The Tibetan pig is one of domestic animals indigenous to the Qinghai-Tibet Plateau. Several geographically isolated pig populations are distributed throughout the Plateau. It remained an open question if these populations have experienced different demographic histories and have evolved independent adaptive loci for the harsh environment of the Plateau. To address these questions, we herein investigated ~ 40,000 genetic variants across the pig genome in a broad panel of 678 individuals from 5 Tibetan geographic populations and 34 lowland breeds.PLA2G12A, RGCC, C9ORF3, GRIN2B, GRID1 and EPAS1, are involved in high-altitude physiology including angiogenesis, pulmonary hypertension, oxygen intake, defense response and erythropoiesis. A majority of these genes have not been implicated in previous studies of highlanders and high-altitude animals.Using a series of population genetic analyses, we show that Tibetan pig populations have marked genetic differentiations. Tibetan pigs appear to be 3 independent populations corresponding to the Tibetan, Gansu and Sichuan & Yunnan locations. Each population is more genetically similar to its geographic neighbors than to any of the other Tibetan populations. By applying a locus-specific branch length test, we identified both population-specific and -shared candidate genes under selection in Tibetan pigs. These genes, such as Tibetan pig populations have experienced substantial genetic differentiation. Historically, Tibetan pigs likely had admixture with neighboring lowland breeds. During the long history of colonization in the Plateau, Tibetan pigs have developed a complex biological adaptation mechanism that could be different from that of Tibetans and other animals. Different Tibetan pig populations appear to have both distinct and convergent adaptive loci for the harsh environment of the Plateau.The online version of this article (doi:10.1186/1471-2164-15-834) contains supplementary material, which is available to authorized users. The environmental condition of the plateau is characterized by the reduced oxygen availability, low ambient temperature, high ultraviolent radiation and amid climate. The unique ecological condition imposes severe physiological challenges on inhabits in the high-altitude region. Native inhabitants like Tibetans have evolved the adaptive mechanism to address the harsh environment during the long history of colonization. Today, a number of researchers have identified particular physiological traits favoring local adaption in Tibetans. The adaptive traits include increased nitric oxide level, elevated resting ventilation, reduced pulmonary vasoconstrictor response and low hemoglobin concentration compared with acclimated lowlanders [The Qinghai-Tibet Plateau, known as the “roof of the world”, is the highest  and largest  in the hypoxia-inducible factor (HIF) pathway have been concordantly shown to carry the adaptive mutations [EPAS1[EGLN1[EGLN1 gene is suggested to be the causal variant for local adaption in Tibetans [EGLN1 and different selection-nominated candidate genes like BHLHE41 have been identified. In non-human species indigenous to the Qinghai-Tibet plateau, the draft genomes of the Tibetan antelope [Although the physiological traits in response to high-altitude environments are relatively well characterized, an understanding of the molecular basis underlying these traits has significantly lagged behind. Recently, population genomics offers an effective approach to characterize the adaptive mechanism of Tibetans and other colonists in the Qinghai-Tibet Plateau. One common approach is involved in genotyping a large number of loci across the genome on divergently differentiated populations, and searching “outliers” (candidate targets of selection) in the extreme tail of the empirical distribution of statistics like FST, 5 and lST–8. To daST, 5, 8–11utations . Both EPs [EPAS1 and EGLN1[EGLN1, 11 variTibetans , 13 and Tibetans , in whicantelope  and Yak antelope  have beeThe Tibetan pig, one of Tibetans’ domestic animals, is originally distributed in farming and farming-pastoral regions at altitudes of 2,900 - 4,300 m in the Qinghai-Tibetan Plateau . Like TiTo elucidate the genetic basis of the altitude phenotypes in Tibetan pigs, we have previously searched for population-differentiated SNPs across the genome between two Tibetan pig populations and 16 lowland breeds using the Illumina 60 K SNP data. We identified several candidate genes that might play a role in high-altitude adaptation in Tibetan pigs . More reHere we genotyped a broad panel of 678 pigs from 5 Tibetan regional populations and 34 lowland breeds using high-density SNP genotyping arrays. By applying population genetic and genomic approaches, we characterize the genetic landscape for Tibetan pigs and reveal both population-shared and -specific candidate genes contributing to local adaptations in the 5 Tibetan populations. Our findings provide novel insights into evolutionary history of Tibetan pigs and the genetic architecture of high-altitude adaptation in highland animals and their owners.To investigate population structure of Tibetan pigs, we first constructed a neighbor-joining (NJ) tree based on genome-wide allele sharing of the 678 pigs from 5 Tibetan populations, 28 Chinese lowland breeds  in all tested pigs. The PCA plot  using a subset of 14,202 SNPs with low linkage disequilibrium (LD) extents r2 < 0. in all tTo assess evolutionary origin and historical admixture patterns of Tibetan pigs in a context of worldwide populations, we further conducted a Bayesian ancestry inference analysis using the program ADMIXTURE .According to the above-mentioned population genetic analyses, we divided Tibetan pigs into three independent populations: Tibet, Gansu and SCYN. To identify population-specific loci under positive selection in Tibetan pigs, we calculated the LSBL value for each of the 41,495 informative SNPs along the genome using a three-group contrasting model: one Tibetan population, one Chinese lowland group and the other two Tibetan populations (See Methods). The three-group test identified SNP outliers that had highly differentiated allele frequencies in one Tibetan population relative to the other two groups. These outlier SNPs are candidate loci (or to be in linkage disequilibrium with selected variants) under selection for adaptation to high-altitude hypoxia.C9ORF3, GRIN2B and GRID1, three functionally plausible genes (See Discussion), exhibited the most significant signals of selection in Gansu, Tibet and SCYN populations, respectively , corresponding to 140, 129 and 124 candidate genes (50 kb up- and downstream of each SNP outliers) in Gansu, Tibet and SCYN populations, respectively , detection of mechanical stimulus involved in sensory perception , artery development , positive regulation of smooth muscle cell proliferation  and glutamate receptor signaling pathway . We also identified 4 and 3 KEGG pathways in Gansu and Tibet populations (Table Pcor = 0.031), circadian entrainment (Pcor = 0.033), nicotine addiction (Pcor = 0.043) and adherens junction (Pcor = 0.045) pathways in the Gansu population, and propanoate metabolism (Pcor = 0.004), sulfur metabolism (Pcor = 0.004) and DNA replication (Pcor = 0.024) in the Tibet population.We sequentially performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on highlighted candidate genes in each Tibetan pig population. GO analysis identified 27, 15 and 17 overrepresented genes in Gansu, Tibet and SCYN populations, respectively . Allele C at the SNP is predominantly presented in Tibetan pigs with average frequency of 0.816 whereas is much rarer in Chinese lowland breeds at an average frequency of 0.207 except for Neijiang (0.750), a breed known for its well adaptability to diverse environmental conditions [HFM1 and ZNF644 in response to hypoxia has not been established and warrants further investigations. The second strongest LSBL SNP (rs81403518) is located at 12 kb upstream of the PLA2G12A gene, a biologically plausible gene (see Discussion). The third strongest outlier is located at ~17 kb downstream of a hypoxia-inducible gene: RGCC.To test if Tibetan pig populations have common adaptive loci for the Plateau, we also looked across the genome to identity signals of selection using the three-group test model with all Tibetan pigs as one group and two Chinese lowland groups (See Methods). A total of 207 SNP outliers were visualized on the top 0.5% of the empirical distribution, corresponding to 122 genes , histone H3-K9 methylation , positive regulation of positive chemotaxis , regulation of cytokinesis , fatty acid catabolism , detection of mechanical stimulus involved insensory perception of pain , and behavioral defense response  (Table Pcor = 0.036), fat digestion and absorption (Pcor = 0.042) and amyotrophic lateral sclerosis (Pcor = 0.046) . The two SNPs showed apparently different variation patterns between Tibetan pigs and low-altitude individuals . ADAM17etan yak . We, thePLA2G12A, RGCC, C9ORF3, GRIN2B and GRID1) are reported for the first time.As shown in Figure The formation of Tibetan present-day pig populations was likely influenced by the migration of their owners: Tibetans. Today, both archaeological and genetic data support an ancient initial colonization of modern humans in the early Upper Paleolithic before the last glacial maximum  and a recent population expansion and gene flow from outside the highland region in the early Neolithic  . The recEPAS1 and EGLN1 at the top list are consistently identified in multiple Tibetan studies, many of these selection candidate genes are unique to each study. Such differences may be at least partly attributed to different demographic histories and genetic differentiation among Tibetan populations that likely have various degree of admixture with different neighboring populations [C9ORF3, GRIN2B and GRID1 genes exhibit population specific signals in the Gansu, Tibet and Sichuan & Yunan populations, respectively . While ulations , 26, 27.y Figure . These cC9ORF3, also known as Aminopeptidase O, encodes a member of the M1 zinc aminopeptidase family that catalyzes the hydrolysis of amino acid residues from the N-terminus of peptide. C9ORF3 is involved in the renin-angiotensin pathway, in which C9ORF3 cleaves angiotensin III to generate angiotensin IV [C9ORF3 is positively correlated with maximal oxygen uptake and the percentage of type 1 fibers in humans [C9ORF3 is associated with newborn respiratory distress syndrome. Therefore, preferential selection on the C9ORF3 gene is likely required for Tibetan (Gansu) pigs to increase oxygen uptake and avoid pulmonary or cerebral vascular hypertension and edema during the long-standing living in the hypoxic highland.ensin IV . It is kensin IV , 30 and ensin IV . Moreoven humans . As predn humans , C9ORF3 GRIN2B encodes a subunit of N-methyl-D-apartate receptor that is the predominant excitatory neurotransmitter receptor in the mammalian brain. The receptor has a central role in memory and cognitive function. Defects in the GRIN2B gene have been found in human patients with mental retardation [GRID1 encodes glutamate receptor delta 1, a subunit of glutamate receptor channels that mediate most of the fast excitatory synaptic transmission in the central nervous system and play key roles in synaptic plasticity [GRID1 gene have been associated with human schizophrenia [ardation . GRID1 easticity . Variatiophrenia , 37. PosPLA2G12A and RGCC in our top-ranking list stand out as strong candidate genes that likely play a role in high-altitude adaptation in Tibetan pigs.In addition to population-specific candidate loci, we highlighted a list of selection-nominated candidate genes shared by all Tibetan pigs. Many of these genes are functionally related to energy metabolism, angiogenesis, melanin synthesis and behavior defense response. Of note, PLA2G12A encodes secreted phospholipase A2, group XIIA enzyme. The enzyme hydrolyzes phospholipids into arachidonic acid and other lipophilic molecules that exert a variety of biological effects. Of note, arachidonic acid plays an important role in regulation of pulmonary vascular tone in lungs of both newborns and adults. In pigs and other species, arachidonic acid can induce dilation in pulmonary arteries. Altered arachidonic acid metabolites have been implicated in the development of pulmonary hypertension in chronically hypoxic piglets [PLA2G12A is a strong candidate gene contributing to high-altitude adaptation in Tibetan pigs. High frequencies of PLA2G12A favorable mutations in Tibetan pigs could be