Charadrius alexandrinus dealbatus), widely regarded as a subspecies of the Kentish Plover . Described as distinct in 1863, its name was consistently misapplied in subsequent decades until taxonomic clarification ensued in 2008. Using a recently proposed test of species delimitation, we reconfirm the phenotypic distinctness of dealbatus. We then compare three mitochondrial and seven nuclear DNA markers among 278 samples of dealbatus and alexandrinus from across their breeding range and four other closely related plovers. We fail to find any population genetic differentiation between dealbatus and alexandrinus, whereas the other species are deeply diverged at the study loci. Kentish Plovers join a small but growing list of species for which low levels of genetic differentiation are accompanied by the presence of strong phenotypic divergence, suggesting that diagnostic phenotypic characters may be encoded by few genes that are difficult to detect. Alternatively, gene expression differences may be crucial in producing different phenotypes whereas neutral differentiation may be lagging behind.Understanding and resolving conflicts between phenotypic and genetic differentiation is central to evolutionary research. While phenotypically monomorphic species may exhibit deep genetic divergences, some morphologically distinct taxa lack notable genetic differentiation. Here we conduct a molecular investigation of an enigmatic shorebird with a convoluted taxonomic history, the White-faced Plover ( Explaining the occurrence and maintenance of phenotypic variation has been a central theme in evolutionary biology for more than 150 years. Darwin Phenotypic characters have been the traditional taxonomic tool of choice and continue to contribute a major share to our current understanding of the Earth's zoological diversity. Starting in the 1980s, however, a molecular revolution in the field of phylogenetics added DNA to the taxonomists' toolkit Corvus crows, domesticated animal breeds and humans Disagreement between phenotypic and molecular characters between closely related taxa can be due to inadequate data or false assumptions in at least one of the data sets, or it can be real and may point to fundamental underlying biological phenomena Charadrius alexandrinus dealbatus), an enigmatic East Asian shorebird that is often thought to be a subspecies of the Kentish Plover . C. alexandrinus is a widespread breeding resident of beaches and salt pans throughout northern temperate to subtropical latitudes that has served as a model organism in ecological and evolutionary research et al.C. nivosus). In large parts of Eurasia, there is limited morphological differentiation in C. alexandrinus, and most populations are considered to belong to the nominate subspecies, apart from an isolated and morphologically distinct resident population in southern India and Sri Lanka, C. a. seebohmi. However, in East Asia, the situation is complicated by the presence of a problematic additional taxon, dealbatus. Over the past few decades, most authorities , to the south of the breeding distribution of alexandrinus, and with unknown dynamics in the area where their breeding ranges come close. Surprisingly for such a phenotypically distinct bird, dealbatus skins have been sitting in the drawers of major museums – unrecognized – for more than a century. Kennerley et al.When dealing with poorly-known taxa, especially those characterized by a history of shifting taxonomy, the availability of solid molecular data is vital in assessing genetic diversity and distinctness and contrasting these with phenotypic characters. Here we investigate the evolutionary history of the White-faced Plover . Two of these (C. ruficapillus and C. peronii) as well as samples from five Asian populations of C. alexandrinus had not previously been available for phylogenetic research on the group dealbatus, we also applied a recently proposed but promising phenotypic test of species delimitation et al.dealbatus is indeed a member of the biological species C. alexandrinus, and, if so, whether it exhibits any population genetic differentiation.Documenting the true distribution and the pronounced phenotypic differences of a “lost” taxon of plover, Kennerley he globe , we usedet al.dealbatus based on the morphometric, ecological, behavioral and plumage characters presented by Kennerley et al.et al.We used the species delimitation criteria recently proposed by Tobias et al.We obtained DNA from toe pad samples from museum specimens and blood samples from breeding and non-breeding populations of 278 plover individuals from across the globe . DNA froFor DNA samples extracted from blood we amplified three mtDNA genes: (1) a ∼400-base-pair (bp) NADH dehydrogenase subunit 3 fragment  . The concatenated Bayesian analysis arrived at a phylogram that corroborated the relationships uncovered by individual gene analyses and refined them with additional resolution . MP analdealbatus mtDNAs were nested within a large and undifferentiated clade of alexandrinus mtDNAs from across Eurasia  versus the score for a topology constrained to keep each dealbatus and alexandrinus monophyletic (−ln L C). Likelihood scores (−ln L B/−ln L C) were evaluated for the best-fit model of each locus  with solid nodal support .We also recovered high nodal support for a monophyletic diverged . C. marg support . The cloST value between alexandrinus and dealbatus was 0.012, indicating limited population genetic differentiation. In contrast, FST values between dealbatus and the three other species available for this analysis  ranged from 0.378 to 0.405, indicating a deeper level of genetic differentiation in agreement with the results of the mtDNA analyses. In addition, FST values among those three other species were pronounced (0.478 to 0.581).The Falexandrinus and dealbatus specimens (not including the other plover species) using the program STRUCTURAMA yielded a probability of 0.99 for K = 1, i.e. that samples do not aggregate into multiple population clusters . Despite the low probability for the presence of two distinct population clusters we used STRUCTURE to compute assignment probabilities for each individual in a 2-cluster scenario  possessed a roughly equal probability of being assigned to the first versus the second cluster, indicating that there was no detectable population-genetic differentiation either between alexandrinus and dealbatus, or across Eurasian sampling sites.Population structure analyses of the microsatellite data of 180 scenario . Even ifet al.Charadrius alexandrinus dealbatus, based on a suite of biometric, ecological, behavioral and plumage characters that set this form apart from nominate alexandrinus. While stopping short of elevating dealbatus to species level, the authors considered it likely that the two taxa do not interbreed and constitute distinct biological species. Our application of a phenotypic species delimitation test confirms these findings and shows that the level of phenotypic differentiation between dealbatus and alexandrinus is on a par with that of known biological species pairs.Kennerley et al.et al.et al.alexandrinus and dealbatus, which are known to co-exist on migration or in the wintering grounds while keeping geographically separate on the breeding grounds. The level to which the breeding grounds of dealbatus and alexandrinus are separated is unknown: while it is unlikely that they breed sympatrically over a wide area, their breeding ranges may abut somewhere in Fujian Province (China) or they may form a narrow hybrid zone. Under these likely scenarios, a score of up to 3 could justifiably be added to the total, which would propel the test diagnostic to 11 and well beyond the gray zone around the threshold of 7. Note that vocal differences were not scored, as none are known. In summary, the application of the phenotypic species test corroborates conclusions that dealbatus and alexandrinus are phenotypically well-differentiated taxa probably worthy of species status The phenotypic species threshold at 7 is based on a calibration using a large set of known avian sister species, but Tobias dealbatus is characterized by little genetic differentiation from alexandrinus  For the mtDNA, the Shimodaira-Hasegawa test was not expected to show a significantly poorer fit for the monophyly-constrained topology if the undifferentiated shallow topology of the alexandrinus-dealbatus clade  The application of the same three mtDNA genes uncovers considerable differentiation among closely related plovers  between closely related sibling species in the C. alexandrinus superspecies complex. While some of our microsatellites are found in conserved genomic regions, others are located in non-conserved ones ST values strongly suggest that our markers were sufficient.Our genetic markers may have been too crude or too few, failing to provide sufficient population-genetic resolution. If available in sufficient quantities, nuclear population-genetic markers can discriminate between recently-differentiated populations us clade  were mer plovers  and othedealbatus and alexandrinus may still occasionally exchange genetic material through introgression Even if they had reached species-level differentiation, dealbatus and alexandrinus may be genuine and extend to large parts of the genome. While more extensive nuclear locus sampling would be needed to rule out that we have not overlooked substantial differentiation based on marker choice, it is likely that dealbatus and alexandrinus join a list of unusual organisms that are characterized by great differentiation in phenotype but not in genotype, such as Darwin's finches, Corvus corone crows, domesticated animals, certain lizards and our own species Homo sapiens .The lack of differentiation in highly variable population genetic markers may indicate unimpeded gene flow between the two forms where their breeding ranges meet or overlap. Thus dealbatus and alexandrinus. It has been shown that speciation processes can be mediated by a select number of key genes, so called ‘genomic islands of speciation’, in the absence of any notable neutral genomic differentiation Little is known about the underlying mechanisms that may account for a conflict between phenotypic and genotypic differentiation as seen in Corvus corone corone and C. c. cornix, respectively) are phenotypically well-differentiated  but display limited genetic differentiation at 25 neutral nuclear introns On the other hand, new research into changes in gene expression e.g.  suggestsdealbatus should (1) focus on candidate loci for plumage pigmentation  ion e.g. .C. nivosus and C. alexandrinus are not conspecific C. peronii – which is not always considered a member of this superspecies  from sites across the whole of Eurasia. We had a large sample size (n>15) for each Spain, Ukraine, Turkey, the Arabian peninsula, and Taiwan , and a moderate sample size (5<n≤15) for the Indian subcontinent, Japan and north-east China, with additional samples from Xinjiang in central Asia , to assess if this extreme genetic homogeneity extends to all populations.Using both microsatellites and mtDNA genes, we sampled 208 individuals of ral Asia . C. alexle mtDNA  or microle mtDNA  differenIn conclusion, we show that morphological and genetic differentiation are decoupled between White-faced and Kentish Plovers. In addition, our work reveals novel insights into the distribution of a cosmopolitan superspecies of shorebird that has served as a model organism in evolutionary and ecological research e.g. . To fullFile S1Contains the following supporting material: Methods; Table S1. Sample identities and localities; Table S2. Evolutionary models and parameters selected for each locus by jModelTest.(DOC)Click here for additional data file.


