high biodiversity and harbors numerous endemic alpine plants. This is probably the result of combined orographic and climate oscillations occurring since late Tertiary. Here, we determined the genetic structure and evolutionary history of alpine oak species (including  Historical processes such as geographic and climate changes have profoundly shaped the population genetic structure and demographic history of extant species Hewitt, . Global Taxus wallichiana and Picea likiangensis supposed to originate in the HHM regions experienced ranges expansion during the Last Glacial Maximum (LGM) rather than during the Last Interglacial (LIG) tropical areas of the Northern Hemisphere , i.e., Quercus spinosa, Quercus aquifolioides, and Quercus rehderiana , the number of haplotypes (NH), the haplotype diversity (Hd), the nucleotide diversity (ϕ), and neutrality test statistics of Tajima's D  , total gene diversity (HT) and the differentiation for unordered alleles (GST) and for ordered alleles (NST) based on 1000 random permutations.Population genetic parameters, including the number of segregating sites  repetitions for cpDNA sequences. We also used maximum parsimony (MP) and ML in PAUP* v4.0 beta 10 .The phylogenetic relationships of cpDNA haplotypes were reconstructed via Bayesian and maximum likelihood (ML) methods with 8 MCMC steps were carried out, with sampling at every 5000 generations, following a burn-in of the initial 10% cycles. MCMC samples were inspected in TRACER v1.5 to confirm sampling adequacy and convergence of the chains to a stationary distribution. The results were visualized using FIGTREE v.1.3.1. Generally, previous studies utilized the cpDNA substitutions rates [1.1 × 10−9 to 2.9 × 10−9 substitutions per site per year (s/s/y)]  approach as implemented in ARLEQUIN v3.5  were estimated by POPGENE v1.31   of genetic clusters with the program STRUCTURE HARVSTER    and we repeated the structure analysis independently except for running K from 1 to 15 for populations occurred in the EH-HM region .The program STRUCTURE v2.3.3  and two-phase model of microsatellite evolution with the proportion of single step stepwise mutation set to 95% and the variance set to 5% with 104 simulations as recommended by Piry et al.  and contemporary migration rates , respectively. We defined four clusters as follows: one for Q. aquifolioides, one for Q. rehderiana, and the other two for Q. spinosa as suggested by the results of STRUCTURE (see results section). We performed maximum-likelihood analyses in MIGRATE-N v3.6 using 10 short chains (104 trees) and three long chains (105 trees); 104 trees were discarded as burn-in. To increase the efficiency of the MCMC, we applied the following parameters: replicated = YES, long chains, randomtree = YES, heating = ADAPTIVE: 1 . In order to ensure the consistency of estimates, we repeated this procedure five times and reported average maximum-likelihood estimates with 95% confidence intervals. When estimated the contemporary gene flow using BAYESASS v3.0, We examined the parameters including migration rates (ΔM), allele frequencies (ΔA) and inbreeding coefficients (ΔF) to ensure the optimal acceptance rates of the three parameters fell within the range of 20–60%. The delta values for the three parameters were 0.26, 0.37, and 0.49, respectively. Following that, we conducted the analyses with 107 iterations after a burn-in of 106 iterations, setting 1000 as the sampling frequency. Ten independent runs were executed to minimize the convergence problem. The result with the lowest deviance was adopted according to the method of Meirmans  option implemented in BEAST v1.7.5. The result of species tree suggested the Q. spinosa was more ancient than other two species. Furthermore, Ma  and population dynamics  of these oak species, We used the approximate Bayesian computation (ABC) procedure in DIYABC v2.04 . All the bioclimatic variables  were downloaded from WorldClim website with a resolution of 2.5 arc-min (http://www.worldclim.org/)  from Chinese Virtual Herbarium (http://www.cvh.org.cn/cms/). We used the default parameters of MAXENT and included 80% of species records for training and 20% for testing the model and the default convergence threshold (10−5) and set the number of maximum iterations to 5000 and the number of replicates to 10. The output format was set to logistic. Models no better than having random predictive ability have an area under the receiver operating characteristic (ROC) curve (AUC) value of 0.5, whereas models with perfect predictive ability have an AUC approximating 1. An AUC score above 0.7 was considered as good model performance . We then used the background similarity test as implemented in ENMTOOLS v1.3 to determine whether the ENMs of different pairs of species are more or less similar than expected from the differences in the environmental backgrounds of the regions where they occur  and Q. rehderiana (C23–C25) had three private haplotypes, respectively, while two haplotypes (C2 and C4) were shared between them. Q. spinosa possessed 15 private haplotypes ; in addition, it shared C6 with Q. aquifolioides and C20 with Q. rehderiana, respectively . Values of NST and GST were both very high in all three species, and in no instance was NST significantly higher than GST  and five private ribotypes (N8-N12), respectively  than that in the other two species. In Q. aquifolioides and Q. spinosa, NST  was significantly larger than GST   indicated that the oak species diverged in Late Miocene  , indicating a Mid-Miocene split between the species and the outgroup. And the coalescent time of all chlorotypes within species  across the 33 populations, and there was no evidence for LD. After Bonferroni correction, significant deviation from HWE induced by homozygote excess was detected in all 12 loci when all samples were treated as a single population within species. However, there were no HWE deviations within each population after Bonferroni correction.HT and HS were 0.699 and 0.673 with a range of 0.279–0.905 and 0.278–0.891, respectively. Population differentiation was significant for all 12 loci . As for the standardized genetic differentiation, due to the high HT and variable nSSR makers used in this study, G'ST was higher than FST across all loci , and the majority (63.72%) of the variation partitioned within populations  from Yunnan Province, those from the east of Mts. Hengduan (except GY and QL) and one population of Q. rehderiana (WN), formed one group; the other group comprised all populations of Q. aquifolioides, six populations of Q. rehderiana (except WN), and the remaining populations of Q. spinosa from Mts. Hengduan and Yungui Platea. .The Wilcoxon test under the SMM and TPM models failed to detect any recent genetic bottleneck in any of the populations, and all populations showed an L-sharp allele distribution when ruling out two populations , and the divergence time between QA and the ancestor of QS1 and QS2 (t2) were 3.24 × 104 and 7.58 × 104 generations ago, respectively. Assuming the generation time to be 150 years, the divergence times of t1 and t2 corresponded to 4.86 Ma and 11.37 Ma, respectively. The estimated median mutation rates and the proportion of multiple step mutations in the generalized stepwise model of microsatellites were estimated to be 1.75 × 10−5 and 0.564, respectively  0.5139–0.5940], much higher than other eight scenarios. The median values of the effective population sizes of QS1, QS2, QA, QR and NA were 3.75 × 10Q. spinosa, Q. aquifolioides and Q. rehderiana, respectively]. As indicated by the contraction-expansion model, the point estimates of the current population sizes for the Q. spinosa, Q. aquifolioides and Q. rehderiana were 5.72 × 105, 5.43 × 105, and 5.72 × 105, respectively, the population sizes between current and ancient were 7.41 × 102, 2.00 × 103, and 1.56 × 103, respectively, and the ancient population sizes were 5.31 × 105, 4.84 × 105, and 4.60 × 105, respectively. The contraction time was 3.17 × 104, 3.22 × 104, and 3.21 × 104 generations ago for the Q. spinosa, Q. aquifolioides and Q. rehderiana, respectively , and the expansion time was 3.03 × 103, 3.67 × 103, and 3.53 × 103 generations ago for the Q. spinosa, Q. aquifolioides, and Q. rehderiana, respectively  (Table Our nuclear microsatellite data for the three closely related oak species were best fitted with the contraction-expansion model [posterior probability = 0.8114 (95% CI: 0.7933–0.8295), 0.7247 (95% CI: 0.7093–0.8401) and 0.6583 (95% CI: 0.6440–0.6725) for the d) Table .Q. spinosa has distinct expansion, there have been little change in the predicted spatial distributions of the other two species despite climate warming  than the predicted scores under the null hypothesis both for Q. aquifolioides vs. Q. spinosa, Q. spinosa vs. Q. rehderiana, and Q. rehderiana vs. Q. aquifolioides , and the estimated differentiation values (GST) were larger than the mean genetic differentiation calculated for maternally inherited plastid markers in white oaks of Europe (0.975–1 vs. 0.848)  may also carry some seeds short distances , implying a Pliocene split. Likewise, molecular dating of cpDNA data revealed dramatic divergence since 5.08 Ma  in the ancestral population of species, which determines the rate of coalescence of lineages, and the time between successive speciation events (Hudson, Generally, numerous studies have revealed shared DNA polymorphisms between closely related species or species complex (e.g., Du et al.,  Hudson, . While t Hudson,  and refeOur results of 2MOD strongly supported the gene flow-drift model, indicating that interspecific hybridization significantly contributed to the observed genetic admixture between the three species. Despite these three oaks have weak boundaries and close phylogenetic relationships Figures , 2, the Our Bayesian cluster analysis Figure  indicateQ. spinosa occurs in low-altitude regions, while Q. aquifolioides grows in high-altitude regions; both are common keystone species in local forests, however, Q. rehderiana occupies moderate-altitude areas with scattered distribution, such distribution patterns usually lead to stronger gene flow from core populations to peripheral ones as suggested by Alleaume-Benharira et al. (Q. rehderiana, giving rise to the current model of gene flow.Our results indicated there were asymmetric contemporary gene flow for five related pairs whereas the historical gene flow for all pairs seemed symmetric Table . A possia et al. . In addiIn general, gene flow is a vital factor for population structure over time, it may reduce local adaptation by homogenizing populations found in differing environments or by spreading detrimental alleles across populations, and it also serves to introduce potentially adaptive alleles to populations, and increases genetic diversity (Sexton et al., Our analyses of the three related species reveal similar demographic history according to ENMs and ABC analyses. The neutral genetic markers did not depict the profile of differentiation entirely due to introgression or incomplete lineage sorting. Geological and climatic changes since Miocene have markedly affected the observed patterns of genetic variation and divergence among and within species. Our study indicates that a combination of orographic and bioclimatic analyses can yield deep insights into the diversification and evolutionary history of species in the EH-HM and adjacent regions.GZ and ZL designed the research; LF and JY collected samples; LF, QZ, and YZ performed experiments; LF and ZQ analyzed data; LF and ZQ prepared figures and tables; LF, ZQ, and ZL wrote the manuscript; and LF, QZ, ZQ, JY, YZ, ZL, and GZ revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Channa argus; Anabantiformes: Channidae) and their subsequent expansion is one of many problematic biological invasions in the United States. This harmful aquatic invasive species has become established in various parts of the eastern United States, including the Potomac River basin, and has recently become established in the Mississippi River basin in Arkansas. Effective management of C. argus and prevention of its further spread depends upon knowledge of current population structure in the United States.The introduction of northern snakehead  loci from genomic DNA extracted from 165 C. argus in the United States. Additionally, information from the SNP loci enable us to begin to calculate the long-term effective population size ranges of this harmful aquatic invasive species. We estimate long-term eN to be 1,840,000–18,400,000 for the Upper Hudson River basin, 4,537,500–45,375,000 for the Lower Hudson River basin, 3,422,500–34,225,000 for the Potomac River basin, 2,715,000–7,150,000 for Philadelphia, and 2,580,000–25,800,000 for Arkansas populations.Analysis of those SNP loci help to resolve existing population structure and recover five genetically distinct populations of C. argus in the United States. The valuable information gained from this study will allow effective management of the existing populations to avoid expansion and possibly enable future eradication efforts.This work provides evidence for the presence of more genetic populations than previously estimated and estimates population size, showing the invasive potential of  Channa argus  is one of numerous fish species that have been introduced to the United States and is threatening native populations of organisms. This large, freshwater, piscivorous fish is native to China, Manchuria, southern Siberia, and Korea  were collected from 165 agencies . This inAlfI. Two site-specific ligation adaptors (NC/NN) were used to employ a AlfI restriction. The reduction scheme was chosen based on an approximate genome size of C. argus of 616–861 Mb to target approximately 2,500 SNP loci . Samples were dual barcoded with unique combinations before being sent for sequencing at HudsonAlpha Institute for Biotechnology Genome Services Laboratory  on an Illumina Hi-Seq 2500 using v4 chemistry, generating 50 bp single-end reads.Genomic DNA preparation followed the 2b-RAD protocol from de novo reference sequence as outlined in https://github.com/Eli-Meyer) and the software package Stacks v.1.35  on SNP data that is then retained to perform a Discriminant Analysis on all PCAs. Optimal clusters (K), likely representing populations, were identified through Bayesian information criterion (BIC) likelihood values from retained principal components. Visualization of the DAPC analyses was performed within the Adegenet package.Discriminant Analysis of Principal Components (DAPC) in the Adegenet v2.0.1 package  in the RK was estimated with the cross-entropy criterion and least squares estimates were used to calculate ancestry proportions  v1.8.1 package in R . K was eportions . AdmixtuNe) of each of the C. argus populations. Initially, SNP data were analyzed assuming two populations (East Coast and Arkansas), based on the geographical distance between the eastern United States and Arkansas. Discrete populations were identified using both DAPC and LEA analyses. These analyses showed that the original seven sampling regions were not distinct genetic populations, but instead Chesapeake Bay and Potomac River were one genetic population (Potomac River basin population), and the Lower Hudson River basin and Chinatown fish were also one genetic population (Lower Hudson River basin population). Additional summary statistics were performed on each of the five putative populations and pairwise Fst differences were calculated to assess genetic differentiation between the newly identified populations. The effective population size of the East Coast and Arkansas populations were calculated using the following