Eruca sativa has a geographically narrow distribution across a steep climatic gradient that ranges from mesic Mediterranean to hot desert environments. These conditions offer an opportunity to study the influence of the environment on intraspecific genetic variation. For this, we combined an analysis of neutral genetic markers with a phenotypic evaluation in common-garden experiments, and environmental characterization of populations that included climatic and edaphic parameters, as well as geographic distribution. A Bayesian clustering of individuals from nine representative populations based on amplified fragment length polymorphism (AFLP) divided the populations into a southern and a northern geographic cluster, with one admixed population at the geographic border between them. Linear mixed models, with cluster added as a grouping factor, revealed no clear effects of environment or geography on genetic distances, but this may be due to a strong association of geography and environment with genetic clusters. However, environmental factors accounted for part of the phenotypic variation observed in the common-garden experiments. In addition, candidate loci for selection were identified by association with environmental parameters and by two outlier methods. One locus, identified by all three methods, also showed an association with trichome density and herbivore damage, in net-house and field experiments, respectively. Accordingly, we propose that because trichomes are directly linked to defense against both herbivores and excess radiation, they could potentially be related to adaptive variation in these populations. These results demonstrate the value of combining environmental and phenotypic data with a detailed genetic survey when studying adaptation in plant populations.In Israel This article describes the use of several types of data to estimate the influence of the environment on intraspecific genetic variation in populations originating from a steep climatic gradient. In addition to molecular marker data, we made use of phenotypic evaluation from common garden experiments, and a broad GIS based environmental data with edaphic information gathered in the field. This study, among others, lead to the identification of an outlier locus with an association to trichome formation and herbivore defense, and its ecological adaptive value is discussed. The East Mediterranean region is considered a biodiversity hotspot, being on the edge of the southern and northern distribution ranges of temperate and desert species, respectively, as well as the distribution center of many others  and their association with geographic and environmental conditions in search for adaptive variation.A considerable amount of research has been focused on the characterization of genetic diversity in the Near East, including the associations between phenotypic variation and environmental factors with local adaptations  and also showed earlier flowering than those in populations of more mesic habitats . We therefore set out to investigate whether patterns of molecular and phenotypic variation in E. sativa populations reflect adaptations to the strong environmental gradient. To explore this possibility, we used amplified fragment length polymorphism (AFLP) markers and characterized phenotypic traits and environmental conditions in populations of E. sativa, in order to address the following questions: (1) what is the role of the environmental and geographical conditions in structuring neutral genetic variation? (2) do these factors affect genotypic and phenotypic variation? (3) do candidate loci for selection associate with local environmental conditions?he south , across he south ; Fig. 2.E. sativa were mapped along the plant's geographical distribution range in Israel: along the Jordan Valley, the east coast of the Sea of Galilee, and the southern slopes of the Golan Heights . The restriction, ligation, and preselective PCR of the AFLP procedure followed Vos et al.  with theElectropherograms were scored automatically with Genmarker 1.75  and corrected manually. Extra care was taken to avoid scoring fragments twice that were labeled with two different dyes, and ambiguities were recorded as missing data. Genotyping error rates were estimated with 15 replicated samples by dividing the number of mismatches between replicates by the total number of phenotypic comparisons , Bet Dagan, Israel , outside the geographical study area . An addiPhenotypic evaluation in the common-garden experiment included recording of the day of bolting (when the first elongated flower stem reached 1 cm in height), the first day of flowering, and stalk height at the end of the growth stage. Qualitative estimation of trichome density on buds and stems was estimated on a scale of 0 – glabrous to 5 – highly dense. Herbivore damage in the common-garden experiment was estimated at the rosette stage on a scale of nil damage (0) to severely damaged (5). Qualitative evaluation of intensity of yellow pigmentation in the flowers was evaluated in a scale of pale cream (1) to bright yellow (4). Examples of qualitative traits are given in Fig. S1. In the net-house, the number of rosette leaves on each plant was counted, and the rosette diameter was measured on the day the plants started their elongation stage. Stalk lengths were measured every 3 days, and the days of bolting and of the first flowering were noted. Trichome density on buds and stems was estimated qualitatively, and the number of inflorescences on each plant was counted.The phenotypic evaluation was performed by one person. All plants used in the phenotypic evaluations were raised from seed pools collected in nature, and thus the possibility of maternal effects cannot be excluded.Climatic environmental data at the sites where populations were sampled were obtained from the Israel Biodiversity and Information System . These data included annual temperatures, precipitation, altitude, and slope  and S1.3) were assessed by measuring the amount of CO2 gas evolved from a 100-mg soil sample in a constant-pressure calcimeter (Machette 2 = 0.949)  with a separation of at least 4 m between samples. The soil samples were dried at room temperature, crushed gently, and sieved to pass a 2-mm screen, in order to qualitatively separate and weigh the stones and the <2-mm fractions. The latter were analyzed as follows for edaphic properties: (1) pH and electrical conductivity (EC), as an expression of salinity, were measured in the supernatant, following shaking 15-g soil samples in 30 mL of demineralized water on a reciprocal shaker for 1 h and centrifuging at 3100 g; (2) Carbonates  HygK = 2–10, where K is the assumed maximum number of clusters in the data. An admixture analysis of individuals from one population was subsequently carried out using BAPS 5.4 to estimate the genetic ancestry of individuals. This analysis was done using predefined clusters based on the initial mixture results. Significance of admixture coefficients was estimated by comparing them to coefficients from 200 individuals simulated from source populations whose allele frequencies matched those of the populations inferred in the preceding cluster analysis . To quantify differentiation, analyses of molecular variance based on band frequencies  method based on Nei's genetic distances Nei , using tFST/(1 − FST) and geographic distances were ln transformed  were used. In keeping with regression methods commonly used in IBD (isolation by distance) analysis, genetic distances were measured as  Rousset . The cli Rousset  and the  Rousset  and yiel Rousset  in R . Significance testing was done with permutation tests. The regression models considered were: (1) Phenotypic distance (net-house) =  and (2) phenotypic distance (common-garden experiment) = . The common-garden and the net-house data were tested separately as data for one locality of each experiment were missing.The effects of the environmental conditions and geographical distances on phenotypic variation were analyzed with multiple regressions. Environmental distances were based on the four first axes of a PCA as above. The same was done for the population means obtained from the phenotypic evaluation and used as the phenotypic distance. Significance of the regression coefficients and correlation coefficients were estimated with the multiple regression on distance matrices (MRM) function in the R-library ecodist   was estimated from the AFLP data using the untrimmed mean, and theta was set to 0.018. Varying theta between 0.009 and 0.09 did not affect the results significantly.To identify candidate loci, potentially influenced by selection, three different methods were used. First, the FST values into a population-specific and a locus-specific component. Departure from neutrality is inferred when the locus-specific component is necessary to explain the pattern of diversity seen in the data. This is done by comparing posterior probabilities of a model with the locus-specific component with those of a model without it. Rather than using P-values, the authors of the program recommend the use of q-values and outlier was regarded significant at a threshold q-value of 5%. A q-value for a locus is the expected proportion of false positives inferred when the locus is regarded as significant.Second, an alternative outlier method implemented in the Bayescan 2.1 program was used   and at higher altitudes on the southern slopes of the Golan heights , all with an average annual rainfall >400 mm. Along the Jordan Valley, populations of E. sativa grow at low altitudes (>165 m below sea level) and in an aridity gradient that ranges from semiarid  to arid  conditions  sites. In general, the southern sites are more saline , but contain smaller stone fractions  (Table S1). The soil LOI did not show a clear pattern, with values being lower in the north and south (Table S1).3) in the populations on the slopes facing the Sea of Galilee  were markedly lower than those of other populations, but no differences were observed between the reactions (PH) of the soils (Table S1). The assumed SSA values in soils originating from populations EG and SU  were similar to that of EH, AE, and NA, but lower than those in the other Jordan Valley soils (>100 m2/g), indicating a higher clay fraction in the latter soils.The contents of carbonates . The first two axes of the PCA accounted for 75.6% of the variation, mostly affecting the differentiation between the southern arid sites of the Jordan valley  and the semiarid sites (BS and EH), and between the latter two and the Mediterranean mesic habitats (EG and SU) in the northern distribution range of the species . The first principal component was dominated by climatic conditions, especially temperature and rainfall, and the second component was correlated mostly with variations in soil parameters, such as soil surface area, calcium carbonate content, LOI, and stoniness, but also with topographic parameters, for example, slope and aspect .The phenotypic characteristics varied significantly among populations for all measured traits; the data are supplied as an appendix Excel file. In general, sites with low vegetation cover, for example, NA, SA, and SU, showed earlier bolting and flowering than other sites, in both the net-house and the common-garden experiments, but no trend was found in the heights of the plants. Trichome density on buds and stems was lower in populations from northern sites than in those from the southern sites in the net-house but these differences were diminished in the common-garden experiment. In addition, the southern populations showed a tendency to be less damaged by herbivory than the central or northern ones. However, many of the traits varied unpredictably among the sites, or showed partially contrasting patterns between the common-garden and the net-house experiments.A principal components analysis of the phenotypic data from the net-house and the common-garden experiments is shown in Fig. S3. In the net-house experiment, the first two axes accounted, respectively, for 44% and 28% of the variation : the first axis correlated mainly with flowering and bolting dates, stalk height, and the number of inflorescences; the second was dominated by stem and bud trichomes and rosette diameter . For the common-garden data, the first two axes accounted for 35% and 30% of the variation : the first axis corresponded mainly to flowering and bolting dates and early herbivore damage; the second to late herbivore damage, stalk height, and trichome density .E. sativa populations. The AFLP analysis, with six different primer combinations, yielded 229 loci that could be reliably scored, of which 81% were polymorphic at the 99% level. The mismatch error rate was low, at 0.78%. Genetic diversity within populations, expressed as unbiased expected heterozygosity (He), was similar in all the sampled populations , a hierarchical AMOVA partitioned 8.26% of the variation between the two