Senecio vernalis, we created propagule mixtures of four levels of genotype diversity by combining seeds across remote populations, across proximate populations, within single populations and within seed families. In a first container experiment with constant Festuca rupicola density as matrix, genotype diversity was crossed with three levels of seed density. In a second experiment, we tested for effects of establishment limitation and genotype diversity by manipulating Festuca densities. Increasing genetic diversity had no effects on abundance and biomass of S. vernalis but positively affected the proportion of large individuals to small individuals. Mixtures composed from proximate populations had a significantly higher proportion of large individuals than mixtures composed from within seed families only. High propagule pressure increased emergence and establishment of S. vernalis but had no effect on individual growth performance. Establishment was favoured in containers with Festuca, but performance of surviving seedlings was higher in open soil treatments. For S. vernalis invasion, we found a shift in driving factors from density dependence to effects of genetic diversity across life stages. While initial abundance was mostly linked to the amount of seed input, genetic diversity, in contrast, affected later stages of colonization probably via sampling effects and seemed to contribute to filtering the genotypes that finally grew up. In consequence, when disentangling the mechanistic relationships of genetic diversity, seed density and microsite limitation in colonization of invasive plants, a clear differentiation between initial emergence and subsequent survival to juvenile and adult stages is required.Genetic diversity is supposed to support the colonization success of expanding species, in particular in situations where microsite availability is constrained. Addressing the role of genetic diversity in plant invasion experimentally requires its manipulation independent of propagule pressure. To assess the relative importance of these components for the invasion of  Arabidopsis thaliana, i.e. seedling emergence, biomass, flowering duration and reproduction. However, establishing empirical relationships between invasion success and genetic diversity is not sufficient to demonstrate a mechanistic link Genetic diversity within species and populations has been proven to have profound impacts on ecosystems by affecting community structure, nutrient fluxes and productivity, thereby affecting biotic interactions, such as resistance to disturbance and invasions Taraxacum officinale) invasiveness. They found sampling effects, i.e. the presence or absence of a particular genotype identity, to be more important for explaining invasiveness of dandelions than genotypic diversity per se.Biodiversity experiments in grasslands have shown that diversity effects may comprise complementarity effects, i.e. reduced competition due to niche partitioning Establishing a mechanistic link of genetic diversity in invasions requires accounting for confounding variables, i.e. further factors that affect both genetic diversity and invasion success. Genetic diversity in invading populations is supposed to be induced by multiple introductions and, in particular, by the number of propagules released into a new range or habitat Apart from seed availability, the availability of sites suitable for seedling establishment is another major determinant of recruitment success in plant populations Senecio vernalis Waldst. & Kit. is a suitable study organism to approach these questions experimentally. Senecio vernalis, spring groundsel, is an insect pollinated therophyte, native to West Asia and expanding to northern, central and Western Europe along railways S. vernalis is characterized by the release of large amounts of achenes per individual S. vernalis in Central Europe. Further investigations have provided evidence that populations of S. vernalis in Eastern Germany differ significantly in seed traits and germination characteristics .In the present study, we aim at quantifying the role of genetic diversity during invasions in interaction with propagule pressure at the local scale and microsite availability. S. vernalis. In particular, we tested the hypotheses that i) high propagule pressure favours the establishment of S. vernalis, ii) high levels of microsite availability increase the establishment of S. vernalis. We, iii) hypothesized that, in both experiments, mixtures with higher levels of genetic diversity display higher establishment success than mixtures with lower genetic diversity. Furthermore, the effect of increased genetic diversity was expected to display interaction effects with propagule pressure and microsite availability, and to be more prominent at low levels of propagule density and microsite availability.We established two fully randomised container experiments to analyse the relationship between 1) propagule pressure and genetic diversity as well as 2) microsite availability and genetic diversity for No specific permissions were required for sampling activities, for we sampled only material at publicly accessible locations. We confirm that these locations are not privately-owned or protected in any way. The field studies did not involve endangered or protected species.seed families (SF), within populations (WP), proximate populations (PP) and remote populations (RP). As a population pool, we referred to 18 populations located in Halle , Saxony Anhalt, Germany, and in its urban periphery, with a maximum distance