equation: π = 4∗Ne∗μ, where π represents nucleotide diversity, Ne represents the effective population size, and μ represents the mutation rate of the SNPs (Nb) was calculated, using the following equation: Nb = Ne(0.485 + 0.758∗log (adult lifespan∕age of first reproduction)) ) .C. argus; quality filtering and SNP calling resulted in 1,007 independent SNP loci being retained in the final dataset.A total of 23,695 single nucleotide polymorphism loci were recovered from 165 C. argus in the United States  for each of the C. argus populations are shown in Ne is the smallest, with an estimated range of 1,840,000–18,400,000 individuals (π = 0.0722 and the SNP mutation rate (μ) is 1 ×10−8–1 × 10−9 mutations per generation). The number of breeding individuals (Nb) is estimated to be 2,287,120–22,871,200 individuals based on an adult lifespan of 10 years and age of first reproduction of one year , and an estimated range of 5,640,113–56,401,125 breeding individuals (Nb). The Ne of Arkansas population is estimated to be 2,580,000–25,800,000 individuals (π = 0.0921) and Nb estimated range of 3,206,940–32,069,400 individuals. The long-term effective population sizes  of the eastern United States introductions. Thorough geographic sampling provides a clear distinction between the East Coast populations and the Arkansas population. The Arkansas population is the one that is the most genetically differentiated from the fish in the Potomac River and Lower Hudson River basins. It cannot be completely ruled out that the Arkansas population originated from an unidentified East Coast population not sampled in our study. Adding samples from C. argus native ranges could help to determine the source of the North American introductions, however, those samples were not available for the current study.The SNP data support the presence of five genetically distinct putative populations of C. argus in the eastern United States, as well as determine the source of the C. argus population in the Upper Hudson River basin. C. argus. The results of this genomic scan study show a more complex story, providing evidence for the presence of five distinct populations of C. argus. Based on the shared ancestry and lack of genetic differentiation in the Potomac River basin population and fish from the Chinatown fish market, the fish in both geographic regions likely came from the same source. One individual sampled from the Potomac River basin shows strong genomic similarity to the C. argus in Philadelphia, as well as one individual from the Hudson River basin, suggesting introduction from one source, and subsequent population expansion. This is not surprising because of the close proximity of the Potomac River basin to Philadelphia (approx. 306 kilometers). However, because of the admixture that has occurred in each of the populations, it is not possible with the current dataset to determine how many introductions occurred.One of the principal goals of this study was to use a higher resolution method to answer the questions that remained unresolved from a previous study using microsatellites that sought to determine the number of genetic populations of Ne based on nucleotide diversity in a population. Since they were first reported in Arkansas in 2008, it’s possible that individuals from the original source population could skew the Ne and Nb estimates if any of the fish used in this study are from the original introduction event. Channa argus has a relatively long lifespan and high fecundity in both native and introduced ranges. Previous studies have used C. argus individuals from the Potomac River basin that were estimated to be as old as 10.1 years , the high genetic diversity in these populations, high reproductive potential, and extensive protection of offspring, it is not surprising that the long term effective population size estimates are so high.An accurate assessment of current population size is crucial for conservation efforts and management, along with making accurate models of population growth. This is the first estimate of long-term effective population size for the Arkansas and East Coast populations using SNP data and these estimates are based off the estimation method used by .1 years  and have.1 years . Adult f.1 years  to 28,60.1 years . Further.1 years . In addine weeks . Landis C. argus has a low overall risk rating for the probability of establishment in the Great Lakes basin because they are currently located in Arkansas in low numbers and known to only occur in one river system. These results show that there are likely more fish in Arkansas than previously known, they may occur in more than one river system, and likely have become established. Despite a decrease in population size following an eradication attempt by the Arkansas Fish and Game Commission in 2009, the Arkansas C. argus effective population size is not the smallest of the five populations. Channa argus is an extremely environmentally tolerant species, and therefore was able to rebound from the eradication attempt, and currently has an effective population size within the range of the other populations, demonstrating its potential to quickly increase in abundance. Furthermore, C. argus establishment and determined that the climate conditions throughout most of the Great Lakes would be suitable for their establishment. These results show that C. argus has established large effective populations along the East Coast of the United States and in Arkansas. Other aquatic invasive species with similar life history traits and habitat preferences, such as grass carp, quickly expanded their ranges after being introduced in Arkansas, spreading into the Mississippi River basin, and the Great Lakes basin within 50 years of their introduction. These results illustrate the potential for C. argus to quickly expand its range, like the grass carp and its relatives. Expansion could have major economic and environmental impacts on commercial and recreational fisheries, and native fish populations (According to the US Army Corps of Engineers , C. arguulations .C. argus expansion and subsequent harm to the Mississippi River and Great Lakes basin ecosystems is enhanced by the results of this study. Source population information would aid in the development of a complete picture and the best resulting management strategies. That information in currently not available, but important conclusions and management recommendations can still be made based on our current dataset. These data provide evidence of gene flow between the Potomac River basin and Philadelphia populations. Previous studies have shown that this will make eradication efforts difficult due to the increased likelihood of recolonization (C. argus population in Arkansas is isolated from the East Coast populations, indicating recent establishment and little to no gene flow between Arkansas and the East Coast. Additionally, the main ancestral genotype present in the Arkansas population is rare in the East Coast populations, providing evidence for a different source population. If, as our data suggest, the Arkansas fish originated from a different source than the East Coast populations, then it is important to monitor and prevent possible means of introduction into Arkansas. Therefore, management efforts should focus on the entire population in Arkansas and should be handled differently than the East Coast populations. The isolation of the Arkansas population increases the likelihood of successful eradication, which is crucial to avoid expansion and subsequent harm to the Great Lakes basin.The ability to develop effective management strategies to avoid further nization . However10.7717/peerj.4581/supp-1Fig. S1Click here for additional data file.10.7717/peerj.4581/supp-2Fig. S2Click here for additional data file.10.7717/peerj.4581/supp-3Table S3Click here for additional data file.