genetic clusters (P = 0.030), and 5.8% was partitioned among populations within each of the two groups (P < 0.001).The mixture analysis  and the R2 = 0.33; P = 0.003; and R2 = 0.19, P = 0.039, respectively), and the variance explained when adding both variables was only a fraction higher (R2 = 0.340). A stepwise regression showed that each of the variables significantly improved the model.Multiple regressions revealed that both the environmental and geographic distances showed significant positive relationships with genetic distances. The environmental distances accounted for more of the variance than geographic distances .Phenotypic data from the net-house and the common-garden experiments showed differing patterns. The PCA distances based on data from the common-garden experiment were positively related to both environmental and geographic distances. However, a stepwise regression selected only environmental distance, whereas geographic distance did not improve the model significantly . In contFST over all loci was 0.067. Two loci had FST values higher than the 99% confidence interval. Repeating the analysis after removal of these two loci revealed one additional candidate locus and, therefore, three loci were potentially affected by divergent selection  was similar to the number of markers with higher P-values (N = 95), confirming that the simulation model was appropriate . Analysis with Bayescan yielded four candidate loci significant at the q-value threshold of 5%, all of which showed signs of diversifying selection. Two of these were also found in the Dfdist analysis and thus a total of five outliers were detected by the analyses  and edaphic conditions , as well as vegetation. Despite this, genetic diversity and structure was not unambigously associated with environment or geography, instead it was best explained by genetic cluster. Also, the finding of a genetic break that divided the investigated populations into two clusters did not coincide with the most striking differences in morphology observed in nature. Indeed, differences in phenotype largely disappeared in the common-garden experiments, which showed that this variation was almost entirely due to plasticity. Nevertheless, regression analysis showed that part of the phenotypic variation measured under field conditions could be attributed to the environment. In addition, several candidate loci for selection were detected by three different methods, suggesting a role for adaption to environmental conditions.This study provides a broad survey of genetic variation along the distribution range of The present results show a moderate but clear genetic break that subdivides the nine studied populations within the narrow distribution range into a southern and a northern cluster Figs. , S4. AltBecause the two clusters were located in a north-south pattern, testing for effects of geographic distance without correcting for this spatial pattern might yield spurious regression coefficients. The same applies to environmental factors if they are correlated with the geographic hierarchical structure. This was exemplified by the significant effects of the geographic and environmental distances on genetic distances in multiple regressions, which do not control for hierarchical structure. Cushman and Landguth  suggesteE. sativa. However, although the existence of IBA (isolation by adaptation) patterns seems relatively common , as well as observed variation in seed germination in response to photo-thermal cues  were identified by more than one method. However, in light of the fact that the programs use different methods to detect candidate loci, it is not surprising that the different programs identified different markers as outliers. Even when only comparing differentiation based outlier methods, several papers have shown that programs perform differently using both experimental and simulated data, for example,  affected the detection of outliers with Dfdist. In this analysis fragments 55GA170 and 54GA254 were again identified, whereas fragment 55GA254 was not . However, fragment 55GA254 showed a pattern of differentiation that was most marked among populations within the genetic groups, therefore its detection should not be affected by the hierarchical structure. This was also true of the two additional outlier loci detected by Bayescan, which suggests to us that the results of the outlier analyses are robust with respect to hierarchical structure.However, the pattern of differentiation in r et al.  which pr3, and SSA, but also to topography. If we include candidate loci with nonsignificant associations, seven out of nine loci were most strongly associated with PC1. However, this is not significantly more than for PC2 ; furthermore, it may reflect the correlation between subclusters and climate, which might influence the results of this analysis. Thus, candidate loci in E. sativa showed no clear trend toward closer association with climatic than with other environmental factors. Most studies that test for associations between marker and environmental data use climatic data, most likely because of their availability. However, our results suggest that incorporating other data such as edaphic conditions might be a promising strategy; and this is not surprising in the light of the importance of edaphic factors in ecotypic variation , we found two correlations that were significant for fragment 54GA254, the only candidate identified by all three methods: (1) stem trichome density ; and (2) late herbivore damage . It is well known that trichome production is induced under herbivore attack (Yoshida et al. Of the five ironment . The thrThere is no doubt that more experiments are needed to determine the local selective pressures and the underlying relationships between genotype, phenotype, and the environment. The use of new approaches that utilize phenotypic data from genotyped individuals within populations in exploratory genome scans (Herrera and Bazaga


In the present study, we investigated the application of 13 short tandem repeat (STR) loci  routinely used in forensic analysis, for delineating population relationships among seven human populations representing the two major geographic groups, namely the southern and northern Chinese. The resulting single topology revealed pronounced geographic and population partitioning, consistent with the differences in geographic location, languages and eating habits. These findings suggest that forensic STR loci might be particularly powerful tools in providing the necessary fine resolution for reconstructing recent human evolutionary history. The present Chinese population of around 1.4 billion is primarily divided by the Yellow River into two large groups, the southern and the northern, with diverse languages and eating habits. There is thus an immense scope to study the processes of anthropological subdivisions and microevolutionary effects in different populations groups of China. However, the traditional structure of Chinese populations is facing the imminent threat of disintegration through urbanization and increasing communication, with the consequential gene flow between subcastes through marriages. Therefore, there is a need for understanding local traditional population structure and its role in shaping human genome diversity.A large-scale survey of autosomal variation in an ample geographic sample of human Asian populations has shown that, apart from geography, genetic ancestry is strongly correlated with linguistic affiliations . A distiBased on the population data of these STR polymorphisms, the forensic parameters of the respective loci were calculated in order to estimate their value in genetic identity testing. Furthermore, genomic affinities among the diverse regional population groups were evaluated. The current study contributed to supplementing the ever-increasing population-information database worldwide.Whole blood was obtained by venipuncture in EDTA-coated vaccutainers from unrelated, consenting donors. Community history and family disease backgrounds were recorded on blood donor cards.Seven geographically targeted populations, encompassing the major biogeographical zones and representing the two main Han populations (southern and northern), were selected. These included 4 southern, the Sichuan , 0.5 μL of a dNTPs mix , and 1U Taq polymerase  in a total volume of 25 μL. A total of 30 cycles were run, with an initial incubation (preliminary denaturation) step at 96° C for 2 min, followed by 10 cycles of 94 °C for 1 min, 60 °C for 1 min and 70 °C for 1.5 min, followed by 20 cycles of 90 °C for 1 min, 60 °C for 1 min and 70 °C for 1.5 min, ending with a final extension at 60 °C for 30 min.PCR amplification was carried out on a thermal cycler, using primers with the same sequences as those in the “PowerPlex 16 System” kit (The PCR product (1.5 μL), as well as GeneScan-400HD-ROX Size Standard (Applied Biosystems) (0.5 μL), were added to 24.5 μL of deionized formamide, and subsequently denatured for 3 min at 95 °C. Alleles were then separated by capillary electrophoresis in POP-4 polymer (Applied Biosystems) with the GS STR POP4 D Module (1 mL), using an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). Samples were injected into the capillaries in batches of 16 samples, directly from the microtitre plate, for 10 s at 3 kV. Electrophoresis was performed at 15 kV and 60 °C for 45 min under routine running conditions. Alleles were identified by means of GeneScan Analysis 3.7 Software (Applied Biosystems), whereupon the analyzed data were automatically genotyped using Genotyper 3.6 Software (Applied Biosystems) and a template specially made for this specific multiplex system. The Peak Amplitude Threshold adopted was more than 150 RFU (relative fluorescence units).i.e., the power of discrimination  (Tables S1-S13). 13 STR loci among seven Chinese populations showed similar trends Furthermore, both genotype and allele distribution were not significantly different among the seven Chinese populations (p > 0.05). These results are thought to reflect the influence of gene flow due to geographic proximity.Despite the wide range of allelic variation in the 13 STR loci, a discernable pattern depicting mutual geographical affiliation is apparent. Generally speaking, frequency was high in only few alleles , as well as the results from the various analytical procedures for testing the correspondence of genotype frequencies with Hardy-Weinberg equilibrium, are shown in Tables S1-S13.All the 13 loci complied with Hardy-Weinberg equilibrium, with no evidence of association of alleles among the 13 loci. The parameters for both forensic efficiency and genetic variability, such as MP, PD, PIC, PE and heterozygosity, were calculated and subsequently listed for each population in the supplementary tables.In order to ascertain relationships among the seven Chinese populations, we have calculated the Nei measure of pairwise genetic distances using allele frequency data from the 13 STR markers. Polish population data (The longest distance (0.0320) was noted between the Fujian (a southern) and Henan (a northern) populations, whereas the lowest (0.0041) was observed between Beijing (a northern) and Tianjin  populations .Based on genetic distance data, population trees were constructed using the UPGMA and Neighbor-Joining methods. As both methods revealed the same pattern, UPGMA results were preferred for display. Bootstrap values for the trees were high . The SicOwing to the several advantages, such as high polymorphism, ease and low-cost, STR markers have been widely used for fine-scale genetic mapping , all theOver the past decades, and based on STR polymorphisms, important information has contributed to elucidating the history of human populations (The following online material is available for this article:Genetic polymorphism at the D3S1358 locus for the seven Chinese population groups.Genetic polymorphism at the D16S539 locus for the seven Chinese population groups.Genetic polymorphism at the TPOX locus for the seven Chinese population groups.Genetic polymorphism at the TH01 locus for the seven Chinese population groups.Genetic polymorphism at the CSF1PO locus for the seven Chinese population groups.Genetic polymorphism at the D7S820 locus for the seven Chinesepopulation groups.Genetic polymorphism at the VWA locus for the seven Chinese population groups.Genetic polymorphism at the FGA locus for the seven Chinese population groups.Genetic polymorphism at the D8S1179 locus for the seven Chinese population groups.Genetic polymorphism at the D21S11 locus for the seven Chinese population groups.Genetic polymorphism at the D18S51 locus for the seven Chinese population groups.Genetic polymorphism at the D5S818 locus for the seven Chinese population groups.Genetic polymorphism at the D13S317 locus for the seven Chinese population groups.This material is available as part of the online article from http://www.scielo.br/gmb.