of 27 km and a minimum distance of 0.22 km to each other (We created seed mixtures of different levels of genetic diversity based on the assumption that genetic diversity increases with increasing geographic distance. We expected lowest levels of genetic diversity within seed families (i.e. seeds sharing a common mother plant), intermediate levels within single populations , and highest levels of genetic diversity within mixtures of seeds sampled across several populations. For the same populations as in the current study, phenotypic variation and genetic diversity was studied as a function of habitat type by Hantsch et al. (unpubl. data). The authors found overall very high phenotypic and genetic variation and only little evidence of habitat type-dependent differentiation. In particular, there was a strong differentiation among populations in germination traits. We created four levels of seed mixtures of increasing diversity in origin and increasing distance labelled ch other , Fig. S1proximate (PP) and remote population (RP). Each of the mixture replicates was composed of five populations. For the level of remote population (RP), each one initial population out of the total pool of 18 populations was selected at random. Subsequent filling of populations was done by random selection of additional four populations out of the sub-pool of those populations with a minimum distance of >13 km to the respective initial population. Accordingly, seed mixtures of proximate populations (PP) referred to eight populations of the total population pool . This restricted pool included populations located in the central part of the total study area, thus, representing the nested core area with a population distance <7 km to each other.For the two container experiments, we set up six population mixtures each for within population level (WP), we included achenes sampled across several individuals within populations. A total of 12 populations was randomly selected as replicates. For the seed family level (SF), we created a total of 18 replicates, referring to each of three populations from the groups of proximate and remote populations with each three individuals within population as replicates. The complete set of all treatments, thus, comprised a total of 42 mixtures. For details on exact composition of seed mixtures by populations and individuals see Supplementary Data (proximate population (PP). As a consequence of random assignment from a larger population pool for the mixtures of remote populations (RP), the overlap between the experiments was much smaller with each three populations being exclusively assigned to RP mixtures of the seed addition experiment  and the microsite experiment , respectively  and remote populations (RP) separately by experiment (see proximate population (PP) than in the mixtures of remote populations  and calculated mean expected germination of ment see . In a twions RP, .S. vernalis. We used containers of 20 cm diameter and a volume of 6 l filled with a substrate mixture composed by compost and sand with a ratio of 2∶1. Each container was planted with seven individuals of Festuca rupicola Heuff (Poaceae) that served as constant matrix in this experiment. Festuca rupicola individuals were raised by seeds from seed material collected in the neighbourhood of S. vernalis populations and planted as three week old seedlings a few days prior to the experiment. For seed addition of S. vernalis, we applied three levels of seed densities with 30, 60 and 90 achenes for each of the 42 seed mixtures. The intermediate level of seed number corresponded to a density of 2000 seeds m−2 following Turnbull et al. In the first experiment, we studied the role of propagule pressure, i.e. seed density, and genetic diversity for establishment of All containers were positioned randomly in one experimental plot in the Botanical Garden of the Institute of Biology/Geobotany  and regularly watered to ensure optimal water availability during the experiment. The experiment ran for 15 weeks from May to September 2009.S. vernalis early life-stage development. For this approach, we proceeded as in the first experiment but manipulated the matrix density of F. rupicola while keeping the seed density level constant at the intermediate level of 60 seeds per container. We created three levels of microsite availability by planting 5, 10 or 15 Festuca individuals per pot. In addition, we used matrix-free containers as control pots for open soil establishment. While all seed mixtures applied were anew chosen at random as described above, mixtures of genetic diversity levels were exactly repeated across all four matrix density levels. Container experiment II lasted 12 weeks from June to September 2009 and included 168 pots.A second experiment was designed for analysing effects of microsite availability and genetic diversity of propagules on establishment and growing success of S. vernalis individuals in each container. In the following we refer to the number of individuals after 1 week and after 12 weeks as “initial abundance” and “establishment”, respectively. Maximum abundance per container was assessed across all monitoring dates. In addition, individuals encountered were assigned to size classes to quantify the demographical development of individuals over time. Individuals were classified either as seedling , as small individuals , or as larger individuals . We calculated log ratios of size classes to assess the demographical development of S. vernalis during the experiment. Log size ratios were calculated as the logarithm to the base 10 of the proportion of summed individuals of size classes 2 and 3, divided by size class 1, i.e. the relative proportion of large individuals to small individuals (at the seedling stage). The more