A. C. concinnus and A. c. talifuenses. In this study, we conducted a genetic analysis at a contact zone of these two lineages to identify whether these lineages either reflect a high level of intraspecific variation in mitochondrial loci or represent incipient speciation. Mitochondrial ND2 and 11 microsatellite loci were used to conduct phylogenetic and population structure analyses.A deep divergence of mitochondrial DNA is common in species delimitated by morphological traits. Several hypotheses can explain such variations, such as cryptic species, introgression, allopatric divergence and ancestral lineage. The black-throated tit harbors several deeply divergent mitochondrial lineages. Two lineages with 5% divergence, but having a high level of gene flow, have been detected in its subspecies, A. c. talifuenses; however, they formed a non-sister relationship when including all available GenBank ND2 sequences. Analyses of microsatellite data indicated no existing population structure and showed a pattern of isolation by distance. Individuals sampled at the contact zone were almost identified as F2 hybrids.ND2 haplotypes actually diverged into two groups within subspecies Isolation for 2.4 Ma, as suggested by a previous study, appeared to be insufficient to develop robust reproductive barriers. Reproductive barriers were weak, or even absent between the divergent lineages, highlighting that incipient speciation was unlikely to be the case. Considering the results from previous studies, the divergent lineages may be better explained by secondary contact after allopatric isolation because of Pleistocene climate changes, but other hypotheses cannot be definitively ruled out because of the lack of representative samples from the other distribution region and its relatives. Considering that divergence in morphology was hardly observed and the particular split in genetics, the two subspecies might be better merged. The current findings also highlight the likely contribution of male-biased dispersal to male-biased gene flow among mitochondrial lineages; more efforts are needed to illustrate the evolutionary history of the black-throated tit.The online version of this article (doi: 10.1186/s12862-017-1114-9) contains supplementary material, which is available to authorized users. Alcippe morrisonia) [Corvus corax) [Phoenicurus phoenicurus) [A growing number of population genetics and phylogeographic studies have noted that a large number of species are genetically structured , 2, partrisonia) , 5, the s corax) , 7, and nicurus) . For thenicurus) , 9; manynicurus) –12. Howenicurus) . Such cynicurus) –15 and, nicurus) –18.http://www.hbw.com). Isolated populations inhabit southern Laos, central Vietnam, Cambodia and the island of Taiwan [The black-throated tit is a small and abundant passerine bird with a broadly continuous distribution in Pakistan, Nepal, Bhutan, Indian, Myanmar, Thailand, Laos, Vietnam and mainland China  in southeastern Iberia showed a narrow and steep hybrid zone, highlighting the different evolutionary trajectories and independent species identity for these two subspecies [Odocoileus hemionus), however, revealed a hybrid swarm at their hybrid zone, indicating a lack of prezygotic and postzygotic barriers despite the dramatic morphological, ecological and genetic differences between the two lineages [Thus, studies of hybrid zones are paramount in assessing taxonomic status and systematics, as well as for providing insight into the mechanisms that result in reproductive isolation and speciation. For example, a genetic analysis conducted at the contact zone between two subspecies of the ocellated lizard  according to the manufacturer’s protocols. The sequence of the ND2 coding gene was amplified with the primers H6313 and L5219 following the methods for polymerase chain reaction (PCR) outlined in . ND2 PCRA. C. manipurensis was included in the latter. The number of segregating sites, haplotype diversity and nucleotide diversity of our sampled individuals, downloaded sequences and both (combined data) were estimated using DnaSP 5.0 [Sequences of ND2 and Cyt B were aligned using the ClustalW algorithm implemented in MEGA 6 . For ND2naSP 5.0 . McDonalnaSP 5.0 . For CytAegithalos caudatus) as outgroups. Substitution models for data sets were selected using jModelTest 2.1.7 based on Akaike’s information criterion [http://www.atgc-montpellier.fr/phyml/). We also applied the algorithm of automatic model selection to construct the phylogeny. The robustness of the phylogeny in the ML tree was tested by a bootstrap test with 100 resamplings. Bayesian inference (BI) was conducted using MrBayes 3.1.2 [For ND2, haplotypes with different lengths (981 and 734) for the combined data were used to reconstruct the phylogenetic relationships using maximum likelihood (ML) and Bayesian methods (BI). ML and BI were also used for constructing the phylogeny of Cyt B haplotypes. We used GenBank sequences of the long-tailed tit (riterion . GTR + Ies 3.1.2 . Four MeE) and observed heterozygosity (HO) for each locus and population. Allelic richness was standardized for sample size by rarefaction in Fstat 2.9.3 [www.genepop.curtin.edu.au/index.html).Micro-Checker  was usedat 2.9.3 . We alsoCT), between sampled populations within lineages (ΦSC) and between all sampled populations (ΦST). We further tested for isolation by distance among all localities by regressing geographical distance against FST. Linear Euclidean distances (in kilometers) between samples were calculated from their metric easting and northing coordinates using the program Geographic Distance Matrix Generator (version 1.2.1) (Ersts Internet). Mantel tests with 10,000 permutations were performed with IBDWS version 3.14 (www.ibdws.sdsu.edu/) [To test for geographic genetic population structure, analyses of molecular variance (AMOVA) with 10,000 permutations were conducted with Arlequin version 3.1 software , 53. Samsu.edu/)  to assesWe used the individual-based Bayesian clustering method in the program STRUCTURE version 2.3.4 , 56 to iWe performed analysis with NEWHYBRIDS v.1.1  to assigP = 0.18).A total of 204 specimens were sequenced successfully for the mitochondrial ND2 gene. These newly generated data, with a length of 734 bp, contained 66 polymorphic sites—59 of which were parsimony informative—and defined 27 haplotypes, whereas the data with a length of 981 bp had 84 polymorphic sites—75 of which were parsimony informative—and identified 32 haplotypes. Sequences available in GenBank had 64 haplotypes based on 734 bp of 208 sequences, while 75 haplotypes were found based on 981 bp of 207 sequences. The combined data with a length of 734 bp defined 84 haplotypes, while 99 haplotypes were found for the combined data with a length of 981 bp , whereas lineage A was the sister to lineage B-C and A. C. iredalei , and a paraphyletic relationship was observed for lineage B-C, A. C. iredalei and lineage A-manipurensis . Sympatlei Fig. . Howeversis Fig. .Fig. 2Pmanipurensis, with iredalei as its sister group. Lineage B-C was the sister group to all three lineages. Most importantly, all nodes had high support in both the BI and ML trees  and 4 sampled populations. No linkage disequilibrium was detected after correcting for multiple tests. A total of 126 alleles were found for 11 loci in our sampled populations. The mean number of alleles per locus was 9.719±0.249 SEM . Allelic richness (Ar) varied across sites (1.537–3.718). HFST showed that most genetic variation (98.39%) occurred within sampled populations. Genetic variation among sampled populations within groups was only 1.59%, with that among groups accounting for only 0.02% of the total , indicating that the structuring of sampled populations exhibited a pattern of isolation by distance , and only 6 individuals had posterior probabilities less than 50%. The highest posterior probability of individuals being one of the putative parental classes was 51%, and only 5 individuals had posterior probabilities higher than 40%. The posterior probabilities of individuals being F1 hybrids and belonging to backcross categories were less than 2% Additional file 2: Fig. S1.Plots of the best K identified by the method of ΔK. (DOCX 163 kb)Additional file 3:Microsatellite data file used for analyses. (XLSX 40 kb)