beneficial for increasing blood flow at low degree of hypoxic pulmonary hypertension. piglets . TherefoRGCC plays a critical role in hypoxia-induced antiangiogenesis. It has a physical interaction with HIF1α and vascular endothelial growth factor (VEGF) that are key mediators in cellular response to hypoxia and ischemia [HIF1α activates the expression of VEGF that is critically important for skeletal muscle angiogenesis. The increased growth of blood vessels has been associated with capillary leak [RGCC gene may inhibit the capillary growth and consequently avoid capillary leak in Tibetan pigs under long-term exposure to the hypoxic environment.ischemia . In respary leak . TherefoEPAS1, the “master regulator” of erythropoiesis in the HIF pathway , also atan pigs . It shouRGCC), pulmonary hypertension remodeling (PLA2G12A), oxygen intake (C9ORF3), neural response (GRID1 and GRIN2B), melanin synthesis  and heart development . Further characterization of these genes will be necessary to identify variants responsible for the observed signals. Both distinct and shared genomic signatures of selection were illustrated in Tibetan regional pig populations. We argue that both independent and shared adaptive variants could be responsible for local adaptation in each Tibetan pig population. Our findings reflect a complex picture of the genetic mechanism underlying high-altitude adaptation in Tibetan pigs. The adaptive mechanism seems to involve a range of genes regulating diverse biological processes. Distinct set of genes including some broadly shared loci act in a highly coordinated manner to offset severe physiological challenges imposed by the harsh hypoxic environment in different Tibetan pig populations.In this study, we identified a total of 489 genes as potential selection targets in all Tibetan pig populations .All animals were genotyped for ~62,000 SNPs on Porcine SNP60 BeadChips   was genotyped on 324 Chinese indigenous pigs by the iPLEX MassARRAY platform  according to the supplier’s protocol. SNP genotype calls were filtered with MAF > 0.05 and genotype call rate > 95%, resulting in 49 informative SNPs. The 49 SNPs were merged into the illumina SNPs to form a common subset of 41,544 SNP data in Chinese indigenous pigs, which were then used to detect signals of selection on the 3 hypoxia genes in Tibetan pigs.A panel of 56 SNP markers at an average interval of 5.4 kb , the proportion of polymorphic markers (PN) and expected heterozygosity (HE) using ADZE [http://beast.bio.ed.ac.uk/FigTree). A subset of 14,202 SNPs with low linkage disequilibrium (r2 < 0.3) filtered by PLINK [A common subset of 25,340 SNPs with MAF ≥ 0.2 in all tested pigs were explored to calculate three measures of genetic variability of each population: allelic richness . Because the range of FST is originally defined between 0 and 1 [ST values that do not have a biological interpretation were set to 0.In the above formulae, n 0 and 1 , negativPopulation structure was analyzed using the Maximum Likelihood approach implemented in ADMIXTURE v 1.20  and the TreeMix  was emplST using the above Reich’s calculation at every SNP position for each two-way group comparison . Next, the pairwise FST values were used to calculate the LSBL at each SNP as described previously [ST were identified as candidate selection loci. LSBL outliers were defined as sites with LSBL statistics surpassing 0.5% of the empirical distributions.LSBL statistics, a robust indicative of selection-nominated loci , were caeviously . With thhttp://ensembl.org/index.html). Furthermore, human orthologous genes were identified by aligning pig gene-associated regions against the human genome using the BLAST-like Alignment Tool [http://www.geneontology.org) was then queried by the ClueGO plugin of Cytoscape [The 50 kb upstream and downstream of significant LSBL loci were operationally defined as candidate regions under selection. Pig annotated genes within candidate regions were first searched against the pig genome assembly 10.2  via the ent Tool . To perfytoscape  using SyThe genotyping data set supporting the results of this article is available in the Dryad database (doi:10.5061/dryad.53j31).Additional file 1: Table S1: Summary of genetic diversity of each tested population. (DOC 110 KB)Additional file 2: Figure S1: The neighbor-joining tree of all tested breeds and Tibetan pig populations based on genome-wide allele sharing. The tree illustrates a clear evolution split between Chinese and Western pigs. It also shows obvious genetic differentiation among Tibetan pig populations that usually group together with their geographic neighbors. Two Chinese synthetic breeds including Sutai and Lulai define intermediate branches between Chinese and Western groups. Such intermediate branches were also observed for Chinese Licha and Kele pigs, corresponding to our previous findings of the historical introgression of Western pigs into the two Chinese populations . Tibet 1Additional file 3: Figure S2: Principle components analysis of all tested breeds and Tibetan pig populations in the present study. Principal component (PC) 1 (x-axis) verus PC2 (y-axis). PC1 clearly discriminates Chinese and Western pigs. PC2 separates Chinese pigs including Tibetan populations in a manner corresponding to their geographic locations. The 5 Tibetan pig populations highlighted in shade exhibit genetic similarity to their geographic neighbors. The two synthetic breeds (Sutai and Lulai) and two admixed breeds (Licha and Kele) show consistent signals of admixture with Western pigs. (TIFF 339 KB)Additional file 4: Figure S3: Population structure of each population revealed by the ADMIXTURE software. At K = 8, the ancestry structures of Tibetan pigs from Sichuan and Yunnan were nearly identical, differing from those of Tibetan pigs from Tibet and Gansu. From K = 2 to K = 5, ~80% of the Western pig genomes were assigned to Western wild boars. Of note, about 20% of Chinese ancestry was consistently observed in Landrace and Large White, suggesting a historical admixture between Chinese and Western pigs. The observation is in agreement with the previous report of a ~35% Asian fraction in Western pigs according to the whole genome sequence data . The abbAdditional file 5: Figure S4: TreeMix plot for residual fit from the maximum likelihood tree without migration events. (TIFF 738 KB)Additional file 6: Table S2: SNP outliers and candidate genes for high-altitude adaptation in each and all geographic populations of Tibetan pigs. (XLSX 132 KB)Additional file 7: Figure S5: A venn diagram showing shared and distinct candidate genes in each and all geographic populations of Tibetan pigs. Numbers indicating how many genes belong to each of Tibetan populations are shown in the graph. (TIFF 488 KB)EPAS1, EGLN1 and ADAM17) names and their corresponding regions are indicated below each panel. (B) Allele frequencies of the two outlier SNPs at the EPSA1 regions in a panel of Chinese indigenous pig populations. The breed codes are identical to those given in the legend of Figure Additional file 8: Figure S6: Patterns of selection signatures within three well-characterized hypoxia genes in Tibetan pigs. (A) Distribution of LSBL values within the target regions. LSBL values are plotted along the y-axis, and the threshold indicating signature of selection is denoted with a dashed grey line. The candidate gene  in 324 Chinese indigenous pigs. (XLSX 122 KB)Additional file 9: Table S3: Genotypes of 56 SNP markers within 3 well- characterized hypoxia genes (


Impatiens glandulifera, an invasive plant in Europe that can have a major impact on native plant communities. We compared populations from the species’ native range in Kashmir, India, to those in its invaded range, along a latitudinal gradient in Europe. For comparison, the results from 39 other studies of genetic diversity in invasive species were collated.Invasive species can be a major threat to native biodiversity and the number of invasive plant species is increasing across the globe. Population genetic studies of invasive species can provide key insights into their invasion history and ensuing evolution, but also for their control. Here we genetically characterise populations of I. glandulifera was established in the wild in Europe at least twice, from an area outside of our Kashmir study area. Our results further revealed that the genetic diversity in invasive populations of I. glandulifera is unusually low compared to native populations, in particular when compared to other invasive species. Genetic drift rather than mutation seems to have played a role in differentiating populations in Europe. We find evidence of limitations to local gene flow after introduction to Europe, but somewhat less restrictions in the native range. I. glandulifera populations with significant inbreeding were only found in the species’ native range and invasive species in general showed no increase in inbreeding upon leaving their native ranges. In Europe we detect cases of migration between distantly located populations. Human activities therefore seem to, at least partially, have facilitated not only introductions, but also further spread of I. glandulifera across Europe.Our results suggest that Impatiens glandulifera across Europe.Although multiple introductions will facilitate the retention of genetic diversity in invasive ranges, widespread invasive species can remain genetically relatively invariant also after multiple introductions. Phenotypic plasticity may therefore be an important component of the successful spread of The online version of this article (doi:10.1186/s12863-015-0242-8) contains supplementary material, which is available to authorized users. Invasive plant species are becoming increasingly common and can threaten biodiversity across the world . Apart fThe successful invasiveness of some species in spite of low genetic diversity is commonly referred to as the genetic paradox of invasive species . It has,Phylogeographic analysis of intraspecific genetic variation can be used to explore the migration history of a species, including species that have recently colonized an area , the Himalayan Balsam, is an invasive species in Europe , No, NoImpatpe  and the introduced range within Europe across a large part of the species’ invaded north – south distribution. The main aims of our study were 1) to investigate the number of introductions into Western Europe, 2) to compare the genetic diversity of the species and its distribution in the invaded and native range, 3) to explore the importance of evolutionary forces, in particular gene flow, between populations in shaping the distribution of genetic diversity in the invasive range and 4) to compare our results with general population genetic patterns in invasive species.Here we assess both local and more large-scale patterns of genetic diversity in I. glandulifera. Originally individuals from 10 populations, some located within the same municipality, along the species’ north – south distribution in Europe were genotyped for eleven microsatellite markers and compared with individuals from three populations from Kashmir in the species’ native range  yielded identical genotypes upon repeated scoring. Genotyping error rate was not estimated for samples with high quality chromatogram markers, but is expected to have been considerably lower than for low quality chromatogram markers. In 75 out of 117 marker - population combinations the frequency of null alleles was estimated to be less than 5 % suggesting that null alleles were not a predominant property in most populations  were found in all populations . Instead populations with both comparatively high and low diversity measures could be found both among more northern and southern populations. Trondheim2, one of the northernmost populations, did, however, stand out as having the fewest number of alleles, lowest expected heterozygosity and most highly negative inbreeding coefficient of all populations . The number of alleles (Table p < 0.05) in Kashmir populations  than in European  as were the number of private alleles . The inbreeding coefficient was significantly higher in Kashmir than in Europe (t-test p < 0.05) and two of the Kashmir populations (1 and 3), but none of the European populations, had significant inbreeding coefficients when calculated across all loci  and the total genetic diversity  were lower in Europe than in Kashmir  as the number of clusters best describing the data , also the number of clusters with the highest repeatability between runs according to CLUMPP H values (0.997), with the