Arvicola amphibius populations have recently experienced severe decline in several European countries as a consequence of both reduction in suitable habitat and the establishment of the alien predator American mink Neovison vison. We used DNA microsatellite markers to describe the genetic structure of 14 island populations of water vole off the coast of northern Norway. We looked at intra- and inter-population levels of genetic variation and examined the effect of distance among pairs of populations on genetic differentiation (isolation by distance). We found a high level of genetic differentiation (measured by FST) among populations overall as well as between all pairs of populations. The genetic differentiation between populations was positively correlated with geographic distance between them. A clustering analysis grouped individuals into 7 distinct clusters and showed the presence of 3 immigrants among them. Our results suggest a small geographic scale for evolutionary and population dynamic processes in our water vole populations.Water vole  The amount of genetic variation within a population has implications for its ability to cope in the face of environmental changes Wright , such asCynomys ludovicianus, American pika Ochotona princeps, and water vole Arvicola amphibius (formerly named Arvicola terrestris) seem to best conform to the metapopulation paradigm . In one of our studied metapopulations, at Sleneset, the eagle owl has one of the highest breeding densities reported for the whole of Europe  along the coast of northern Norway , Slesenet (N = 5), Lovund (N = 4), and Myken (N = 1). A higher level of dispersal is expected to occur within each of these groups of islands.The breeding season of this species in our study area extends from April to August . The islands are geographically clustered in 4 groups of islands among which we might expect little dispersal to occur, from south to north: Lånan  and probability of recapture (ci). Model M0, assumes equal capture probability for all animals on all trapping occasions; Mb assumes different capture and recapture probabilities; Mt assumes that capture probability varies with time; Mh assumes different capture probabilities for different individuals. The minimum adequate model was selected by comparing the Akaike Information Criterion corrected for small sample sizes . Accordingly, we believe that the sample sizes reflect real differences in population sizes and are not an artifact due to differences in sampling effort. Population size for each population was also estimated with the software MARK 6.1 (White and Burnham N of traps on each island = 20–80). Each capture session continued until we reached a 40% cumulative recapture rate . The longest session lasted 10 days. The traps were checked at least twice a day, but more often when the weather was very cold or very warm to prevent any negative effects of trapping on the population. About 40% of the capture consisted of individual classified as “adults” according to their size and 43.7% of the capture were males. For details about the capture and measurement methods, see Melis et al.  and 3 μL DNA (approx. 20 ng/μL). The final concentration of each F and reverse (R) primer in the multiplex PCR was 0.0625 μmol/L. The touchdown PCR profile had an initial denaturation step at 94°C for 15 min, then 12 cycles starting with 30 sec at 94°C and 1 min 30 sec at temperature 62–50°C, decreasing 1 degree pr cycle, and an extension step at 72°C for 1 min. This was followed by 23 cycles with 30 sec at 94°C, 50°C for 1 min 30 sec, and 72°C for 1 min. The PCR ended with an elongation step at 60°C for 5 min and then the PCR products were stored at 4°C. After amplification, the PCR products were diluted 1:2 by adding 10 μL ddH2O to the products. 1 μL of the diluted PCR product was then mixed with 10 μL of a mix of Hi-Di formamide  and GeneScan 600 LIZ (Applied Biosystems) size standard (for one sample: 9.524 μL Hi-Di formamide and 0.476 μL 600 LIZ). Electrophoresis and separation of alleles  were conducted on an ABI3130xl Genetic Analyzer (Applied Biosystems). Individual alleles on each microsatellite locus were scored using the software GeneMapper 4.0 (Applied Biosystems).All the individuals were genotyped at the 14 loci in 10 μL reactions. Each reaction contained 2 μL primer-mix, 5 μL Qiagen multiplex PCR solution (containing na), allelic richness corrected for minimum sample size (AR), observed (HO) and expected (HE) heterozygosities for each locus, as well as inbreeding coefficients (FIS) for each population and overall FST, were calculated in FSTAT version 2.9.3.2  on the basis of individual data. In STRUCTURE, we ran five iterations for each K = 1–20  using the admixture model, correlated allele frequencies and no prior information of the sampling locality. Furthermore, we followed the procedure described by Evanno et al.  were both negative and positive, none differed significantly from zero when correcting the P-value for the number of simultaneous tests  among populations was high (FST = 0.396 ± 0.042 (SE)). Accordingly, there was strong genetic differentiation between nearly all pairs of populations  and at the level of localities . Moreover, according to the clustering analysis using both the method of Pritchard and Wen  to test statistically if their sex ratio was biased toward one sex. Dispersal was detected only within the Sleneset and Lovund localities. However, it is likely that we underestimated dispersal because we did not sample all the island populations at each locality i.e. not all possible sources of immigrants have been assessed. Moreover, for example, at Lånan, the island populations were grouped by STRUCTURE into a single cluster, making it impossible to detect immigrants among them. The 7 distinct population clusters reflected the geographic structure of the populations. Moreover, the analysis with HIERFSTAT identified not only a strong level of genetic differentiation between localities, but also strong genetic differentiation between islands nested within localities. The localities Myken and Lånan are quite distant from the others . But even Lovund and Sleneset, which are not so far apart (ca. 9 km), were identified as separate clusters and no immigrants were detected between them. The age of the identified immigrants suggests that dispersal occurs at juvenile stage in water voles, before reproduction has occurred. This is similar to what has been found based on capture-mark-recapture data (Lambin et al. The inbreeding coefficients In Norway, water voles are perceived as pests and their conservation is currently not a concern Bevanger . However


Despite the limited genetic heterogeneity of Spanish populations, substantial evidences support that historical African influences have not affected them uniformly. Accounting for such population differences might be essential to reduce spurious results in association studies of genetic factors with disease. Using ancestry informative markers (AIMs), we aimed to measure the African influences in Spanish populations and to explore whether these might introduce statistical bias in population-based association studies.p-values for the differences was adequately controlled by correcting for genetic ancestry estimates derived from a reduced number of AIMs.We genotyped 93 AIMs in Spanish (from the Canary Islands and the Iberian Peninsula) and Northwest Africans, and conducted population and individual-based clustering analyses along with reference data from the HapMap, HGDP-CEPH, and other sources. We found significant differences for the Northwest African influence among Spanish populations from as low as ≈5% in Spanish from the Iberian Peninsula to as much as ≈17% in Canary Islanders, whereas the sub-Saharan African influence was negligible. Strikingly, the Northwest African ancestry showed a wide inter-individual variation in Canary Islanders ranging from 0% to 96%, reflecting the violent way the Islands were conquered and colonized by the Spanish in the XV century. As a consequence, a comparison of allele frequencies between Spanish samples from the Iberian Peninsula and the Canary Islands evidenced an excess of markers with significant differences. However, the inflation of Although the African influences estimated might be biased due to marker ascertainment, these results confirm that Northwest African genetic footprints are recognizable nowadays in the Spanish populations, particularly in Canary Islanders, and that the uneven African influences existing in these populations might increase the risk for false positives in association studies. Adjusting for population stratification assessed with a few dozen AIMs would be sufficient to control this effect. Populations inhabiting the Iberian Peninsula have been influenced by the same major human prehistoric migrations that have affected the rest of European populations, regardless of the extent to which Neolithic expansions from the Near East have influenced their genetic makeup Despite this, several lines of evidence support the existence of identifiable unequal African influences in populations from Spain, both from the Iberian Peninsula and the Canary Islands. The excess of diversity observed in Southwestern Europeans for genome-wide autosomal haplotypes was interpreted as resulting from direct migrations from North Africa across the Mediterranean These major documented historical influences from divergent populations such as those from the nearby North Africa Ancestry informative markers (AIMs), i.e. genetic loci showing large allele frequency differences between populations, allow accurate apportioning of genetic influences in populations p-value = 0.00054), only two markers departed significantly from HWE: rs1073321 in Canary Islanders and rs7277342 in Northwest Africans. Given that not a single marker deviated from HWE after this correction in all three genotyped samples, and that genotyping was performed simultaneously for all samples obtaining similarly high completion rates (≥97%), we interpreted that HWE departures were more likely to be related to chance and retained all 93 EuroAIMs for further studies. On average, genetic differentiation levels (FST) obtained for the comparison of the 93 EuroAIMs between Northwest Africans and Iberians, Northwest Africans and Yoruba Nigerians (YRI), and Iberians and YRI were 0.0422, 0.100 and 0.255, respectively  ST genetic distances revealed three well separated clusters of populations (p = 6.23E-17) and PC2 differentiating Northwest Africans from SSE Europeans (p = 3.88E-17). Spanish populations were assigned at intermediate positions in the NNW-SSE axis of European differentiation, with Canary Islanders clustering in their vicinity, albeit showing a slight shift towards Northwest Africans (p = 8.93E-17)  , as woulST distances of Canary Islanders to Iberians and to a Spanish sample without information of sampling location ST distances were obtained for different partitions of the Iberian sample (not shown), including a separation on an East-West axis as noted in a previous study Among Spanish, the FTo verify that population relationships were not affected by selection processes, we re-assessed population and individual-based analyses excluding the EuroAIMs with potential effects on gene function according to the SNP Function Portal Taken together, these observations support the genetic discontinuity observed between Northwest Africans and their closest European neighbors As there is not a formal way to recognize if minor individual ancestries are accurate measurements or artifacts of the methods used, we first assessed the influences at population level with the only purpose of deciding which populations to include in STRUCTURE analyses. For that, we evaluated African influences in Canary Islanders as, among Spanish populations, it would be more likely to detect such influences in this population based on the historical records. Given that results from a three-population model of genetic contributions in Canary Islanders indicated that a direct sub-Saharan African influence in this population was negligible , and thap = 1.00E-6) (p = 0.488), nor among the samples from the different islands (ANOVA p = 0.213)  . In addi= 0.213) . Note, h= 0.213) . With fep<0.05 (2 test p = 0.0375). To explore if this effect was controlled by using the ancestry information derived from EuroAIMs, we adjusted these comparisons using logistic regression models including as covariates either the STRUCTURE Northwest African individual ancestries or the PCA scores. Adjusting for STRUCTURE estimates, based on the full marker set or any of the subsets of markers ranking higher for different measures of ancestry informativeness, barely decreased the number of significant tests , increased from p = 0.0002 with the unadjusted test (the Cochran-Armitage trend test) to p = 0.006 after the adjustment. The first p-value will remain significant after a Bonferroni-like correction for the multiple comparisons, but the second will not. Nevertheless, the adjustment of comparisons using the PC1 scores not only resulted in reduced significance levels but also in an effective control of false positives at 5% rate . Strikingly, a parallel geographical clustering of the Northwest African influence in Iberian populations was recently revealed with Y-chromosome binary markers It was not unexpected to find a low level of bidirectional gene flow (5%) between Northwest Africans and Iberians given the results from previous studies The substantial Northwest African ancestry found for Canary Islanders supports that, despite the aggressive conquest by the Spanish in the XV century and the subsequent immigration, genetic footprints of the first settlers of the Canary Islands persist in the current inhabitants. Paralleling mtDNA findings In view of the disparate estimates of the African influences obtained for the two Spanish populations analyzed, we anticipated an increase of false positive results in population-based association studies of genes with disease simply due to systematic differences in such influences. Using PC scores and, alternatively, individual admixtures obtained by means of STRUCTURE, we have shown that there is a potential benefit of correcting for population stratification in Spanish studies including samples from Canary Islanders. If no adjustment was done, the number of false positive associations resulted in more than twice the expected amount under the null hypothesis. Thus, even small levels of population admixture can undermine an association study and lead to false positive results We warn that because we used markers showing large allele frequency differences between populations In conclusion, we have extended the use of EuroAIMs to allow distinguishing the contrasting Northwest African influences existing among the Spanish populations. We have also demonstrated that these differences might increase the risk for false positives in genetic epidemiology studies that can be effectively controlled using a reduced number of EuroAIMs.This study was approved by the Hospital Universitario N.S. de Candelaria and Hospital Universitario Dr. Negrín Ethics Committees. Written informed consent was obtained from all participants involved in the study.www.bancoadn.org), consisting on 77 samples assigned to different Iberian localities based on the individual's self-reported grandparental origin: Andalusia (n = 15), Murcia (n = 5), Extremadura (n = 5), Castile-La Mancha (n = 4), Valencia (n = 7), Castile and Leon (n = 9), Madrid (n = 1), Catalonia (n = 2), Galicia (n = 4), Cantabria (n = 3), Navarre (n = 4), La Rioja (n = 4), Asturias (n = 1), and mixed Iberian origin (n = 13). The Canary Island population was represented by DNA samples from 104 unrelated healthy donors with at least two generations of ancestors born in the Canary Islands collected for the study , and 15 additional samples from Gran Canaria obtained from the Spanish National DNA Bank. Additionally, 68 DNA samples from unrelated healthy individuals with at least two generations of Northwest African descent were studied: 25 from Casablanca, 25 from Rabat, and 18 from other regions of Morocco. Details of laboratory procedures can be found in the DNA samples from individuals of the general Iberian population were obtained from the Spanish National DNA Bank . Nine SNPs that gave poor quality data on this platform were finally determined using SNaPshot® Multiplex Kit reactions   . GenotypST genetic distance InAllele counts, exact tests for HWE departures ST genetic distances, to assess PCA of individual samples, and to determine the ANOVA statistics for population differences along each of the PCs. Multidimensional scaling was used to represent pairwise population FST genetic distances in two dimensions using SPSS ver.15 .EIGENSOFT R least-squares estimator, that ignores sampling and genetic drift in populations W maximum likelihood estimator, that allows estimating genetic contributions taking into account the effects of sampling and genetic drift in all populations and the differentiation between parental populations before the admixture event In order to obtain initial estimates of the genetic contribution of populations in Canary Islanders, particularly the influence of sub-Saharan Africans, population-based estimates were calculated using LEADMIX Individual ancestry estimates were assessed using an unsupervised assignment of individuals to populations by means of STRUCTURE 2.2 In and δ values, given that FST and In values were highly correlated (r2 = 0.99). STRUCTURE was run for each subset using the same parameters as described above and all correctly identified K = 2. A cut-off membership of 0.70 was used as a criterion for the individual inclusion in the self-reported population. Pearson's correlations between ancestry estimates derived from each subset and those from the 93 EuroAIMs set were computed.With the purpose of exploring the ability of reduced EuroAIMs subsets to estimate population membership and ancestry proportions, we selected three subsets containing the most informative 23, 46 and 69 markers. For that, EuroAIMs were ranked based on the average of ranks for To explore whether allele frequencies of the EuroAIMs differed significantly among the Spanish samples, we treated each individual EuroAIM marker as a candidate locus for association in a mock study where Canary Islanders were taken as “cases” and Iberians were considered “controls”. Differences were first tested using the Cochran-Armitage trend test, which is similar to the allele count-test albeit not relying on the assumption of HWE Figure S1STRUCTURE results based on EuroAIMs. This analysis used data from Iberians (IBE), Northwest Africans (NWA) and Yoruba Nigerians (YRI) from HapMap (TIF)Click here for additional data file.Figure S2p-values (as −log10P) for marker allele frequency differences between Spanish populations. Upper panel: adjustments based on STRUCTURE estimates; Lower panel: adjustments based on PC1 scores. Closed circles: trend test statistics; Open circles: statistics adjusted for estimates based on 93 EuroAIMs; dark grey circles: statistics adjusted for estimates based on 69 markers; triangles: statistics adjusted for estimates based on 46 markers; light grey circles: statistics adjusted for estimates based on 23 markers. The discontinuous line indicates the null distribution.Quantile-quantile plots of (TIF)Click here for additional data file.Table S1Summary statistics for EuroAIMs used in the study.(DOC)Click here for additional data file.Table S2ST genetic distances.Pairwise population F(DOC)Click here for additional data file.Table S3Functional annotation of EuroAIMs within RefSeq genes.(DOC)Click here for additional data file.Text S1Ancestry informativeness of EuroAIMs in samples from HGDP.(DOC)Click here for additional data file.Text S2Ancestry informativeness of EuroAIMs subsets.(PDF)Click here for additional data file.Text S3Details of laboratory procedures.(DOC)Click here for additional data file.