Ambrosia artemisiifolia L., (common ragweed), is an annual invasive and highly troublesome plant species originating from North America that has become widespread across Europe. New sets of genomic and expressed sequence tag (EST) based simple sequence repeats (SSRs) markers were developed in this species using three approaches. After validation, 13 genomic SSRs and 13 EST-SSRs were retained and used to characterize the genetic diversity and population genetic structure of Ambrosia artemisiifolia populations from the native (North America) and invasive (Europe) ranges of the species. Analysing the mating system based on maternal families did not reveal any departure from complete allogamy and excess homozygosity was mostly due the presence of null alleles. High genetic diversity and patterns of genetic structure in Europe suggest two main introduction events followed by secondary colonization events. Cross-species transferability of the newly developed markers to other invasive species of the Ambrosia genus was assessed. Sixty-five percent and 75% of markers, respectively, were transferable from A. artemisiifolia to Ambrosia psilostachya and Ambrosia tenuifolia. 40% were transferable to Ambrosia trifida, this latter species being seemingly more phylogenetically distantly related to A. artemisiifolia than the former two. Inferring recent demographic history and contemporary evolutionary processes are major goals in the field of population genetics. Climate change, human disturbances of natural habitats and human-aided dispersal can cause dramatic shifts in the distributions of natural species, and biological invasions are increasingly prevalent worldwide. Analyzing the genetic diversity and population genetic structure of native and introduced populations of an invasive species allows recovering pathways of invasion and identifying founding events and/or admixture events among invasive populations. All these processes affect the demographic success and future expansion of the invasive species and determine its potential for adaption to new environmental conditions. Their understanding is invaluable for devising appropriate management strategies ,2.The molecular markers used for population genetics studies are currently essentially of two kinds: genome-wide Single Nucleotide Polymorphisms (SNPs) identified by next-generation-sequencing-based techniques such as Restriction site Associated DNA sequencing (RAD-seq) or genotyping-by-sequencing , and SimAmbrosia in the Asteraceae family includes at least 51 species collectively known as “ragweeds” and mainly distributed in North America [A. artemisiifolia L., A. trifida L., A. psilostachya D.C. and A. tenuifolia Spreng.) occur in Europe but are native from America [A. artemisiifolia is an annual herb mostly known as a successful invasive and a highly allergenic plant causing severe rhinitis and asthma [th century by the import of contaminated grain and forage [A. artemisiifolia has colonized different types of habitats such as railways, riversides and wastelands, as well as cultivated fields where it is now a noxious weed competing with several summer crops [A. artemisiifolia, reliable and polymorphic molecular markers are needed. To date, only a few gSSR markers have been developed from French A. artemisiifolia populations [A. artemisiifolia [The genus  America . Four di America ,14. A. ad asthma ,16. It hd forage . A. arteer crops . To inveulations ,19. Thesulations –25, Nortulations  and Chinulations . In addiulations –26. Excesiifolia –23.A. artemisiifolia following three different approaches , (b) investigate the genetic diversity, population structure and mating system of A. artemisiifolia using populations sampled in North America and Europe, and (c) assess marker transferability to A. trifida, A. psilostachya and A. tenuifolia.The present study had three purposes: (a) develop new nuclear SSR markers for A. artemisiifolia individuals were sampled from 11 populations spanning the invasive range in Europe and 5 populations in North America  [A. psilostachya and A. tenuifolia have the same basic chromosome number as A. artemisiifolia but variable ploidy levels [A total of 321  America , Fig 1. ribed in . All thr2n = 36, , 29,30).2n = 36, , we coun2n = 36,  in 10 ply levels , 29, 32.A. artemisiifolia individuals was isolated using the DNeasy Plant Mini Kit  and processed by GenoScreen, . A SSR enriched DNA library was obtained as described in [For the SSR-enriched gDNA library approach, total gDNA from 8 ribed in . BrieflyA. artemisiifolia individual was extracted using the DNeasy Plant Mini Kit . Sequencing was performed at GENTYANE . An Illumina paired-end shotgun library was prepared by shearing 2 μg DNA using a Covaris M220 ultrasonicator  and following the standard Illumina TruSeq DNA Library Kit protocol . Sequencing was conducted on the Illumina MiSeq with 250 bp paired-end reads.For the Illumina gDNA sequencing approach, total gDNA from one A. artemisiifolia transcriptome 454 sequence data were downloaded from Genbank Sequence Read Archive [For the EST public data use approach, the two existing sets of  Archive . They coFor each of the three sequence datasets, stringent sequence quality control and filtering were performed using the ShortRead package in the Bioconductor software . BrieflyA. artemisiifolia individuals from three populations . PCRs were performed in 10-μL as previously described [SSRs were identified with QDD version 3.1 . Only 2-escribed . CyclingA. artemisiifolia were used to genotype 384 individuals, including 321 A. artemisiifolia (16 populations), 20 A. trifida (two populations), 22 A. psilostachya (one population) and 21 A. tenuifolia (one population) individuals  or unexpectedly low . We used data from A. artemisiifolia and the software Bayescan [All SSR loci were screened for evidence of selection based on an FBayescan . This prBayescan , we usedA. artemisiifolia was investigated using five gSSR markers  in six additional French populations sampled in 2014 and located within a few kilometres around population GEN13.03 , expected heterozygosity (HS) and the genetic differentiation (FST) were calculated using Fstat [FST values was based on 1000 bootstrap resampling over loci. Inbreeding coefficient (FIS) were estimated with INEST 2.1 [FIS = 0) based on the Deviance Information Criterion (DIC). Genetic diversity and differentiation parameters for A. artemisiifolia were calculated over all populations, over North American populations and over European populations.The allelic richness per locus and per population using a rarefaction method (ng Fstat . SignifiNEST 2.1  using a K) best fitting the data. The length of the burn-in period was 100,000 runs followed by 500,000 Markov Chain Monte Carlo. Ten iterations were performed for each value of K from 1 to 15. K was determined graphically based on log likelihood values as previously described [K. Finally, Clumpp 1.1.2 [Population structure was assessed using Structure 2.2 . The admescribed  using thescribed . In addiescribed  was usedFST values based on the F-model [FST values, and two models that used either latitude or longitude as explanatory variables. In addition to the study of genetic divergence patterns, we investigated the signature of recent