second highest ΔK and CLUMMP H values for the K = 4 model . As our primary aim was to evaluate how the violation of STRUCTURE assumptions affected the clustering we compared the results from the STRUCTURE analyses with the highest support to those from the DAPC analyses with the same number of clusters. The DAPC results showed a high degree of correspondence with the outcome of the STRUCTURE analyses suggesting that the effect of LD and deviation from HWE on the analyses had been minor.We additionally evaluated our data for genetic structuring using discriminant analysis of principal components (DAPC), which is free of the assumptions of HWE and no LD present in STRUCTURE. The number of DAPC clusters best describing the different data sets was not clear-cut for the full and European data sets . The north – south clustering found in the STRUCTURE analysis was not apparent in the PCA  of the full dataset clearly separated the Kashmir (black and grey) from the European populations (in colour) Fig.  along thPCA Fig. .Fig. 3PC0.05 = 292, q0.95 = 3220) and 4850 generations  respectively. Similar estimates of separation time were obtained when only the European populations were analysed, where the time back to separation of the southern and northern European regions had a median value of 342 generations . In both cases ABC modelling supported a separation of the two European regions predating their introduction in Europe during the 19th and 20th centuries although a postintroduction separation was not fully excluded by the analysis of European populations only.We postulated that the two regional clusters detected in the STRUCTURE and DAPC analyses, southern and northern Europe, could be the result of independent introductions into Europe. In our approximate Bayesian computation (ABC) modelling, posterior probability values Table  consisteAnalysis of molecular variance (AMOVA) showed significant genetic structure among the 13 populations and higher hierarchical levels Table . SignifiST values . FST values were lower between pairs of Kashmir populations  than between pairs of European populations , and the difference was not driven by the larger distances covered in the European sampling. This was shown by the fact that FST values only for within-municipality pairs of populations  were also significantly higher than FST values for the Kashmir populations (Wilcoxon rank sum test p < 0.05).Pairwise Fes Table  between ST and RST values in the form of FST/(1-FST) and RST/(1-RST) respectively (Table ST/(1-FST) and genetic distance  but not to the proportion of shared alleles or RST/(1-RST) .We evaluated isolation by distance within Europe using four different measures of genetic differentiation between pairs of populations: pairwise genetic distance, proportion of shared alleles and pairwise Fly Table . GeograpST/(1-FST) or RST/(1-RST) , while the southern populations showed signs of isolation by distance when genetic similarity between populations was measured as RST/(1-RST) . The presence of isolation by distance among the northern populations was mainly created by the large genetic distances between the single Stockholm population and all three Trondheim populations and did not persist when Stockholm was removed .Looking at the regional clusters detected by STRUCTURE, the northern populations showed signs of isolation by distance when genetic similarity between populations was measured as FST values did in most cases not differ from the FST values  suggesting that mutation has been of limited importance in differentiating populations both within and between continents. A limited role of mutation in Europe was further supported by the fact that only four private alleles were found in the ten European populations, compared to the 20 private alleles that were found in the three Kashmir populations studied.Pairwise Res Table  (Amiens1p > 0.05). However, two of the northernmost populations, Trondheim1 and Trondheim2, showed the shifted mode indicative of a recent bottleneck. The proportion of migrants into a population, assessed using the software BayesAss, was in most cases less than 1 %, and only a few populations showed indications of more than 10 % of the individuals being migrants from other populations . A diversity in the invasive range similar to that of the native range was, however, not uncommon. In the 41 comparisons that identified a number of introductions, the majority, 32, suggested multiple introductions and only five a single origin of the invasive species . Although small population sizes in newly introduced species could lead to an increase in the amount of inbreeding in a species, there was no significant difference in the inbreeding coefficients of the native and invasive ranges of species reported in the literature to be outbreeding .The species reviewed did not have significantly higher FThe distribution of genetic diversity within and among populations, as analysed by AMOVAs, showed that within each species similar amounts of variation were present within and among populations in the native and invasive ranges  of the European I. glandulifera most likely separated from the Kashmir populations at least a couple of hundred years before the species was introduced in Europe [I. glandulifera. A wider sampling of I. glandulifera, preferably from its full native range, will be needed if possible sources for the populations in Europe are to be identified.The confident identification of the true source population(s) of any invasive species typically requires a wider and denser sampling of the native range than the one in the present study. Our ABC modelling suggests that the Kashmir populations sampled in this study are not the direct source of the European populations studied. The separation time between the Kashmir n Europe . IndicatI. glandulifera in Europe limits our ability to make inferences about colonisation processes, such as the exact amount of loss of genetic diversity during colonisation. We note, however, that all but four of the alleles detected in the European populations were also present in the Kashmir populations suggesting that the alleles present in Kashmir populations to a large extent represent those of the source of the European populations. While we sampled a large number of individuals from few populations in the species native range, Nagy and Korpelainen [I. glandulifera have yet to be studied genetically, but our Kashmir populations most likely sufficiently well characterize populations in the species’ native range for us to draw tentative conclusions regarding the genetic differences between I. glandulifera in its native and introduced ranges.The fact that the Kashmir populations in this study are not the source population of pelainen  sampled pelainen  and Kashpelainen  suggestepelainen  it is liI. glandulifera in Europe was reported from gradually more northern locations  Finland . We founAlthough our PCA showed a gradual transition from more southern to more northern populations Fig.  this wasI. glandulifera show that it most likely predates the species introduction in Europe. If the regional division is an artefact of isolation by distance we expect populations in the two regions to have separated from each other only after the species colonised Europe. Although the ABC modelling produced a large range for the estimates of the time since separation and a divergence after the introduction in Europe is possible from the analysis of European data only, a separation pre-dating the introduction in Europe is more likely and suggests at least two independent introductions. The fact that Stockholm individuals cluster away from all other European individuals in the PCA  than that detected in many invasive species so far, with an average diversity reduction of 11 % ST – RST comparisons, mutation seems not to have played a major role in the differentiation of European populations during the evolutionary short time since their introduction, nor in the differentiation of the Kashmir populations. Of the six cases where FST differed significantly from RST four involved the population Kashmir2. Interestingly, we found significant differences also between Kashmir2 and the two other Kashmir populations, located only a few kilometers apart, even though it seems unlikely that these populations have been separated long enough for mutation to cause significant amounts of differentiation.From our FST values in the invasive range is consistent with some invasive species, invasive plants in general were found not to differ in FST values or the distribution of genetic diversity as shown by AMOVAs  suggested that gene flow may be higher between the Kashmir populations than the European ones. Although higher Fulations . It shouulations  was baseulations .ST values from Lund and Amiens were partially the reason why within-municipality FST values were higher in Europe than in Kashmir and it is possible that man-mediated dispersal to single populations in these municipalities has contributed to the high FST values observed. The ornamental qualities ascribed to I. glandulifera [I. glandulifera in such isolated locations in Britain such as the Isles of Scilly, Shetland and Orkney [I. glandulifera.We see our estimates of migration rates primarily as an indication of populations more likely to have experienced gene flow. As could have been expected, most of the migrants detected were from neighbouring populations . The longer migrations detected (600 km or more), if not a consequence of few markers and low diversity, should be the result of man-mediated dispersal. The latter seems a likely scenario between Lund1 and Amiens1 where as many as eight individuals were identified as possible first generation migrants. High within-municipality FI. glandulifera represent a smaller proportion of the genetic diversity of the native range than what is typically found for outcrossing invasive species. Relatively low genetic diversity is thus possible in widespread invasive species even after multiple introductions. This suggests a possible role for phenotypic plasticity in facilitating the spread of I. glandulifera across Europe.In conclusion, we find that invasive populations of I. glandulifera  with at least 30 flowering individuals were sampled in 2011 from six municipalities, the local area around a town or city, along a 1600 km latitudinal gradient in Western Europe. Populations were also sampled from Kashmir, India , following the manufacturer’s recommendations. Eleven already published microsatellite loci , 100 werST [CH, the Cavalli-Sforza and Edwards genetic distance [ST and DCH for the presence of null alleles did not affect the conclusions drawn and these measures are therefore not reported. DS  and only the former is reported.The expected heterozygosity under HWE (h) was calculated with a purpose-written Perl script (available upon request). GenePop , 86 was ST  and DCH,distance . Adjustidistance ) and RSTn model, , 91 weren model,  and diffIS and tests for LD (using 10000 permutations) were calculated and carried out using Arlequin v 3.5.1.3 [Hierarchical AMOVA, F 3.5.1.3 . For AMO 3.5.1.3  was used 3.5.1.3  was usedWe examined geographic clustering of the genetic diversity using a combination of different approaches. STRUCTURE v 2.2 , 77 was Adegenet package, and PCA. The number of copies present in an individual  for each allele at each locus was treated as independent variables in the PCA. In addition, R was used to test for correlations between latitude and genetic diversity and the two main principal components in PCA respectively as well as between geographic distance and pairwise FST, RST, number of alleles and genetic distance respectively, using Pearson’s product–moment correlation. R was also used to carry out t-tests, or alternatively the Wilcoxon rank sum test if the underlying assumptions of parametric tests were not met.Geographic structure was further explored using R v 3.0.3  to carryI. glandulifera in Europe. We compared two models, one where all European populations first separate from the Kashmir populations followed by a later split into a southern and a northern regional cluster, and one with a southern regional cluster first splitting from the remaining populations, followed by a second split between a northern cluster and the Kashmir populations (Additional file We used the ABC approach implemented in DIYABC  to inferIn addition to literature reviewed in Bossdorf et al.  and DlugThe data set supporting the results of this article is available from the Dryad repository http://dx.doi.org/10.5061/dryad.gp2tc .