Thymallus thymallus) populations have been well studied in most of its native habitat; however the southernmost populations of the Balkan Peninsula remain largely unexplored. The purpose of this study was to assess the genetic diversity of Serbian grayling populations, detect the impact of stocking and provide guidelines for conservation and management.The structure and diversity of grayling . The mitochondrial DNA control region  and the ATP6 gene (630 bp fragment) were sequenced for 20 individuals . In addition, all individuals were genotyped with 12 microsatellite loci. The diversity and structure of the populations as well as the recent and ancient population declines were studied using specialized software.We detected three new haplotypes in the mtDNA CR and four haplotypes in the ATP6 gene of which three had not been described before. Previously, one CR haplotype and two ATP6 gene haplotypes had been identified as allochthonous, originating from Slovenia. Reconstruction of phylogenetic relations placed the remaining two CR haplotypes from the River Danube drainage of Serbia into a new clade, which is related to the previously described sister Slovenian clade. These two clades form a new Balkan clade. Microsatellite marker analysis showed that all four populations are genetically distinct from each other without any sign of intra-population structure, although stocking of the most diverse population (Drina River) was confirmed by mtDNA analysis. Recent and historical population declines of Serbian grayling do not differ from those of other European populations.Our study shows that (1) the Ibar, Lim and Drina Rivers grayling populations are genetically distinct from populations outside of Serbia and thus should be managed as native populations in spite of some introgression in the Drina River population and (2) the Rzav River population is not appropriate for further stocking activities since it originates from stocked Slovenian grayling. However, the Rzav River population does not represent an immediate danger to other populations because it is physically isolated from these. Thymallus thymallus) extends westward to France and Great Britain, northward across Fenoscandinavia and northern Russia, eastward to the Ural Mountains near the Kara River  and 162 bp of the flanking tRNA were amplified using the LRBT-25 and LRBT-1195 primers . The com2O (21 μL H2O for ATP6), 3 μL 10 × PCR buffer, 0.75 μL 10 mM of each primer, 1.2 μL 25 mM MgCl2 (1.8 μL 25 mM MgCl2 for ATP6), 1.5 μL 0.2 mM dNTP, 0.2 μL Fermentas Taq polymerase and 1 μL of template DNA (~100 ng of genomic DNA); the cycle parameters were as follows: initial denaturation  followed by 32 cycles of strand denaturation , primer annealing  and DNA extension . All PCR amplifications were performed in a programmable thermocycler GeneAmp® PCR System 9700 (Applied Biosystems). Amplified DNA fragments were run on a 1.5% agarose gel and subsequently isolated from the gel using the QIAEX II gel Extraction Kit (QIAGEN).The following PCR conditions were used: each reaction mixture (30 μL) contained 21.6 μL HAll sequencing reactions were prepared using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's recommendations. The 3'end of the 595 bp fragment of the mtDNA CR with the 74 bp of flanking tRNA were sequenced using primer LRBT-1195 . The 5'e2O, 1 μL 10 × PCR buffer, 0.25 μL 10 mM of each primer, 0.6 μL 25 mM MgCl2, 0.5 μL 0.2 mM dNTP, 0.075 μL Fermentas Taq polymerase and 1 μL of template DNA (~100 ng of genomic DNA); the cycle parameters were as follows: initial denaturation  followed by 30 cycles of strand denaturation , primer annealing  and DNA extension . Fragment analysis was performed on a 3130xl Genetic Analyzer and genotyped using GeneMapper v4.0.Twelve microsatellite loci were isolated and characterized as previously described i.e. BFRO004 , BFRO005a) using MEGA version 2.1 [DNA sequences were aligned using the computer program ClustalW . Sequencsion 2.1 .Aligned haplotypes were imported into the program PAUP Version 4.0b10  for phylRelations among haplotypes were also determined using the TCS 1.2 program  with a cE), non-biased (Hn.b) and observed (HO) heterozygosities were calculated with GENETIX 4.04 [ST values, all based on 1000 permutations. Genetic relationships between individuals were estimated as the proportion of shared alleles at each locus, i.e. allele sharing distances (DAS) [AS was used to construct Neighbour-Joining trees of individuals and populations with POPULATIONS software [Microsatellite allele frequencies, expected (HTIX 4.04 . FSTAT 2TIX 4.04  was usedes (DAS) . A matrisoftware .e-4Ne generations ago) can be detected with BOTTLENECK 1.2.02 [8 iterations.Recent population declines . Haplotype Soc18 had already been described in the population of Soča River in Slovenia  while inReconstructing phylogenies by both Neighbour-Joining and parsimony methods revealed that haplotype Da25 and the Slovenian haplotypes from the Sava River drainage area form a sister clade of the new clade containing haplotypes Da27 and Da29 from the Danube River drainage area of Serbia , observed (0.47) and expected (0.49) heterozygosities were detected in the Drina River samples, while those of the Rzav and Ibar Rivers had very low levels of heterozygosity. No deviations from Hardy-Weinberg equilibrium were detected Table .Recent bottleneck was detected in the Rzav River (SMM and TPM), P < 0.05. Mode-shift test also revealed distortion from L-shape allele frequency distribution in the same river. Coalescent analyses assuming an exponential population growth/decline estimated that the population decline started 1000 - 10 000 years ago with the present population sizes representing 0.03-0.44% of the historical sizes  on the basis of microsatellite data [Within the Balkan clade, the division between northern (Slovenian) and southern (Serbian) populations is obvious. This northern/southern differentiation has also been recorded in another Danubian salmonid species i.e. huchen  in the Balkan clade defined by the synapomorphy at position 34 of the ATP6 gene , populations from this study are comparable to other populations ,51. FST drainage ,52. ReceSerbian locations represent the furthest grayling habitats from the maximal extent of ice sheets  and possThe Rzav population shares 19 out of 21 alleles (90%) with Slovenian populations , while the Drina, Lim and Ibar populations share 47, 56 and 53% of alleles with Slovenian populations, respectively. The two alleles in the Rzav population not shared with Slovenian populations are found in other Serbian populations, which means that there is no Rzav specific allele. This is not surprising since the river was not naturally inhabited by grayling. While stocking of the Rzav River is confirmed both by the mtDNA haplotypes (100% Da25) and the 90% alleles shared with Slovenian populations, the situation in the Drina River is different. Although 40% of the Drina River samples had the Da25 haplotype, hybridization with non-native grayling was not detected by nuclear markers. The percentage of shared alleles with the Slovenian populations was lowest in Drina River (47%), most likely because the population had already the highest genetic diversity prior stocking. Influence of stocking in the Drina River was not detected in its tributary i.e. the Lim River, which is in accordance to the generally low migration rates for the species ,53. WhilSerbian grayling populations are genetically distinct from Slovenian and other European populations. In order to preserve their overall genetic diversity and integrity, further stocking of non-native fish from other regions or from allochthonous populations in the Rzav River should be stopped. Populations from the Ibar and Lim Rivers (which show no signs of introgression of non-native grayling), as well as the population from the Drina River should be regarded as native and subject to proper management. The population from the Drina River is the most diverse in this study and the only one with the mtDNA haplotype Da29. It probably represents the most valuable genetic resource in the region. However, any future management such as supplementary stocking of hatchery-reared Drina River grayling should take into consideration genetic testing prior formation of brood stock, because introgression has been detected. Since the area studied here represents only a minor part of the Balkan Peninsula, genetic polymorphism of the grayling within the region as a whole may be even higher, because grayling from the countries adjacent to Serbia  have not been studied so far.The authors declare that they have no competing interests.SM performed the laboratory work and wrote the manuscript with assistance from AR and PS. AR conducted the data analyses. VN organized the logistic for the fieldwork, participated in the collection of data and helped to draft the manuscript. PS coordinated and supervised the study. All authors read and approved the final manuscript.Variable nucleotide positions for mtDNA CR haplotypes defined in this study (underlined) and haplotypes from other European clades. Positions are homologous to the T. thymallus haplotype Da1 (Accession number. AF522395) and correspond to the control region (430-1024), and the tRNA phenylalanine gene (1025-1098) Asterisks refer to base pair deletions or insertions, dashes represent concordance with the Da1 haplotypeClick here for fileVariable nucleotide positions for mtDNA ATP6 gen haplotypes defined in this study. Positions are homologous to the T. thymallus haplotype Soc18 (Accession number. AY779004) and correspond to the ATP6 gene (14-643); dashes represent concordance with the Soc18 haplotypeClick here for fileIndividual microsatellite marker data. Individual microsatellite marker data in each population for Tables 2 and 3 and Figures 4 to 6Click here for file