bottleneck events using the Wilcoxon test for excess expected heterozygosity implemented in INEST2.1 and based on the method of Cornuet and Luikart [Genetic divergence is likely to vary across populations because of differences in population effective sizes and local migration rates. This is especially the case when recent founder effects have occurred, such as during range expansions. Patterns of genetic divergence were estimated for the invasive range (Europe) by calculating population-specific  F-model . We used F-model  implemen Luikart . AnalyseA. artemisiifolia  than other markers, but a very high within-population genetic diversity (Hs = 0.776), suggesting balancing selection at or near the locus. This locus was therefore discarded for further analyses.Genetic polymorphism was assessed using 16 ividuals . After cividuals  was unamividuals . SSR86 sversus 4.748) but the difference was not significant  .All 26 retained loci were polymorphic and revealed high levels of genetic diversity . The fre= 0.778) . The meaA. artemisiifolia and large numbers of pollen donor parents.Thirty-six maternal progenies sampled from six French populations were analysed with five gSSRs to estimate mating system parameters. In addition, direct evidence for the presence of null alleles was sought by considering the progenies from maternal plants apparently homozygous at one locus. If a null allele was present, the maternal plant would actually be heterozygous . Its progeny would thus contain some plants apparently homozygous for alleles different from the maternal one, but actually carrying one maternal null allele and one paternal detectable allele. Evidence for the presence of null alleles was obtained for all five markers. Depending on the marker considered, from 25% (3 out of 12) to 35% (8 out of 23) of the progenies from plants scored as homozygotes contained non-matching genotypes. Mating system parameters were estimated after excluding these progenies. The maternal inbreeding coefficient was non-significant (F = 0). Multi-locus outcrossing rates (tm) were high and not significantly lower than 1 in all populations . The ratFIS values were on average higher in the native range than in the invasive range (ST in the native range was low (0.042) but significant. FST in the invasive range was higher (0.071) and also significant. The difference in FST values between the two areas was significant based on 99% bootstrap confidence intervals.We compared patterns of genetic diversity between the native range (North America) and the invasive range (Europe). Allelic richness within population and mean expected heterozygosity within population were slightly higher in North America than in Europe . Howeverve range , althougK, whereas the Evanno method (49) suggested that the most likely number of genetic clusters was six  in comparison to the null model with no explanatory factor (posterior probability: 0.28) or the model including longitude (posterior probability: 0.04). Population-specific FST values increased with latitude  was greater in the invasive range. A similar trend had also been observed previously [FST values may arise simply because only a small area of the native range was sampled (five American populations) in our study and in [Genetic diversity within population was similar in North America (the native range) and in Europe, but genetic differentiation among populations  could simply reflect the elevated genetic divergence of some populations. Indeed, Structure analyses are known to be biased towards inferring extra genetic clusters when some populations have undergone strong recent genetic drift; in that case, and contrary to the assumptions of the admixture model, not all genetic clusters are ancestral sources for the present populations [The main pattern of population structure we observed in Europe was a west-east gradient. Differentiation between the western and eastern parts of the European invasive range had previously been observed  and attrulations . Populatulations . This, tulations . PopulatA. artemisiifolia  were transferable to A. psilostachya and A. tenuifolia, whereas only 40% were transferable to A. trifida. The genus Ambrosia is composed of many, not clearly delineated species [A. artemisiifolia, A. psilostachya and A. tenuifolia as related species belonging to one same group, while A. trifida was classified in a separate group [A. artemisiifolia, A. psilostachya and A. tenuifolia but n = 12 for A. trifida, [A. artemisiifolia and A. psilostachya was suggested [Most SSR markers developed for  species  for whicte group . This istrifida, ,29,30,32trifida, . The sucA. psilostachya and A. tenuifolia are perennial species that reproduce both sexually and clonally [A. artemisiifolia and A. trifida, being less widespread and invasive, our SSR markers will be useful for assessing vegetative versus sexual reproduction, as well as for identifying colonization sources and relatedness among populations. However, given that several ploidy levels were reported in these species [A. artemisiifolia are transferred to A. psilostachya and A. tenuifolia. A. trifida is a noxious annual weed, very widespread in its native area [A. artemisiifolia and the low success rate of markers transferability, we recommend that additional SSR markers are specifically developed for A. trifida.clonally , 32, 62. species , 29, 32,ive area  and intrive area ,65. DespA. artemisiifolia, providing useful new resources for genetic studies of this highly noxious invasive weed. All markers were highly polymorphic. EST-SSRs revealed as many alleles as gSSRs and yielded similar genetic diversity estimates. The genetic patterns revealed for a set of American and European populations confirmed results from previous studies by showing a high within-population genetic diversity in both the native and invasive ranges. A geographical gradient of genetic variation in Europe was consistent with at least two major colonization events in Western and Eastern Europe, respectively. Secondary founding events were identified, especially in Central Europe. In addition, we settled a former controversy by demonstrating that inbreeding observed within populations is attributable mostly to the presence of null alleles rather than to selfing. Last, most SSRs were transferable to three other Ambrosia species. These SSRs can readily be used for studying key aspects of the biology and population dynamics of the two species most closely related to A. artemisiifolia (A. psilostachya and A. tenuifolia).Large sets of genomic SSRs and EST-SSRs were developed and validated in S1 FigThe red arrows indicate the chromosomes. Gx100.(TIF)Click here for additional data file.S2 FigResults of Bayescan FSToutlier analysis on 14 gSSR (a) and 13 EST-SSR loci (b). The vertical bars correspond to threshold P-values of 0.05 (solid line) and 0.01 (dashed line) for the neutral model. (a) Data from all 16 populations. (b) Data from 11 European populations.(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 TableA. artemisiifolia.Loci in bold represent the markers selected in (DOCX)Click here for additional data file.S2 TableA. artemisiifolia.Loci in bold represent the markers selected in (DOCX)Click here for additional data file.S3 TableA. artemisiifolia.Loci in bold represent the markers selected in (DOCX)Click here for additional data file.S4 TableN: number of individuals genotyped, A: average allelic richness after rarefaction, HO observed heterozygosity, HS expected heterozygosity, FIS inbreeding coefficient estimated taking into account the presence of null alleles. * FIS estimates significantly greater from zero (using the Bayesian model comparison based on Deviance Information Criterion implemented in INEST2.1).(DOCX)Click here for additional data file.