Varanus niloticus) is a commercially valuable species in the international leather industry, with the highest levels of exploitation concentrated throughout Sahelian Africa. In this study, we examined the fine-scale genetic patterns of V. niloticus populations in the Sahel, with the expectation that the genetic structure would correspond to riverine drainage basins. The analyses were based on genotypes at 11 microsatellite loci for 318 individuals, spanning three separate watersheds throughout the Sahel.Overexploitation of wildlife populations results in direct consequences, such as extinction and local extirpation, as well as indirect effects including genetic diversity loss and changes in genetic structure. A clear understanding of the underlying genetic patterns of harvested species is necessary for sustainable management. The Nile monitor (FST = 0.47). Contrary to our expectation, the largest genetic break occurred within a single watershed, the Niger basin, rather than between watersheds. However, other localities displayed evidence of reduced gene flow between watershed boundaries. Across methods, the westernmost population exhibited lower estimates of Ne as well as lower levels of genetic diversity compared to the other inferred populations. While we did not detect evidence for recent population bottlenecks, our analyses indicated historic population declines around 1,000–1,800 years ago.Our analyses identified four genetic clusters throughout the region, one of which (the westernmost population) exhibited very high levels of genetic differentiation  contains supplementary material, which is available to authorized users. Ne), and thus the genetic diversity, without affecting the overall population size [Overexploitation has been identified as the main source of decline in many threatened species worldwide . Direct ion size . Even noion size .Varanus niloticus) is the second most heavily traded varanid species in both the leather and the pet industries [V. niloticus is also commonly used as a source of food in some regions [V. niloticus is entirely carnivorous, preying upon a large variety of organisms from snails and insects to small mammals [The Nile monitor  and relatively early sexual maturation (around two years of age), intense exploitation could be affecting their populations [V. niloticus individuals collected near Lake Chad after years of intense harvesting were significantly smaller than those collected in a similar location years earlier when harvesting was minimal. Furthermore, the combined effects of harvesting V. niloticus for food, leather, and the pet trade are likely leading to local extirpation of subpopulations [Although ulations ,13. Highulations . A studyulations  additionulations . HoweverVaranus niloticus is protected under the Convention on International Trade in Endangered Species of Wild Fauna and Flora , little is known about the population size and genetic structure of this species, which hinders effective management decisions [V. niloticus populations throughout Sahelian Africa, where harvesting is heavily concentrated. Due to the semi-aquatic behavior and habitat requirements of this species [V. niloticus to correspond to river drainage basins throughout the region.While ecisions . This st species , we expeAll tissue samples used in this study were collected and transported in compliance with the Convention on International Trade in Endangered Species of Fauna and Flora.Varanus niloticus tissue samples collected from 18 separate localities throughout Sahel  are located along tributaries , observed heterozygosity (Ho), and number of alleles (Na) with GenAlEx v.6.5 [AR) and private allelic richness (PAR) were calculated with HP-Rare 1.1 [The microsatellite data were examined for null alleles and mis-scoring using MICRO-CHECKER . Hardy-WEx v.6.5  and alleRare 1.1  which usVaranus niloticus populations in Sahelian Africa. We measured genetic differentiation (FST) among sample localities in ARLEQUIN 3.5.1.3 [FST values was conducted in GENALEX v.6.5 [We used multiple approaches to examine the genetic partitioning among  3.5.1.3  using 10EX v.6.5  and plotEX v.6.5 ,25 to viK value, from 1 to 10, with an initial burn-in of 106 iterations and an additional 107 iterations after the burn-in. The optimal number of clusters (K) was estimated by examining the Ln P(X|K) [K [K value analyzed, the results were averaged across runs using CLUMPP v.1.1.2 [7 iterations, 100 reference individuals, and 1,000 iterations of reference individuals [K values and additional ad-hoc analyses. Results from both analyses were visualized with DISTRUCT v.1.1 [Bayesian clustering analyses were performed using STRUCTURE v.2.3.3  and BAPSn P(X|K)  as well (X|K) [K  with the(X|K) [K . For eac v.1.1.2 . For com v.1.1.2 , followeividuals . BAPS diCT v.1.1 .A hierarchical analysis of molecular variance (AMOVA) was carried out in ARLEQUIN v.3.5.1.3  to deterNe) as well as assesses historical demographic changes by applying a Bayesian coalescent-based technique. Ten separate runs were conducted for each of the inferred populations using different starting points and 2.0x104 thinned updates with a thinning interval of 1.0x104. Following de Buffrénil and Rimblot-Baly [Ne with NeESTIMATOR v.2 [For the following set of analyses, we grouped samples into their inferred populations based on the results from the clustering methods. We implemented MSVAR v.1.3  for our lot-Baly , the genlot-Baly ,25 to inATOR v.2  using thATOR v.2 .To test for evidence of recent population declines, we used BOTTLENECK v.1.2.02  which te7 iterations, a burn-in of 106 and sampling every 2,000 iterations.To assess the degree of gene flow among the inferred populations, we used BAYESASS v.1.3  with 107FST/(1-FST) calculations, following Rousset [www.worldclim.org/bioclim), and pairwise environmental distances were calculated as the pixel value difference between each locality.Lastly, we examined whether differences in gene flow among localities were correlated with river distance and environmental distance. Pairwise river distances between localities were measured in ArcGIS 10.1 . We perf Rousset , across  Rousset .To assesK23 and VARSA10) were found to significantly depart from Hardy-Weinberg equilibrium  for western sites  did not begin to plateau until K = 4. We interpreted these results to mean that while the largest genetic break occurred at K = 2, the optimal number of genetic clusters was four. This decision was bolstered by the BAPS result which also produced four clusters.The clustering analyses showed a hierarchical pattern of structuring across o et al. , the larK = 2, separated the two westernmost localities from the remaining sites  from the eastern portion . Lastly, at K = 4, genetic differentiation between the Niger and Lake Chad drainage basins becomes apparent and is more clearly demarcated in the BAPS analysis. Showing patterns consistent with the PCoA, the STRUCTURE analysis produced additional sub-structuring in the Lake Chad drainage basin.The largest degree of genetic partitioning, occurring at  Figures  and 4. CHierarchal analysis of molecular variance (AMOVA) confirmed that the largest amount of genetic variation can be explained by grouping localities into west versus east of the Inner Niger Delta. Groups based on genetic cluster assignment as well as drainage basins also explained a significant amount of the total genetic variation Table .Table 3P = 0.04234); however other mutation models and statistical tests did not produce significant results  and Niono (site 2), central population = Mopti (site 3) and Niamey site 4), Lake Lere population = Lake Lere (site 5), and Lake Chad population = Lake Chad watershed (sites 6–16). Only one population, Lake Chad, showed evidence of a recent population bottleneck under the infinite allele model and the standardized differences test  estimates varied between methods, but showed a general trend of the western population having the smallest Ne and Lake Chad having the largest Ne, while the central and Lake Lere populations exhibited intermediate sizes (based on 95% CI)  Table . These dI) Table . Differees Table .Table 4Varanus niloticus populations have undergone demographic changes. Non-overlapping peaks in log(Ne) for the western, central, and Lake Lere populations . The level of intraspecific differentiation found among V. niloticus populations throughout the Sahel is comparable to proposed distinct evolutionary lineages in other reptile species [V. niloticus from respective localities , alleviating concerns of possible misidentification. Additionally, all sample localities are outside of the published distribution of the ornate monitor, V. ornatus, a morphologically similar species restricted to the forested regions of western and central Africa [V. ornatus museum material , further increasing our confidence in the sample identification. Future studies are necessary to determine whether other ecological, behavioral, or chromosomal factors are contributing to the reproductive isolation of the western population.Although managed as a single taxonomic unit, the results of this study clearly show that  species ,43. Mitol Africa ,45. For Varanus niloticus dispersal, and populations isolated in riverine refugia differentiated from one another.Contrary to our expectations, the largest genetic break occurs within a single drainage basin, near the Inner Niger Delta in Mali, an area characterized by floodplains and reticulating river networks. The Inner Niger Delta represents a suture zone for other Sahelian species, separating western and eastern lineages ,47. ThesVaranus niloticus. Individuals within the Senegal and Niger watersheds were assigned to the same genetic cluster. This low degree of genetic differentiation could be a reflection of the historic connection between the Senegal and Niger drainage basins, which occurred until the late Pleistocene [FST value between these localities suggests that gene flow might be slightly reduced, possibly due to the currently separate watersheds.Other evidence suggests that historic drainage patterns could have played a large role in shaping the present-day genetic patterns within istocene , and likFST values and the spatial clustering analysis with BAPS. In contrast to BAPS, the STRUCTURE results showed a higher level of admixture and