In addition, we examine the distribution of DXS1225-DXS8082 haplotype diversity in Azoreans and mainland Portuguese. Allele distribution has demonstrated that the São Miguel population is genetically very diverse; haplotype analysis revealed 100% discriminatory power for X- and Y-markers and 94.3% for HLA markers. Standardized multiallelic D' in these three genomic regions shows values lower than 0.33, thereby suggesting there is no extensive LD in the São Miguel population. Data regarding the distribution of DXS1225-DXS8082 haplotypes indicate that there are no significant differences among all the populations studied, . Moreover, in these as well as in other European populations, the most frequent DXS1225-DXS8082 haplotype is 210-219. Even though São Miguel islanders and Azoreans do not constitute isolated populations and show LD for only very short physical distances, certain characteristics, such as the absence of genetic structure, the same environment and the possibility of constructing extensive pedigrees through church and civil records, offer an opportunity for dissecting the genetic background of complex diseases in these populations.Studies on linkage disequilibrium (LD) across the genome and populations have been used in recent years with the main objective of improving gene mapping of complex traits. Here, we characterize the patterns of genetic diversity of HLA  Certain factors which generate LD variance, as for example genetic drift and admixture, are population specific. Others, such as recombination rate, gene conversion and natural selection, are specific to genomic regions (Linkage disequilibrium (LD), the nonrandom association of alleles at different th century, the latter being populated mainly with individuals from the other islands.The Azores is a Portuguese archipelago composed of nine islands distributed among three geographical groups: the Eastern group with two islands, São Miguel and Santa Maria; the Central group which includes five islands, Terceira, Pico, Faial, São Jorge and Graciosa; and the Western group, with Flores and Corvo islands. The Portuguese explorers, who discovered the archipelago in 1427, only started the settlement in 1439 through a long and difficult process. Historical data report that the Portuguese crown was compelled to cede both land and privileges, not only to the Portuguese, but also to foreigners, so as to attract people to the islands. Thus the Azorean population received a significant contribution from people with genetic backgrounds other than Portuguese. This included individuals of Flemish, Spanish, French, Italian, German, Scottish and Jewish origins, as well as Moorish prisoners and black slaves from Guinea, Cape Verde and São Tomé . The firSão Miguel is the largest island of the Azores, with a population of 131,609 inhabitants  derived from around 27 generations. Several studies , 2008c hThe sample set was composed of healthy blood donors living in São Miguel Island and obtained from the “anonymous” Azorean DNA bank located in the main Hospital of the Azores archipelago, Portugal  algorithm, an iterative procedure from multilocus genotype data, with the unknown gamete phase implemented in Arlequin v3.0. Evaluation of standardized multiallelic disequilibrium coefficient, D', was performed using the Haploxt application from GOLD software. This program calculates disequilibrium statistics from haplotype data. An estimation of average D' values in each genomic region, was executed with a simple mathematical mean, for all values obtained for each marker pair. Since the extent of the three regions varied widely, a second analysis, taking into consideration a smaller genetic distance of around 3 Mb, was undertaken, so as to give further insight into LD patterns over short distances, and to guarantee the truthfulness of the drawn conclusions.loci varied from 0.821 for -DQB1 and -DPA1 to 0.934 for -B. Considering HLA loci, the overall gene diversity for São Miguel islanders was 0.843 .An analysis of the HLA alleles in 106 individuals from São Miguel Island  revealedas 0.843 . HLA allHaplotype analysis reveals a total of 200 HLA haplotypes, corresponding to 94.3% discriminatory power. Only the three most representative HLA-A-B-DRB1 haplotypes where evaluated (data not shown and provided as supplementary information by the authors upon request). A*01-B*08-DRB1*03 is the most frequent haplotype in São Miguel (8%) followed by A*02-B*44-DRB1*04 and A*02-B*44-DRB1*07, both with 1.4% frequency. Comparison with other European and African populations , we observed the same trend. Nevertheless, values had increased for both Xq13.3 and NRY. The value for HLA had diminished due to the cut-off genetic distance value being smaller than that evaluated previously. The average D'2 0.247;  was not loci, in Several studies  in Azoreloci may be under the same evolutionary forces. In general, the data corroborate previous studies . This may be explained by the difference in number of analysed loci and by the fact that Spinola et al. (2005a) used a high-resolution methodology for the HLA genotype. In addition, the same authors , do not imply strong LD for this region . The distribution of LD among Y-linked alleles is accepted as being substantially larger than that of X-linked markers, since, Y-alleles constitute/have only one-fourth of the effective population size. This assumption is confirmed by data obtained herein. Nevertheless, the highest peak identified in Figures The study of DXS1225-DXS8082 haplotype diversity in Azorean and mainland Portuguese populations, has contributed to understanding how these populations are mutually related. The X-chromosome is an important tool for historical research, since there is but one copy in males, thus facilitating the determination of haplotypes. This feature permits accurately determining LD extension, and also allows for inferring population “maternal lineages“. It is clear that, since this chromosome undergoes recombination, direct maternal lineages may not be obtained. Nonetheless, on studying DXS1225-DXS8082, which has a very small probability of recombination , we could come to some interesting conclusions. Taking into account the frequency ≥1% proposed by loci in the São Miguel population, as well as in Azoreans, will require very high marker density, where data from the HapMap project (There is some controversy regarding the amount of useful LD for mapping studies. According to  project  will mos


Artemia franciscana, transporting cysts (diapausing eggs), and favouring rapid range expansions from introduction sites. Here we address the impact of bird migratory flyways on the population genetic structure and phylogeography of A. franciscana in its native range in the Americas. We examined sequence variation for two mitochondrial gene fragments (COI and 16S for a subset of the data) in a large set of population samples representing the entire native range of A. franciscana. Furthermore, we performed Mantel tests and redundancy analyses (RDA) to test the role of flyways, geography and human introductions on the phylogeography and population genetic structure at a continental scale. A. franciscana mitochondrial DNA was very diverse, with two main clades, largely corresponding to Pacific and Atlantic populations, mirroring American bird flyways. There was a high degree of regional endemism, with populations subdivided into at least 12 divergent, geographically restricted and largely allopatric mitochondrial lineages, and high levels of population structure (ΦST of 0.92), indicating low ongoing gene flow. We found evidence of human-mediated introductions in nine out of 39 populations analysed. Once these populations were removed, Mantel tests revealed a strong association between genetic variation and geographic distance . RDA showed that shared bird flyways explained around 20% of the variance in genetic distance between populations and this was highly significant, once geographic distance was controlled for. The variance explained increased to 30% when the factor human introduction was included in the model. Our findings suggest that bird-mediated transport of brine shrimp propagules does not result in substantial ongoing gene flow; instead, it had a significant historical role on the current species phylogeography, facilitating the colonisation of new aquatic environments as they become available along their main migratory flyways.Since Darwin’s time, waterbirds have been considered an important vector for the dispersal of continental aquatic invertebrates. Bird movements have facilitated the worldwide invasion of the American brine shrimp  Since Given that continental aquatic invertebrates are unlikely to be in migration-drift equilibrium , recent Artemia franciscana (Crustacea: Anostraca), the most widely distributed brine shrimp in America, occurs in hypersaline habitats from Canada to Chile and many Atlantic islands , confirms shorebirds as an effective agent of dispersal in North America  its distribution encompasses three continental-scale bird migratory flyways spanning both North and South America , but is highly fragmented due to its habitat requirements , (2) its habitats are frequented by migratory shorebirds; Artemia is an important prey of these and other waterbirds and its cysts can be readily quantified in waterbird excreta A. franciscana throughout its known native range  using sequence variation for two mitochondrial genes (COI and 16S). Our results indicate a high level of genetic structure and endemism at a continental scale, identify the impact of human introductions and suggest a direct link between bird migratory routes  and the historical colonization of A. franciscana throughout the Americas, revealing a key role for birds in initial founder events.Here, we carry out the first comprehensive phylogeographic study of A. franciscana populations across its American geographical distribution, from Canada to Chile and Argentina, including Caribbean islands and a population from Cape Verde (We obtained samples from 39 pe Verde . Most saArtemia-specific primers 1/2COI_Fol-F and 1/2COI_Fol-R (c Oxidase Subunit I gene (COI). We also amplified and sequenced a 535 bp fragment of the 16S ribosomal RNA gene for a subset of individuals carrying different COI haplotypes to facilitate comparison with other published sequences using primers 16Sar-5′/16Sbr-3′  and 0.5 µM of each primer. PCR conditions were as follows: 94°C for 3 min, followed by 35 cycles of 45 s at 94°C, 60 s at 45°C (60–64°C for 16S locus), and 60 s at 72°C, followed by 5 min at 72°C. PCR products were purified for sequencing using ExoSAP-IT® , cleaned with Sephadex®-G50 , and labelled using the BigDye Terminator Sequencing Ready Reaction v3.1 kit (Applied Biosystems). The resulting fragments were separated on an ABI 3130xl genetic analyzer. Sequences were checked, edited, and aligned using Sequencher® v4.5 . All sequences were deposited in GenBank [accession numbers KF662951–KF663043 and KF725843–KF725869]. Available published sequences of the same gene fragments, to which we could assign a geographic origin, were also included in our phylogenetic analyses .DNA extractions were carried out on individual cysts (previously rinsed in distilled water), whole nauplii or partial adults using a HotSHOT protocol . We usedOI_Fol-R  to ampli16Sbr-3′ . PCRs wehttp://www.phylo.org), optimising free model parameters and executing 