Euchiloglanis fish species complex as a model to demonstrate the compound effects of the Tibetan Plateau uplift and Pleistocene glacial oscillations on species formation in this region. The genetic structure and geographical differentiation of the Euchiloglanis complex in four river systems within the Hengduan Mountain Region were deduced using the cytochrome b (cyt b) gene and 10 microsatellite loci from 360 to 192 individuals, respectively. The results indicated that the populations were divided into four independently evolving lineages, in which the populations from the Qingyi River and Jinsha River formed two sub‐lineages. Phylogenetic relationships were structured by geographical isolation, especially near drainage systems. Divergence time estimation analyses showed that the Euchiloglanis complex diverged from its sister clade Pareuchiloglanis sinensis at around 1.3 Million years ago (Ma). Within the Euchiloglanis complex, the divergence time between the Dadu–Yalong and Jinsha–Qingyi River populations occurred at 1.0 Ma. This divergence time was in concordance with recent geological events, including the Kun‐Huang Movement (1.2–0.6 Ma) and the lag time (<2.0 Ma) of river incision in the Hengduan Mountain Region. Population expansion signals were detected from mismatched distribution analyses, and the expansion times were concurrent with Pleistocene glacier fluctuations. Therefore, current phylogeographic patterns of the Euchiloglanis fish complex in the Hengduan Mountain Region were influenced by the uplift event of the Tibetan Plateau and were subsequently altered by paleo‐river transitions during the late Pleistocene glacial oscillations.The uplift of the Tibetan Plateau caused significant ecogeographical changes that had a major impact on the exchange and isolation of regional fauna and flora. Furthermore, Pleistocene glacial oscillations were linked to temporal large‐scale landmass and drainage system reconfigurations near the Hengduan Mountain Region and might have facilitated speciation and promoted biodiversity in southwestern China. However, strong biotic evidence supporting this role is lacking. Here, we use the  This complex is part of a group of demersal freshwater catfish (Siluriformes: Sisoridae) that is distributed in the upstream region of the Yangtze River basin. The genetic divergence and population structure of the fishes are easily influenced by geographical events because of their weak mobility. Thus, the Euchiloglanis species complex is an ideal subject for investigating how paleo‐drainage shifts affect speciation in connection with the historic uplift of the Tibetan Plateau. However, morphology‐based taxonomy or molecular‐based phylogeny techniques have previously failed to recognize these fishes correctly  gene and 10 microsatellite markers. Our goals were to: (1) infer the genetic structure and geographical differentiation of populations belonging to the Euchiloglanis species complex throughout the Hengduan Mountain Region; and (2) verify a vicariant speciation hypothesis  based on geological evidence of massive scale paleo‐drainage shifts that are related to the uplift of the Tibetan Plateau.We analyzed the geographical differentiation of the 22.1Euchiloglanis fish specimen sampled from the Dadu River is shown in Figure Samples were collected using fishhooks from 2012 to 2015. In all, 360 samples were gathered from 11 populations across four river systems, with 155 specimens being collected from the Dadu River, 117 from the Yalong River, 62 from the Jinsha River, and 26 from the Qingyi River , 1.5 μl 50 mM MgCl2, 2.0 μl 2.5 mM dNTP, 1 U Taq DNA polymerase , 1 μl 10 μM of each primer, 2–4 μl genomic DNA (50 ng/μl), and double‐distilled water added to make a final volume of 25 μl. The following conditions were used for PCR reactions: (1) pre‐denaturation at 95°C for 3 min; (2) denaturation at 95°C for 30 s; (3) annealing at 54–56°C for 30 s; (4) elongation at 72°C for 1 min (repeated 2–4 stages 35 times), and a final elongation at 72°C for 10 min. Negative controls  were used in each PCR run, to test for contamination and artifacts. Reactions were performed in a Veriti Thermal Cycler . PCR products were tested via electrophoresis through 1% agarose gels and were purified with the Qiagen Gel Extraction Kit (Qiagen). Both strands of each product were sequenced using PCR primers.Genomic DNA was extracted from fin tissues using the Qiagen DNeasy Kit , according to the instructions of the manufacturer. The E. kishinouyei and were selected for genotyping analyses  were specifically developed for 2.3cyt b sequences were manually assembled using CONTIGEXPRESS version 3.0.0 . A multiple sequence alignment was performed with MAFFT version 6  and haplotypes (H), haplotype diversity (h), and nucleotide diversity (π), were calculated using DNASP. For microsatellite data, genetic diversity indices, including the number of alleles (NA), expected (HE) and observed (HO) heterozygosities, and the F‐statistics indices (FIT and FIS), were assessed using POPGENE. Allelic richness (Rs) was computed using FSTAT version 2.9.3 ] to the geographical distance (ln·km) across populations for the cyt b gene and microsatellite loci. For each analysis, 100,000 randomizations were calculated. Moreover, analysis of molecular variance (AMOVA) was also computed in ARLEQUIN. Population groups were also divided according to phylogenetic analyses.Genetic variation in the 2.5cyt b sequences of three Pareuchiloglanis sinensis individuals were amplified and used as outgroups because the species is the sister taxon of Euchiloglanis. Glyptosternon maculatum (DQ192471) was also chosen as an outgroup to avoid any bias. Before reconstructing the phylogenetic trees, an optimal DNA substitution model (GTR + I + G) was obtained based on model‐averaged parameters using the Akaike Information Criterion (AIC) in JMODELTEST version 2.1.4 , neighbor‐joining (NJ), and maximum parsimony (MP) were performed to reconstruct the phylogenetic tree among the K value). The lengths of the MCMC iterations were set to 50,000 with a burn‐in period of 5,000. The K value range was set to 1–7, and each K was replicated 20 times. The most likely K value was