hierarchical partitioning among these populations; however, at higher K values (not shown) the distinctiveness of the Lake Lere population became more evident. The STRUCTURE analysis also showed additional sub-structuring among Varanus niloticus localities within the Lake Chad watershed. Sample sites along tributaries displayed differing proportions of ancestry than localities within Lake Chad itself. This could reflect source-sink dynamics of individuals dispersing downstream along tributaries and intermixing in Lake Chad, where the rivers converge.The genetic structure of the central, Lake Lere, and Lake Chad populations supports our hypothesis of reduced gene flow across present-day watershed boundaries. Our analysis of migration rates indicated a high degree of gene flow between the central and Lake Lere populations, located in the western and eastern drainages of the Niger basin, respectively. Although Lake Lere is in close proximity to locations within the Lake Chad watershed (shortest distance = 209 km), our analyses indicated reduced gene flow between these populations, evidenced by significant Ne) and lower genetic diversity compared to other Varanus niloticus populations in the region. This could be a result of the limited gene flow that we detected. Additionally, the prolonged intensive harvesting of V. niloticus in Mali [Across methods, the western population showed evidence of having a small effective population size ( in Mali  could beVaranus niloticus populations that occurred around 1,000–1,800 years BP corresponds to the drastic expansion and organization of human civilizations in West Africa [2 in 800–900 AD before eventually declining [V. niloticus populations in Sahelian Africa likely have a longer history than previously thought.The decline in t Africa . Specifieclining . This aneclining . Archaeoeclining . TherefoVaranus niloticus populations. Although listed on Appendix II of CITES, managing international trade of V. niloticus without population size and genetic structure information could be detrimental to the species. The four genetic groups that we identified in Sahelian Africa represent separate demographic units and possibly a distinct lineage based on private allelic richness and differing allele frequencies. These genetic groups provide an objective and useful delineation for managing populations of V. niloticus throughout Mali, Niger, and Chad. In the future, a genetic assessment of V. niloticus should be expanded to the full range of this species to enable more thorough monitoring of international trade and the ability to source harvested individuals from other locations.Our findings have important implications for the management of Sahelian This study highlights the utility of genetic methods for informing management practices and conservation decisions. Many CITES-listed species are currently managed without empirical data and could greatly benefit from species- and population-level assessments of genetic structure and diversity . MonitorWhile biodiversity data is limited throughout the Sahel, recent studies in this region have uncovered a high degree of endemism and species richness . Similar


Gasterosteus aculeatus) of four Swiss lake systems each harbouring parapatric lake-stream ecotype pairs. We demonstrate significant differences in infections within and between the pairs that are driven particularly by the parasite taxa transmitted to fish from benthic invertebrates. The magnitude of the differences tended to correlate positively with the extent of neutral genetic differentiation between the parapatric lake and stream populations of stickleback, whereas no such correlation was found among allopatric populations from similar or contrasting habitats. This suggests that genetic differentiation is unrelated to the magnitude of parasite infection contrasts when gene flow is constrained by geographical barriers while in the absence of physical barriers, genetic differentiation and the magnitude of differences in infections tend to be positively correlated.Spatial heterogeneity in diversity and intensity of parasitism is a typical feature of most host-parasite interactions, but understanding of the evolutionary implications of such variation is limited. One possible outcome of infection heterogeneities is parasite-mediated divergent selection between host populations, ecotypes or species which may facilitate the process of ecological speciation. However, very few studies have described infections in population-pairs along the speciation continuum from low to moderate or high degree of genetic differentiation that would address the possibility of parasite-mediated divergent selection in the early stages of the speciation process. Here we provide an example of divergent parasitism in freshwater fish ecotypes by examining macroparasite infections in threespine stickleback ( Parasitism is an important selective factor in natural host populations, but the diversity and intensity of infections is often spatially heterogeneous. Such heterogeneity may emerge as a consequence of spatial aggregation of infected hosts that release parasite infective stages (i.e. infection hot spots) or because of ecological specialization, i.e. differences in preference of host individuals and populations towards habitats or diets with variable risk of infection –6. DespiGasterosteus aculeatus species complex) in Switzerland in relation to the magnitude of neutral genetic differentiation between the populations and in comparison to allopatric population contrasts.An important factor underlying variation in possible adaptive divergence among populations under parasite-mediated divergent selection is the magnitude of gene flow between the populations. Gene flow can either constrain –28 or faSeveral freshwater fish taxa have undergone rapid adaptive radiations in northern latitudes after the end of the last glaciation –40. In tG. aculeatus and G. gymnurus respectively [We focused on four lake systems in Switzerland each harbouring independently evolved parapatric pairs of lake and stream stickleback ecotypes. The Constance and Geneva systems have distinct stickleback lineages originating from drainages separated by considerable geographical distance. These lineages are also phenotypically highly distinct and belong to different nominal taxa, ectively . The othectively . A recenectively . This suDiplostomum commonly include several co-infecting species or cryptic species that are morphologically indistinguishable [Diplostomum species found in the eye lenses are different from those in the eye humour [Diplostomum spp. according to their site of infection, lens or humour of the fish eye. The lens-infecting species of Diplostomum cause cataracts in eyes of fish [Sticklebacks of both lake and stream ecotypes were sampled from each of the four lake systems . Using GenoDive 2.0 [ST [GenoDive.DNA was extracted for a total of 272 stickleback individuals which were in some cases caught earlier from the study sites . ExtractDive 2.0 , deviati 2.0 [ST  between Differences in the mean abundance of parasites  between the lake systems and between ecotypes within the lake systems were analysed using generalized linear models (GLM) with negative binomial probability distribution and log link function. This was done to account for the aggregated distribution of parasites, i.e. distributions skewed to the left with few host individuals harbouring a high fraction of the parasite population. Lake system, habitat and fish sex were used as fixed factors, and fish standard length as a covariate. To account for the statistical non-independency of the two lake-stream pairs from the Geneva system, the analysis was run twice including one of the pairs at a time.ST calculated from variation at nine microsatellite loci were used  and in allopatry . To account for the effect of geographical distance in the analyses, distance (direct line “as the bird flies” between two points) was determined for all population pairs. Direct distance measure was used since waterway distances in allopatric combinations would result in arbitrarily large distances because we compared populations between systems draining into different oceans while the colonisation clearly happened by crossing the water sheds at the headwaters within Switzerland [ST values and FST residuals from linear regression of FST against geographical distance. We do not report p-values for these correlation statistics as we only had four independent parapatric data points and in allopatry the pairwise comparisons constituted to an incomplete matrix of statistically non-independent values. However, we calculated one p-value for the complete matrix of pairwise contrasts, including the four parapatric pairs and 24 allopatric combinations, to obtain a conservative estimate of statistical significance of the correlation in allopatry. This analysis was done using partial Mantel test controlling for geographical distance with 9999 permutations of the data. All statistics were performed in IBM SPSS Statistics 20 and statistical package R version 2.15.2.To test if differences in parasite species composition and abundance within habitats across the lake systems and between habitats within and across the lake systems correlated with the extent of neutral genetic differentiation among the stickleback populations, pairwise Fere used . Proporttzerland . HoweverAll fish of the study were euthanized in MS-222 or clove oil solution, commonly used fish anesthetics. All necessary permits to sample sticklebacks for the described field studies were obtained from the cantonal fishery authorities of cantons Bern, Vaud, St. Gallen and Valais. Fish collection followed the Swiss veterinary legislation in concordance with the federal food safety and veterinary office (FSVO) and the cantonal veterinary office in Bern (Veterinärdienst des Kantons Bern).Diplostomum (Trematoda) transmitted to fish as free-swimming cercaria larvae from aquatic snails and glochidia larvae of bivalves (Mollusca) were among the most prevalent parasite taxa, but this depended on the host population. For example, these taxa were found in all lake populations, but they were absent from the stream populations in the Constance and Geneva systems. The acanthocephalan Acanthocephalus lucii, transmitted trophically to fish from isopods, was