1000 rapid bootstraps. Average genetic distances between the main COI lineages – corrected by the K2P + G substitution model – were carried out using MEGA5. Additionally, to identify lineages in our COI dataset, we used the Automatic Barcode Gap Discovery (ABGD) approach (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html).Neighbour Joining (NJ) and Maximum Likelihood (ML) phylogenetic trees were inferred for both COI and 16S gene fragments. NJ trees were constructed using evolutionary distances computed using the Maximum Composite Likelihood method and 1000 bootstrap replicate tests in MEGA5 . The besapproach  using thHs (standardized haplotype diversity) for COI was computed using the program RAREFAC . A geographic distance matrix was then computed using Geographic Distance Matrix Generator v.1.2.3 . We used a population geographic distance matrix  was tested using Mantel tests on IBDWS v.3.21 . Prior te matrix  and a poST values using Kimura 2-Parameter as the evolutionary model for COI data. Environmental variables were whether the flyway overlapped with the Artemia population  and introduction history (0 or 1). We modelled spatial variables using latitude and longitude (x and y).We used canonical Redundancy Analysis (RDA) in CANOCO  to estimhttp://www.birdlife.org/datazone/userfiles/file/sowb/flyways/). These data were used as a proxy for bird movements between locations. This approach is likely to be a rough approximation to the probability of bird migration between locations, given that more precise estimates cannot be obtained due to the absence of sufficient shorebird ringing data for the whole of the Americas. Even if extensive ringing data was available, this would only estimate current bird movements, whereas climate changes since the Pleistocene are likely to have strongly affected bird movements (We used the overlap of sampled populations with the three main migratory flyways in America based on two sources (1) ovements .http://dx.doi.org/10.5061/dryad.7kb11).RDA analyses were carried out using each set of migratory flyway data  as suggested by the program, to 1.0 and used Kimura 80, identifying 12 groups with a prior intraspecific distance of = 0.0028 . When the prior intraspecific distance increases to 0.0046 (not high by any standards) then the number of partitions reduces again to 1.Introduced populations below). In addition, lineage 1 contained six closely related haplotypes only found in Mono Lake – which harbours a population often considered as a separate species, A. monica – and haplotypes found in three Colombian populations , five Mexican populations, and the Jamaican population. Two other lineages were distributed across the continental E-W divide, creating contact zones between lineages. In lineage 12, most of the haplotypes were found in Argentinean populations, but four of these haplotypes (three of them shared across populations) were found in a Chilean Altiplano population . Lineage 11, mainly Caribbean, has a few haplotypes in two Mexican populations from the Pacific side . Finally, although both lineages 1 and 11 are found in Colombia, they were not found together in any of the populations sampled. The genetic divergence between the 12 main lineages ranged from 1.8% (between lineages 1 and 4) to 6.0% (between lineages 2 and 9)  and the other split between two sub-branches along the Pacific Rim (lineages 1–7) and in Central Canada (lineage 8). Overall, there were at least 12 mtDNA lineages, most of them well supported . With th2 and 9) .A. franciscana populations. Interestingly, 16S haplotypes from Jesse Lake (Nebraska) belong to the Central Canadian lineage (lineage 8) together with Little Manitou, Muskiki, Meacham and Chaplin haplotypes.The 16S alignment contained 408 bp from 122 individuals, which collapsed into 59 haplotypes. There were two singleton indels, 63 variable sites and 43 parsimony informative sites. In contrast to the COI analysis, the NJ and ML reconstructions were poorly resolved, especially the basal branches, but most of the lineages recovered by the COI analyses were also recovered for the 16S data, with variable levels of support see . COI linH, and details of the haplotypes present in each population). The number of haplotypes per population ranged from one to seven. The highest standardized gene diversity (Hs) was found in the Mexican population MexH, whereas five populations  were fixed for a single haplotype. Most haplotypes were found within single countries, except for several haplotypes shared between some countries and the two commercial USA populations SFB and GSL , with ΦST being highly significant between all populations except for three lakes from Central Canada, plus one pair from Chile/Argentina .Mantel tests on pairwise genetic and geographic distances for populations ranging from Chile-Argentina to Canada, excluding those inferred to be introduced, revealed a strong IBD pattern  with a hp < 0.02 for all the correlations with genetic distance, whether or not geographical distance was controlled for). The effect of using different flyway data was quite small. Flyway explained 18.7–21.2% of variance in population genetic distance, depending on the flyway dataset used  division reflecting the recognized zoogeographic regions and the long isolation of the continents  extending into new breeding areas becoming available in the north and new wintering sites in the southern extreme of the continent  ntinents  – strongdent see , althougontinent . These rterbirds  and the terbirds . Althougterbirds , the hypA. franciscana agree with other studies in North America pointing to an effect of bird movements on the genetic structure of passively dispersed aquatic invertebrates , and those for A. salina in its native range  the time frame of their fragmentation can be approximated roughly using a COI molecular clock for other shrimp taxa , for which evidence of nuclear DNA (nDNA) introgression among lineages exists . FurtherNatural spread of lineages from refugial areas is likely to result in contact zones between lineages, which we expect to be sharp, as we found between lineages 1 and 11 in Colombia, where despite two lineages being present in the area, there are no sites where both co-occur.A. franciscana, some of them, like lineages 9 (Puerto Rico) plus 10 (Cape Verde) compared to all the others, or lineages 11 (Circum Caribbean) plus 12 (Argentina-Chile) compared to all the others, have genetic divergences of over 5%. Surprisingly, reproductive isolation – due to ecological specialisation and local adaptation – has only been reported between Mono Lake and San Francisco Bay populations  and distant populations as a criterion of recent human mediated introduction. Using this criterion, we identified nine populations where genetic evidence pointed to putative human introductions into Mexico, Cuba, Jamaica, Brazil and Chile. For four of these populations, the occurrence of intentional introductions in the same or nearby sites during the 1970s could be documented. Therefore, our genetic data confirms that the established A. franciscana populations in these locations are, at least partially, of introduced origin, and validates our criterion. As for the impact of introductions on native populations, four Mexican populations contained additional private haplotypes not closely related to the introduced ones, which suggests the presence of a pre-existing population before the introduction and the likely introgression of both populations with persistence of native haplotypes, although nuclear loci would be necessary to confirm this. In two populations, we found a discrepancy between our genetic results and previously published ones. The first case is the MEXLC population, which we regard as introduced (lineage 1), whereas in A. franciscana is known to hybridize even with the genetically divergent A. persimilis (Daphnia) from populations where commercialised strains were introduced.Given the strong phylogeographic structure of rsimilis . This farsimilis . DespiteThe high genetic richness found in our COI analyses, and the presence of private haplotypes belonging to lineage 1 in distant populations away from SFB and GSL, including three Colombian and five Mexican populations likely to be native, indicates that the natural distribution of lineage 1 extended further than the two commercialized populations before any human introductions took place. This was also in evidence from our 16S analyses, which included more extended geographic coverage of NW America, and revealed further sites within lineage 1 in the W USA and British Columbia harbouring private haplotypes not found in SFB or GSL. This is also consistent with the presence of private and closely related haplotypes at Mono Lake. The peculiarity and fragmentation of the habitats used by the species, the potential of salinity and varying ionic composition to act as a strong selective agent, the capacity to produce massive quantities of resting eggs that can be readily dispersed by birds, combined with the apparent limitation in modern gene flow, makes this group an ideal system for further studies testing the role of local adaptation and mass effects on reducing gene flow between populations.A. franciscana across the world, and the high genetic and ecological richness of its native populations, further population translocations should be highly discouraged, and the use of native strains as a source of cysts should be encouraged even within the Americas.Given the impact of the invasive A. franciscana phylogeography in its native range was shaped by (1) Pliocene/Pleistocene climate fluctuations, which contributed to changes in the areas available to the species, (2) historical bird-mediated colonization along migratory flyways, which shaped the East-West population division, (3) strong and persistent founder events, facilitated by high population growth rates and large population sizes, preventing further gene flow despite ongoing bird-mediated dispersal, and (4) human introductions coupled with regional bird dispersal, explaining the large but localised geographic range of the lineages derived from the commercially exploited North American populations. Our findings suggest that, at a continental scale, bird-mediated transport of invertebrate propagules does not result in substantial ongoing gene flow, but instead determines species phylogeography, facilitating the colonisation of newly available aquatic environments along bird flyways.Our analyses suggest that 10.7717/peerj.200/supp-1Table S1Click here for additional data file.10.7717/peerj.200/supp-2Table S2Click here for additional data file.