chosen according to peak value of the mean log likelihood [Ln P(X/K)] and the Delta K statistic for a given K  and (2) the divergence of P. sinensis from E. davidi (1.7 ± 0.3 Ma). The MCMC chain was run for 1 × 108 generations and was sampled every 1,000 generations. The first 10% were burn‐in. TRACER version 1.5   was 0.02, which sufficiently pre‐dated the formation of the lake where speciation likely happened . Overall, haplotype diversity (h = 0.9521 ± 0.0059) and nucleotide diversity (π = 0.01360 ± 0.00059) were relatively high. Among the 11 populations, the values of h and π of the GZ, BZL, and DW populations were lower than those observed in the remaining eight populations. h and π ranged from 0.3580 to 0.9643 and from 0.0006 to 0.0090, respectively (Table HO was 0.602 (presented in the TQ population), and the lowest mean value was 0.178 (presented in the LB population). The mean values of HO were lower than those of HE in all populations, with the exception of the XL population (Table S1), suggesting a deficit in heterozygosity.The 1,137‐bp cFST analyses were conducted to further investigate the genetic differentiation among populations. For the mtDNA data, a significant difference was observed in all samples , indicating a high degree of geographical population divergence. Pairwise FST results suggested significant differentiation between any two populations (p < .001), except the BZL and DW populations and the GZ and DF populations (p > .05)  and 0.467 (p = .0461) for mitochondrial and microsatellite data, respectively, when evaluating the genetic diversity and geographical distance in the Euchiloglanis populations  were shared by two or more populations from the same river. Moreover, the remaining haplotypes were restricted to one population, with 96 haplotypes being singletons (Table S2). These results indicate extensive genetic differentiation among populations. Pairwise 3.2Euchiloglanis complex was monophyletic  formed one lineage, while all haplotypes from the Yalong River  formed another lineage. Sichuan haplotypes from the Jinsha and Qingyi rivers were clustered into a single lineage with two sub‐lineages: (1) the haplotypes of the LB population in Sichuan Province, and (2) the haplotypes of the TQ population. The remaining lineage consisted of all Yunnan haplotypes from the Jinsha River (BZL and DW)  Figure . The hap) Figure .K . The mean values of FIS and FIT were 0.0081 and 0.4632, respectively, based on microsatellite data (Table S6).Bayesian cluster analyses showed that the results of the structure analysis based on microsatellite loci were consistent with those of the phylogenetic analyses and haplotype networks based on mtDNA data. The whole population was split into four genetic clusters with a diverse maximum Δ3.3P. sinensis at 1.3 Ma (95% HPD = 0.9–1.7). The split in the Dadu and Yalong Rivers was at 0.7 Ma (95% HPD = 0.5–1.0). The LB lineage from the Jinsha and Qingyi Rivers diverged at 0.4 Ma (95% HPD = 0.2–0.7). The divergence time of the LB lineage from the Jinsha–Qingyi Rivers and the BZL and DW lineages from the Jinsha River was also at 0.7 Ma (95% HPD = 0.4–1.1). Finally, the Dadu–Yalong lineage and Jinsha–Qingyi lineage diverged at 1.0 Ma  and 0.5–3.5 Ka, respectively. The corresponding fluctuation time of the Qingyi River was 0.5–13 Ka.Bayesian skyline plots analyses showed a comparatively clear demographic history for the five divided clades Figure , indicat44.1cyt b haplotypes indicated four independent evolutionary lineages, with one lineage being split into two sub‐lineages (Qingyi River and Jinsha River of LB). These results suggest that geographical isolation within the same drainage systems shaped the phylogenetic architecture of the populations. For instance, the Dadu River and Yalong River groups initially formed sister relationships and were subsequently clustered with the Jinsha River and Qingyi River groups. However, within the Jinsha group, samples from Yunnan (BZL and DW) did not cluster with the Jinsha River samples from Sichuan (LB). Moreover, the LB individuals from the Jinsha River had close relationships with individuals from the Qingyi River. This phenomenon might be caused by geographical isolation that allowed diverse populations to evolve in independent directions. The results of the Mantel tests based on both genetic markers suggested that the genetic distance was significantly correlated with the geographical distance of Euchiloglanis. Alternatively, the limited number of LB individuals might be the source of these differences. Thus, because of the topographic complexity and unique geological history of the Tibetan Plateau, it is essential to collect more specimens to reconstruct the relationship between geological events and the evolutionary history of endemic species. Moreover, the results of the phylogenetic analyses showed that individuals from the Dadu and Yalong rivers were not completely isolated. Thus, Dadu River group and Yalong River group might have originated from a single ancestral population, which subsequently separated because of crustal movements and river captures . The Qingyi River underwent an expansion at 0.005–0.013 Ma, which was after the EGP (0.5–0.17 Ma), and possibly earlier than the LGM (0.021–0.017 Ma). Therefore, the results of this study provided evidence of the exceptional phylogeographical architecture of the Euchiloglanis in the Hengduan Mountain Region, which was initially shaped by the uplift event of the Tibetan Plateau. Furthermore, the results highlight the importance of paleo‐river connections, which were likely complicated by glacial movements in the late Pleistocene ice age. In conclusion, the divergence detected between the lineages in this study suggests that a number of speciation events might occurred in the Hengduan Mountain Region; however, to confirm these splits, large DNA datasets are required to resolve the phylogenetic relationships within the Euchiloglanis fish complex.Furthermore, the molecular clock results indicated that the divergence time was congruent with the Kun‐Huang Movement (1.2–0.6 Ma) and the extensive glacial period . Zheng, Xu, and Shen  stated tNone declared. Click here for additional data file.