common only in the Biel system whereas the other prevalent taxa, trematode Cyathocotyle sp. (transmitted from snails) and acanthocephalan Pomphorhynchus sp. , were found only in the Wohlen system. Lens-infecting Diplostomum spp. was the most abundant parasite taxon accounting for 59.4% of all parasite individuals. Coverage of cataracts caused by these parasites reached 100% of the lens area in some stickleback individuals. The highest coverages of cataracts were detected in the Wohlen system that also had the highest abundance of lens-infecting Diplostomum spp.A total of 12 different parasite taxa were found from the 303 sticklebacks studied from the lake and stream populations . The higp > 0.05, results not shown). Parapatric lake and stream stickleback populations differed at neutral genetic markers in the Constance  and in one of the two Geneva pairs , but not in the Wohlen  and Biel systems  nor in the second Geneva pair  so that the Wohlen and Biel systems had the highest parasite abundances . The lakST (Diplostomum spp. abundance between lake and stream stickleback (Pearson correlation only for the difference in abundance of Diplostomum spp. in the parapatric pairs: r = 0.490). Difference in prevalence of the lens-infecting Diplostomum spp. tended to correlate with the magnitude of genetic differentiation too (r = 0.638).Correlations between the magnitude of differentiation at neutral genetic markers, and difference in parasite species composition and parasite abundance between parapatric lake and stream populations, were strongly positive , indicating that parasite infections tended to become more strongly differentiated when stickleback ecotypes were more strongly differentiated genetically . These cST . This paST and pairwise difference in parasite species composition and abundance in allopatric lake-lake, stream-stream, and lake-stream population combinations were generally weak and inconsistent in direction ; r = 0.129, p = 0.199 (difference in parasite abundance)).Correlations between pairwise Firection , Fig 3, The role of parasite-mediated divergent selection in ecological speciation and adaptive radiations of freshwater fishes has attracted considerable attention in recent years . HoweverDiplostomum spp., are nevertheless known to occur in these water systems . Similarly, the differences in infections of trophically transmitted parasites such as cestodes and acanthocephalans may reflect the previously shown dietary differences between the ecotypes in this system  and Gyrodactylus monogeneans (not studied here) are known to be associated with fitness reduction and adaptation in resistance genetics in stickleback [In addition to ckleback , 78, 79.ckleback . This suST values between populations occupying the different drainage systems in Switzerland reflect variable colonisation times and histories  rather than variable extents of divergent adaptation and reproductive isolation [While the magnitude of differences in parasite infections tended to be positively correlated with the magnitude of neutral genetic differentiation in the parapatric host population pairs that we studied, there was no such relationship among allopatric stickleback populations, no matter whether they occupied different or similar habitats. This was expected because parasite-mediated divergent selection is not expected to act on alleles at neutral microsatellite loci and because variable Fsolation . The fousolation , 82.To conclude, our results are consistent with other data that suggest that the magnitude of gene flow between parapatric populations of stickleback occupying contrasting habitats is mediated by variation in the extent of specialization and adaptation to lake or stream habitat. This would also mediate variation in the extent of differences in parasite infection, probably reflecting differences in parasite exposure. This pattern was evident both in terms of parasite species composition and infection abundance. Our data thus demonstrate potential for parasite-mediated divergent selection between populations occupying contrasting habitats already at the incipient stages of differentiation, while habitat-specific divergent adaption of the ecotypes to parasite infections remains to be demonstrated experimentally. On the other hand, variation in the extent of genetic differentiation between population pairs with similar magnitude of infection divergence from the same lake system emphasizes that data on many more ecotype pairs is needed to address the generality of our findings. Our results also imply that relatively high similarity of the parasite fauna can be maintained across distant populations owing to a common ‘core’ of parasite taxa. This suggests that it may be possible to detect parallel infection divergence and possibly parallel adaptation in pairs occupying similar habitat contrasts at large geographical scales.S1 Original Data(XLSX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Lumnitzera racemosa across the Indo-West Pacific region. Our results revealed pronounced genetic differentiation in this species between the populations from the Indian Ocean and the Pacific Ocean, which may be attributable to the long-term isolation between the western and eastern coasts of the Malay Peninsula during sea-level drops in the Pleistocene glacial periods. The mixing of haplotypes from the two highly divergent groups was identified in a Cambodian population at almost all 88 nuclear genes, suggesting genetic admixture of the two lineages at the boundary region. Similar genetic admixture was also found in other populations from Southeast Asia based on the Bayesian clustering analysis of six nuclear genes, which suggests extensive and recent secondary contact of the two divergent lineages in Southeast Asia. Computer simulations indicated substantial migration from the Indian Ocean towards the South China Sea, which likely results in the genetic admixture in Southeast Asia.Systematically investigating the impacts of Pleistocene sea-level fluctuations on mangrove plants may provide a better understanding of their demographic history and useful information for their conservation. Therefore, we conducted population genomic analyses of 88 nuclear genes to explore the population dynamics of a mangrove tree  The climatic oscillations that characterized the Pleistocene glaciations have deeply affected the geographical distribution and evolutionary trajectory of plants on the earth123581110131415161718192021Rhizophora species based on microsatellite markers and simulations of ocean circulation patterns2627The habitats of mangrove plants during the glacial periods would be submerged by the rising sea levels in the interglacial periods, and mangrove plants had to move with the shifting of the coastline916Lumnitzera (Combretaceae), a genus consisting of two species L. racemosa and L. littorea, is an important component of mangrove forests in the IWP regionL. racemosa is widely distributed from East Africa through Indo-Malesia to Tonga29L. racemosa produces tiny fruits with a fibrous portion in the fruit wall. The seeds are protected by a hard layer of sclerenchyma tissue inside the outer corky layer of the fruit wall and could generally keep in seawater for more than 20 days and disperse well with ocean currents29L. racemosa based on inter simple sequence repeat (ISSR) markersL. racemosa in the IWP region. 88 nuclear genes were chosen regardless of their functions and sequenced for five pooled population samples using the Illumina platforms, a cost-effective and efficient method in population allele frequency estimation, to obtain the genetic structure of L. racemosa at the genomic level. Because Illumina sequencing is based on pooled population samples, population we used for Illumina sequencing and analysis in this paper is different from the conventional “population” concept. Six of the 88 genes from 12 additional populations were sequenced by the Sanger method to assess the general pattern of genetic diversity in the IWP region. We used IMa2 analysis to estimate the splitting time, population size and pairwise migration rate parameters between populations from different regions. Based on these results, we sought to determine whether 1) glacial sea-level drops caused the genetic divergence between populations from the eastern and western coasts of the Malay Peninsula and 2) genetic exchange occurred from secondary contacts of previously isolated populations of L. racemosa after rising sea levels. A better understanding of these questions will shed light on the evolutionary history of L. racemosa and provide useful information for the conservation of mangroves and other coastal communities that face the threat of rising sea levels under global warming.In this study, we used population genomic analyses to assess the impacts of sea level oscillations and ocean currents on the population history of −3 and θ = 0.077 × 10−3 in QH; −3 and θ = 0.080 × 10−3 in SY). The two populations from Malaysia  had slightly higher polymorphism than those from China. The Cambodian population (KP) showed the highest level of genetic diversity (−3 and θ = 2.434 × 10−3), which was an order of magnitude higher than those in other populations. Relative to the diversity at the population level, L. racemosa showed a higher level of genetic diversity at the species level (−3 and θ = 2.291 × 10−3).Using the Illumina platforms, a total of 11.45 to 14.53 million 75 (QH and SY) or 90 bp pair-end reads  were generated and aligned to 84 to 88 genes with mean depth ranging from 5127× to 6061× . The nuc−3 to 7.000 × 10−3 with an average of 5.488 × 10−3 and θ ranged from 1.120 × 10−3 to 2.710 × 10−3 with an average of 2.093 × 10−3. At the population level, populations from China , Australia (DW and QL), Malaysia (JH and KD) and Sri Lanka (BT and RK) had no or extremely low levels of polymorphism; however, populations from Cambodia (KP), Thailand , Indonesia (BL) and Singapore (TP) harboured relatively high levels of polymorphism  from 295 individuals in 17 populations (including five populations used in the Illumina sequencing) across the IWP region using the Sanger method. At the species level, π ranged from 3.010 × 10morphism . These rUsing the linkage information of SNPs in each pair of reads, the haplotypes were inferred by an Expectation-Maximization