Coregonus albula) in a major northern European subarctic watercourse has resulted in a labile ecological situation in the receiving community. The ecological impact of the invasion has been thoroughly documented, but the genetics of the invasion remains to be explored. We analyzed the genetic diversity and divergence patterns among the two possible source populations from southern Finnish Lapland and three colonists populations within the Inari-Pasvik watercourse using ten microsatellite loci in order to (i) identify the most likely source of the invasion, (ii) reveal the dispersal pattern and genetic structure of the secondary expansion, and (iii) to investigate whether the initial introduction and the secondary expansion were associated with founder effects. We revealed that repeated translocation of vendace from Lake Sinettäjärvi into a tributary lake of L. Inari in 1964–1966 is the most plausible source for the invasion. Both the initial introduction and the secondary expansion were found not to be associated with significant founder effects. The secondary expansion followed a stepping stone pattern and the source and colonist populations of this expansion have undergone rapid genetic divergence within a period of 15–35 years (ca. 8–17 generations). The rapid divergence may be contributed to lack of gene flow among the source and colonist populations due to the extensive hydroelectric damming in the watercourse. Multiple introductions and substantial genetic variation in combination with the boom-and-bust population development of the species thus likely counteracted the founder effects as well as fueled the rapid establishment and expansion of this species within the Inari-Pasvik watercourse.Species invasions can have wide-ranging biological and socio-economic effects and are generally unwanted by legislation. Identification of the source population as well as the ecology and genetics of both the invader population and the receiving community is of crucial importance. The rapid invasion of a small coregonid fish vendace ( Introductions and invasions of exotic species represent a problem of global extent  following the manufacturer instructions. The individuals were genotyped at 10 microsatellite loci  arrangedNA), expected (He) and observed (Ho) heterozygosity, and the fixation index (FIS) were estimated in GenAlEx 6.41  following Rice (NRA) and private allelic richness (NRPA) per population were determined, accounting for differences in sample-sizes, using the rarefaction procedure for the smallest sample size (100 genes) as implemented in the software HP-RARE 1.0  and nodes were tested for robustness by 1000 bootstraps using Populations 1.2.32 (Langella N = 20) obtained from northern Germany was used to assist the clustering of the neighbor-joining tree. Genetic divergence between the possible source populations and the introduced L. Inari population as well as among the colonist populations were estimated by pair-wise FST ) grouping was visualized using STRUCTURE HARVESTER  in L. Inari that led to successful colonization of the Inari-Pasvik watercourse, a neighbor-joining tree was build using Nei et al.  genetic Langella  and viewLangella . A sampling Rice . Bayesiaing Rice , was useing Rice . All runNe). Changes in the effective population size will indicate the relative contribution of genetic drift  with geographic distance and years from translocation to occurrence. This was performed to reveal spatial and temporal patterns of the mechanism underlying the founder event and to estimate the time of the possible expansion from L. Inari downstream the Pasvik watercourse. We expected increased differentiation (pair-wise FST's) and difference in genetic diversity (ΔHe) the longer the distance and time since divergence. The correlation of genotype data and geographical distance  from the source population to each of the populations in the Inari-Pasvik watercourse were compared with the corresponding correlations for the three possible expansion times. The time from introduction to the founding of the L. Inari population was in all estimates set to 8 years, whereas the expansion time from L. Inari downstream the watercourse was tested for initial sighting of vendace larvae (1973), total cover of lake (1980), and peak (1989)  . We usedk (1989) , to testWe did not identify any mismatch between the original individual multilocus genotypes and the re-extracted 3–4% replicates within the present dataset. Heterozygote deficits were indicated by MICRO-CHECKER at BWF1 , C2-157 (L. Skrukkebukta), Cocl-lav06  Cocl-lav10 , Cocl-lav49 (L. Skrukkebukta), ClaTet06 (L. Kelujärvi), ClaTet13 (L. Sinettäjärvi and L. Skrukkebukta) all indicated as caused by the presence of null alleles. To test whether the loci with potential null alleles may affect the results the STRUCTURE analyses were performed as described in the Material and Methods but without the loci showing the most heterozygote deficits across populations . Neither the number of inferred clusters nor the population structure was found to differ when comparing the results with and without these loci . Given tNA) per locus per population varied from 2 to 25 and expected (He) and observed heterozygosity (Ho) per locus per population varied from 0.019 to 0.916 and 0.019 to 0.895, respectively. All populations, except L. Kelujärvi, showed significant departures from HWE associated with heterozygote deficits. Linkage disequilibrium was identified for 4 out of 45 pair-wise locus tests across populations but all returned non-significant after sequential BFC. The individual locus tests displayed 18 out of 50 and 7 out of 50 significant deviations from HWE before and after BFCs, respectively  compared to L. Kelujärvi (NRA = 4.6)  compared to L. Kelujärvi   . Private= 0.369) .FST estimates of genetic differentiation between the two possible source populations (L. Kelujärvi/L. Sinettäjärvi) and L. Inari suggest that L. Sinettäjärvi was the source population as this population pair display the lowest, although significant, differentiation . Within the Inari-Pasvik watercourse, the introduced population (L. Inari) was significantly different from the populations from the secondary expansion (L. Vaggatem and L. Skrukkebukta), whereas the populations from L. Vaggatem and L. Skrukkebukta could not be significantly discriminated  L. Kelujärvi and (ii) L. Sinettäjärvi, L. Inari, L. Vaggatem, and L. Skrukkebukta  ± SD = −7209 ± 1; Da) provided support for L. Sinettäjärvi being the most likely source for the vendace in the Inari-Pasvik watercourse. Moreover, the neighbor-joining tree also provided support for divergence between the founder population of L. Sinet-täjärvi and the introduced population in L. Inari (bootstrap support of 100%) as well as between L. Inari and the populations from the secondary expansion , which is also supported by the low, but significant FST values. Finally, the Bayesian clustering and the neighbor-joining tree grouped L. Vaggatem and L. Skrukkebukta together supporting the non-significant FST value estimated between these populations.The Bayesian clustering revealed that the individuals could be partitioned into two genetic clusters  than equilibrium heterozygosity (Heq) in the analysis of bottlenecks assuming a TPM. Similar results were revealed when assuming a SMM. All estimates of effective population sizes were associated with overlapping CIs (None of the populations showed significantly higher expected heterozygosity (ping CIs . HoweverHe), suggesting that the secondary expansion downstream the watercourse occurred late in the invasion process and that it was associated with genetic constrains. This is also in accordance with the observation of vendace in catches in Pasvik; vendace were first observed in the upper Pasvik in 1989, and then successively downstream the watercourse , capelin , and Atlantic cod (Gadus morhua), it is well known that large population sizes with high genetic variation and no obvious barriers to gene flow effectively hamper the build up of reproductive isolation (Knutsen et al. The results revealed that the populations resulting from the secondary expansion downstream from L. Inari have diverged genetically from the introduced population in a stepping stone pattern. Such a dispersal pattern maintains genetic variation and limits divergence compared to other expansion patterns (e.g., Ibrahim et al. Identifying the source population and reconstructing the routes of biological invasions are crucial for handling and managing invasive species, as well as for gaining knowledge of the ecology behind successful colonization events. However, invasions also offer the possibility to study evolutionary processes such as natural selection and life history changes as they unfold. For example, invaders may evolve in response to an altered selection regime compared to their native range (Facon et al.