algorithm. When the distance between two proximal SNPs at some genes was longer than the covered length of paired reads, these genes were cut into two segments for haplotype inference. Finally, we constructed haplotype networks for 88 polymorphic segments from 85 polymorphic genes across the five populations . For mosWe also constructed haplotype networks of six nuclear genes from 17 populations across the IWP region using Sanger data . Again, ST) for each gene segment was calculated to reveal the levels of genetic differentiation between populations. The averages of pairwise FST values  of the six genes sequenced by the Sanger method . The strThe Bayesian clustering algorithm-based STRUCTURE analysis revealed that the optimum K value was 2 when using the Sanger dataset of six nuclear genes from 295 individuals in 17 populations . For K =L. racemosa across the IWP region under the isolation-with-migration (IM) model based on six nuclear genes from Sanger sequencing. Three populations from China (QH), western Malaysia (JH) and Cambodia (KP) were selected to represent populations from the Pacific Ocean, the Indian Ocean and the Indian-Pacific boundary, respectively (−9 substitutions per site per year (s/s/y) based on the neutral mutation rate of 6.1 × 10−9 in vascular plants−9 s/s/y, and 0.002 Mya under 9.0 × 10−9 s/s/y, respectively. The estimated divergence time between JH and the most recent common ancestor (MRCA) of QH and KP was 5.729 (95% HPDI: 4.271–7.430), corresponding to 0.650–1.949 Mya under different mutation rates. The effective population sizes of QH, KP and JH were estimated to be 0.033 (95% HPDI: 0.007–0.129), 0.105 (95% HPDI: 0.023–1.709) and 0.023 (95% HPDI: 0.003–0.119) in units of θ. All the estimated migration rates were very low. Based on the likelihood ratio (LLR) test, only gene flow from JH to KP, among all the pairwise comparisons, was statistically greater than zero , and gene flow in this direction was two orders of magnitude higher than in the opposing direction . Moreover, the numbers of bidirectional migrants per generation between QH and KP (2NMQH −> KP = 0.004 and 2NMKP −> QH = 0.043) were higher than those between QH and JH (2NMQH −>JH = 0.0002 and 2NMJH −> QH = 0.0007).We performed the IMa2 simulation runs to infer the population history of ectively . The effL. racemosa in the IWP region. In most of the 88 genes, strong genetic differentiation is identified between populations from the Indian Ocean and the Pacific Ocean, suggesting long-term glacial isolation between these two oceanic regions due to steep sea-level drops. It should be noted that these two lineages co-occurred within populations from the coasts of Southeast Asia, which provides compelling evidence for secondary contact of the two divergent lineages. Moreover, re-colonization processes after the last glaciation should be the major reason for the secondary contact at the boundary region. Our study provides new insights into the population dynamics of L. racemosa and enriches our current understanding about the evolutionary history of mangrove plants.The objective of this study was to use population genomic analyses to investigate the genetic diversity and genetic exchange in populations of ST and Kxy)  . Our BARracemosa . During L. racemosaCompared to the high divergence between populations from the Indian Ocean and the Pacific Ocean, the populations from China and Australia are fairly closely related, which is consistent with a previous study in Our study identified apparent haplotype mixing in the populations from Southeast Asia, at the Indo-Pacific boundary. In the Cambodian population (KP), almost all genes showed a combination of the two highly divergent groups . SimilarL. racemosa, the haplotype networks were dichotomous with missing intermediate haplotypes between the two distinctly common haplotype groups. Although disruptive selection or balancing selection could generate two highly divergent groups without intermediate haplotypes within one population, it is unlikely that almost all 88 randomly chosen genes throughout genome are under the selection effect, suggesting that the pattern observed in L. racemosa should not be attributed to the retention of ancestral polymorphisms. Rather, secondary contact of two oceanic lineages from the Indian Ocean and the Pacific Ocean is the most plausible explanation for the haplotype mixing in the populations from Southeast Asia. As described before, the fruits of L. racemosa are very tiny and light (<1 cm and ~0.1 g) and they usually do not anchor immediately around the parent trees. Instead, they flow with seawater and wait to colonize until coming into suitable sediment. This could facilitate long-distance dispersal of the seeds of L. racemosa by sea currents and promote the successful genetic exchange between populations from different regions. We also observed that several populations in Southeast Asia exhibited an indication of genetic admixture barely in part of the six genes. The discrepancy between the different genes may be attributable to strong genetic drift after secondary contact. Small effective population sizes are expected for these populations in Southeast Asia, which were sometimes sparse and fragmented following sea level changes. After initial mixing, alleles from one lineage might have been randomly and quickly fixed within some genes in these small populations due to strong genetic driftSome previous studies about mangroves identified the presence of identical chlorotypes in different populations916L. racemosa appears to be asymmetrical between the Indian Ocean and the Pacific Ocean. The estimated gene flow from the western Malaysian population (JH) to the Cambodian population (KP) was two orders of magnitude higher than in the opposite direction  and Sanya (SY) in China, one population from Kep (KP) in Cambodia and two populations from Johor (JH) and Kedah (KD) in Malaysia for Illumina sequencing. Leaves of 17 to 100 individuals each from the five populations were collected for DNA isolation (L. racemosa from 17 populations (including the five populations mentioned above) from China, Australia, Cambodia, Thailand, Indonesia, Singapore, Malaysia and Sri Lanka for Sanger sequencing  and 200 clones were sequenced from the library. Based on these sequences, we designed primers for the intron-containing genes and used them for PCR amplification. We validated these primers with one individual from the Qionghai, China population (QH). To ensure homology, 88 genes meeting the following three criteria in each case were used for the five pooled population samples: 1) only one single electrophoretic band was amplified, 2) clear sequence was obtained using Sanger sequencing, and 3) two pairs of primers anchoring different locations for each gene produced completely identical sequences in the overlapped region. The sequences of those 88 genes were employed as reference sequences for further short read mapping. We followed the methods of Zhou, et al.To assess the genetic differentiation and secondary contact in et al.MAQ version 0.6.8ST) to measure the levels of genetic differentiation between populations. GenGIS version 2.4.0http://kiwi.cs.dal.ca/GenGIS/MapMaker)Using the linkage information of SNPs in each pair of reads, we inferred haplotypes and their frequencies in each population using an Expectation-Maximization algorithm45https://www.r-project.org), in which each of the haplotypes was initially assigned into one group, and then the closest two groups were joined together at each stage until only two groups were left. The two groups were denoted as group A and B, respectively. The frequency distribution of group A was calculated in each of the five populations  containing more than two SNPs or 2) containing two SNPs but having only two haplotypes, we employed the hierarchical cluster analysis of complete linkage methodulations .ST values among populations. The Illumina sequencing technology successfully produced 85 matrices of pairwise FST for all five populations; therefore, we assessed the robustness of the inferred barriers using these data and considered them as reliable if supported by at least half of these genes.Additionally, the genetic boundaries between the populations were inferred by the Monmonier’sSix of the 88 nuclear genes  were sequenced by the Sanger method. The sequences of the six nuclear genes were aligned with the ClustalX program6 Markov chain Monte Carlo (MCMC) iterations after a burn-in of 200,000 steps and used the admixture model with the assumption of correlated allele frequencies among clustersThe individual-based genetic structure and population admixture were evaluated based on the 93 polymorphic sites of the six nuclear genes by Bayesian clustering using STRUCTURE version 2.3.3L. racemosa in the IWP region, the effective population sizes, population divergence times and bi-directional migration rates between populations were estimated under the isolation-with-migration (IM) model using the IMa2 program with the Sanger data of six genesL. racemosa and other plants in the Combretaceae family, we assumed a mutation rate ranging from 3.0 to 9.0 × 10−9 substitutions per site per year (s/s/y) based on the neutral mutation rate of 6.1 × 10−9 in vascular plants6 steps and 2.0 × 107 steps with 40 chains under the HKY model. To check for the convergence of the simulations, we performed three independent runs with different seed numbers. The posterior distributions of parameters were estimated using 200,000 genealogies sampled from well-mixed runs and the parameters selected to represent the demography were obtained from the peaks of the posterior distributions and 95% of the highest posterior density intervals (HPDI)α = 0.001)To infer the evolutionary history of Accession codes: Sequences obtained by Sanger method were deposited in GenBank with accession numbers KF477441–KF477511, KF496922–KF496926, KF553911–KF553922, KJ476444–KJ476454 and KT246108–KT246120. Raw short reads of five population samples were deposited in the NCBI Sequence Read Archive with the accession number of SRP038914 and SRP044209.How to cite this article: Li, J. et al. Pronounced genetic differentiation and recent secondary contact in the mangrove tree Lumnitzera racemosa revealed by population genomic analyses. Sci. Rep.6, 29486; doi: 10.1038/srep29486 (2016).


Xenopus laevis) populations have become established worldwide. In this study, we investigate the geographic origins of invasive X. laevis populations in France and Portugal using the phylogeographic structure of X. laevis in its native South African range. In total, 80 individuals from the whole area known to be invaded in France and Portugal were analysed for two mitochondrial and three nuclear genes, allowing a comparison with 185 specimens from the native range. Our results show that native phylogeographic lineages have contributed differently to invasive European X. laevis populations. In Portugal, genetic and historical data suggest a single colonization event involving a small number of individuals from the south-western Cape region in South Africa. In contrast, French invasive X. laevis encompass two distinct native phylogeographic lineages, i.e., one from the south-western Cape region and one from the northern regions of South Africa. The French X. laevis population is the first example of a X. laevis invasion involving multiple lineages. Moreover, the lack of population structure based on nuclear DNA suggests a potential role for admixture within the invasive French population.Due to both deliberate and accidental introductions, invasive African Clawed Frog ( Xenopus laevis  and nuclear DNA (nDNA) sequences  and Xenopus Express , has been assessed  of invasive  species . The ori markers . Similarassessed . These sh Africa , althougX. laevis was introduced at Bouillé-Saint-Paul (Deux Sèvres) from a nearby breeding facility where X. laevis was bred from the 1950s until 1996 (X. laevis may have escaped repeatedly and was probably released when the facility was definitively closed in 1996 (X. laevis populations occupy an area of approximately 200 km2 near the city of Saumur (Maine-et-Loire)  . The intf Lisbon . Nowadayf Lisbon  (Rebelo f Lisbon .X. laevis in France (FR) and Portugal (PT) were captured. For comparison with previous work of X. laevis, 21 specimens were sampled. Seven specimens came from two localities in the south-western Cape (SA1) i.e., Cape of Good Hope nature reserve and a single dam at Jonkershoek, the location from which animals were shipped for international trade from the 1940s to 1970s . 14 specimens came from five localities around Rooikrantz Dam near the historical Pirie hatchery nearby King Williams Town, Eastern Cape (SA6) . These new data were supplemented by data on 164 South African specimens available on GenBank. One individual of Xenopus gilliXenopus laevis nas ribeiras do Concelho de Oeiras”. Animals from the native South-African populations were sampled under the permits 0056-AAA007-00092  and CRO 109/13CR  provided by the Department of Economic Development, Environmental Affairs and Tourism and with ethics approval from the Research Ethics Committee: Animal Care and Use (protocol number: SU-ACUD14-00028).In total 80 individuals from 32 localities, covering the known area invaded by ® tissue kit  according to the manufacturer’s protocol. Five genomic DNA fragments (circa (ca.) 2,040 bp) representing two mitochondrial and three nuclear gene fragments were amplified using PCR. These genes were selected in order to enable comparison with previously published work on native  and microtubule associated serine/threonine kinase-like protein  were amplified and sequenced with the primer pairs Exon4_for1/Exon4_rev2, XLAR_for_40/XLAR_rev_431 and Exon13_fora/Exon13_reva .Invasive specimens were euthanized with a lethal injection of sodium pentobarbital. Muscle tissue was dissected from invasive specimens, while native wild caught specimens were toe-clipped to obtain tissue samples. Genomic DNA was extracted by means of a NucleoSpinn native  and invapecimens . Fragmenpecimens . Fragmenn13_reva . The nucn13_reva . PCR amplgorithm  in MEGA EGA v. 6 . For comh), the number of segregating sites (S), nucleotide diversity  and haplotype/allelic diversity .The following indices of genetic diversity were estimated using DnaSP v. 5 : numbersX. laevis mtDNA sequences were reconstructed with Bayesian inference (BI) and Maximum Parsimony (MP). Three alignments were created: two separate Cytb and 16S alignments including only unique alleles and a concatenated Cytb-16S alignment including only unique haplotypes. In all analyses, X. gilli was used as outgroup. Parsimony informative sites were calculated with DnaSP v. 5  and representatives of all populations.Phylogenetic relationships among invasive and native aSP v. 5 . BI analaSP v. 5 . Cytb, 1aSP v. 5 . BI analaSP v. 5 . A totalaSP v. 5  in R 3.1aSP v. 5 . A set oaSP v. 5  with neaaSP v. 5  and TreeaSP v. 5 . Differe 3.5.3.1 . The popFst values were computed based on the allele frequencies to show to what extent alleles are differently sorted among populations . Native populations were defined based upon the geographical regions mentioned in ‘Taxon sampling.’ Individuals from France and Portugal respectively were treated as two populations. Pairwise Fst values were computed from the allele frequencies with 999 permutations to generate the probability that a random value would be greater than or equal to the observed data using GenAlEx v. 6.5  in GenAlEx v.6.5.The minimum number of recombination events within alleles were estimated in DnaSP v.5 . Given pX. laevis with 42 variable positions of which 34 were parsimony informative. No Cytb sequences were obtained of specimens from the regions SA2, SA3 and SA4. In contrast, all geographic regions were represented in the 16S alignment, which involved 15 alleles of X. laevis with 25 variable positions of which 17 were parsimony informative. The concatenated Cytb-16S alignment involved seven unique X. laevis haplotypes with 43 variable positions of which 39 were parsimony informative (Online Resource 2: https://figshare.com/s/44b71de473fc11e5829d06ec4b8d1f61). The concatenated alignment comprised only specimens from the invaded regions PT, IT and FR and the native regions SA1 and SA6. The BI and MP trees for Cytb and 16S are shown in X. laevis alleles from the geographic regions (1) SA5, SA6 and FR, (2) SA7 and (3) SA1, PT, FR, IT. Additionally, the phylogeny generated from the 16S data strongly supported (1) SA1, SA2, SA3, FR, PT and IT and (2) SA3-SA4 as well-supported X. laevis clades. BI and MP trees for the concatenated data (Online Resource 2) showed two highly supported clades comprising haplotypes from (1) FR, PT, IT, SA1 and (2) FR and SA6. Pairwise population Fst values among all geographic regions based on 16S were significant except among (1) SA1, SA2 and PT, (2) SA5 and SA6 and among (3) FR and SA6 (Online Resource 4).Mitochondrial alignments were constructed for Cytb and 16S involving 101 and 181 individuals respectively. The concatenated Cytb-16S alignment involved 64 individuals. None of the alignments showed gaps. The Cytb alignment involved16 unique alleles of n = 121) showed eight polymorphic positions of which seven revealed heterozygous genotypes. The maximum number of heterozygous positions observed within a single AR sequence was three but that was only observed in two individuals. The two most frequent AR alleles i.e., hap_1 and hap_2 differed by two nonsynonymous substitutions. The Mastl alignment (n = 180) showed 37 polymorphic positions and 108 individuals were heterozygous at 3 ± 2 positions on average. The Prmt6 alignment (n = 176) showed 20 polymorphic positions and 107 individuals were heterozygous at on average 2 ± 1 positions. MJN revealed high allelic diversity at Mast1 and Prmt6 and low allelic diversity at AR . The first two PCoA-axes explained 75% of the total variance from the pairwise Fst values. There was a clear separation among SA7 and the remnant populations along the first PCoA axis . Negative values along the second PCoA axis  were linked to the northernmost up to the north-eastern native populations (SA4–SA7), while positive values were linked to the invasive  and southernmost native populations (SA1–SA3)(AR sequences (ty at AR . In the ty at AR . When co(SA1–SA3). Native (SA1–SA3).X. laevis was on average 0.60 ± 0.20 and 0.45 ± 0.18 respectively. Mitochondrial nucleotide diversity within the native regions was either rather low  or high . As mentioned previously, mtDNA and nDNA variation was geographically structured within South Africa .Haplotype diversity of nDNA and mtDNA within the native range of h Africa . The monh Africa . Furtherπ < 0.00105, 0 ≤ Hd < 0.294, π = 0.00213, Hd = 0.605, X. laevis populations from the south-western Cape SA1 (Cytb: hap_12) and from the Cape regions SA1–SA3 (16S: hap_2). Identical AR, Prmt6 and Mastl alleles were found in respectively the following native regions (1) SA1–SA3, (2) SA1–SA4 and SA7 and (3) SA1–SA7. Pairwise Fst values based on 16S were not significant among Portugal and native regions SA1 and SA2 (Online Resource 4). PCoA and MJN revealed that nuclear allele frequencies within Portugal were most similar to those of native region SA1 (π = 0.0007 ± 0.0009) across all loci was much lower than in native populations (π = 0.0039 ± 0.0035). Similarly, the mean Portuguese haplotype diversity (Hd = 0.191 ± 0.261) across all loci was lower than in the native populations (Hd = 0.558 ± 0.323).Nucleotide and haplotype diversities in Portugal were extremely low for all markers . Haplotype diversities for nDNA ranged from 0.50 for AR up to 0.80 for Mastl and Prmt6 (https://figshare.com/s/44b71de473fc11e5829d06ec4b8d1f61). No significant difference was observed when comparing nDNA allele frequencies among individuals representing the two different mitochondrial groups , however sample sizes were low i.e., 49 and 8 individuals representing northern South Africa-Rooikrantz Dam mtDNA (SA5–SA6) and south-western Cape mtDNA (SA1–SA2) respectively. In comparison with the native geographic regions, mean nucleotide diversity across all loci in France was higher (π = 0.009 ± 0.008 versus π = 0.0039 ± 0.0035) while French haplotype diversity across all loci was comparable (Hd = 0.528 ± 0.287 versus Hd = 0.558 ± 0.323).MtDNA haplotype diversity for each marker was low within the French population  that the Portuguese populations may be derived from the latter and (2) that the Portuguese population derives from a single colonization event involving a small number of individuals, most likely stemming from one and the same source population in the south-western Cape region. This is in line with what one would expect from the historical data which attributes the introduction of X. laevis in Oeiras  to a single accidental flood of a basement of a research institute in 1979 . The other French individuals had mtDNA identical to native individuals from the south-western Cape (SA1–SA2). Haplotype and sequence diversity in the French population were relatively high and comparable to those in native South African regions. The mtDNA data thus suggest that two distinct phylogeographic lineages i.e., south-western Cape and northern lineage, contributed to the invasion of X. laevis in France. Although the nDNA data are consistent with this suggestion, they solely indicate the Laingsburg region (SA3) and the south-western Cape (SA1–SA2) as possible source areas of the French nDNA alleles. This might be explained by either a scenario of a single South African source area where south-western and northern lineages admix or a scenario in which France was invaded by individuals from several distinct South African source areas followed by admixture of the colonizing animals. In the case of the first scenario, the most likely source population based upon the nDNA data would be the Laingsburg region which is considered as an admixture zone among the south-western Cape regions SA1-SA2 and the Beaufort West region SA4 . Unfortunately, information concerning export from the latter region is limited. In contrast, there is some export data from the Western Cape province in the period that the French breeding facility was active (1950–1996)  . Until 1r bodies . Around r bodies . Taking X. laevis population is the first example in which two geographically non-overlapping phylogenetic lineages participated in a X. laevis invasion. As mentioned previously, two distinct mtDNA lineages, related to on the one hand the northern regions of South Africa and on the other hand the southwestern Cape regions, are present in the French population. However, no population structure could be found in the invasive French populations based on nDNA. In contrast, within the native South African range these mtDNA lineages are significantly divergent based on nDNA. The latter observation suggests a potential role for ongoing admixture within the invasive French population. Likewise, admixture might have occurred within breeding facilities among specimens from different source populations preceding the French invasion. The combination of genetic variation from multiple phylogeographic lineages might explain the high level of genetic diversity within France, which was comparable to, or even higher than, the diversity observed in native regions. An increase in genetic diversity within invasive population relative to native populations due to multiple introductions of genetically divergent source populations has also been demonstrated in invasions of other organisms elsewhere (X. laevis, it is clear that single introduction events even with low levels of genetic diversity have proven to be highly successful in invading non-native areas such as Chile and Sicily. However, in order to predict the species’ potential range expansion the intraspecific variation should be taking into account (X. laevis in its native South African range allowed us to investigate the geographic origins of invasive X. laevis populations in France and Portugal. The current analyses showed that native phylogeographic lineages are not equally represented in invasive European X. laevis populations.To the best of our knowledge, the French lsewhere . Moreovelsewhere . Yet, a lsewhere . Concern account , especia