positive the log size ratio, the higher the abundance of large individuals compared to small individuals. For calculating log size ratios all values were added on by 1 to avoid division by zero.In both experiments, during the first four weeks, monitoring occurred at weekly intervals, but with increasing monitoring interval lengths thereafter. At each monitoring date, we assessed the total abundance of S. vernalis samples were dried for 48 h at 60°C for dry mass determination to yield overall productivity of mixtures.At the end of the experiments, containers of both studies were harvested. proc GLIMMIX), using Gaussian error distribution and identity link functions for both data sets and included the class variables diversity and seed density in the first container experiment and diversity and microsite availability in the second experiment as fixed factors. Calculated expected germination of a particular seed mixture applied was included as covariate into the model to account for genotype identity effects on maximum abundance calculated across all monitoring dates. In addition, we tested for effects of presence/absence for those populations in the RP mixtures that were exclusively assigned to either the Container experiment I or II. We included Pop 1, Pop 5, Pop 13 and Pop 3, Pop 4, Pop 6 in the models testing for additional covariate effects in the seed density experiment and the microsite availability experiments, respectively.Statistical analyses were done with SAS 9.2 S. vernalis. Covariation due to repeated measurements was modelled with the random statement in proc GLIMMIXWe ran an additional analysis on abundance data of all monitoring dates including ‘time’ as additional random factor to analyse cumulative establishment success of The tests of fixed effects are based on type III SS. We are aware that with applying the same model to different response variables we performed multiple tests. However, we did not apply Bonferroni correction for controlling potential type I errors, given some debate on the conservatism of this correction method Senecio vernalis individuals  displayed a more balanced and significantly higher proportion of large than of small individuals compared to seed applications from separate seed families  which displayed an intermediate log size ratio. In addition, variation in initial abundance and establishment increased with increasing diversity levels as indicated by larger Whisker ranges in There were only a few effects of genetic diversity. Log size ratio was affected by genetic diversity ; mixturelies SF; . Thus, wThere were no significant interaction effects between levels of seed density and genetic diversity in any of the variable studied.Presence/absence of exclusive populations in the RP mixtures affected the overall results only marginally, but presence of single populations accounted for additional variation depending on the variable tested .Festuca density in the recipient matrix  in the course of the experiment might hint at the possibility that positive and negative density effects have compensated each other although we cannot rule out the possibility that a wider range of seed density levels might have provided stronger effects. However, we tested only one beneficial environment, while the direction of density effects will become apparent only across a gradient in environmental adversity The initial high abundance of S. vernalis should strongly depend on the competition intensity brought about by the different Festuca matrix density treatments. However, in our experiment, initial abundance was completely unaffected by the treatments, thus displayed no evidence of microsite limitation. Moreover, at later stages, seedling abundance suffered from high mortality in the open soil, i.e. without Festuca. In contrast, these fewer individuals displayed higher individual performance and grew up to larger sizes finally resulting in increased total exotic biomass. Open soil situations, apparently, reflect more suitable sites for individual growth of S. vernalis, whereas initial facilitation for survival within the Festuca environments seems to be at the expense of competitive constraints at later stages. The sensitivity to such competitive changes during life stages, as found for S. vernalis, corresponds to theories on temporal shifts in plant interactions Acer negundo seedlings by increased survival in non-Acer stands S. vernalis abundance and performance was dependent on availability of bare ground, as provided by disturbance events. Numerous studies on invasive plant species confirm the role of disturbance on initial seedling emergence, such as the seed addition experiments with two non-native weeds of North American pastures and roadsides, Carduus nutans and C. acanthoidesS. vulgaris, congeneric to our study species, that the rate of spread was sensitive to both the size and distribution of gaps in swards S. vernalis to colonize such gap-dominated ruderal habitat types.We expected that the effect of seed addition of S. vernalis with, however, changing relative importance across subsequent early life stages. Initial abundance was mostly triggered by propagule pressure and less by microsite characteristics. Similarly, genetic diversity mattered only to a minor degree at the earliest life stages, such as for germination. However, effects of genetic diversity apparently seemed to be more decisive with regard to the probability of continued establishment, in combination with selection effects of best performing genotypes for consecutive growth. It would be promising to test for these effects on more species in replicated regions to strengthen generalization on these shifts in mechanisms. The differentiation of invasion processes into a first stage of predominant importance of propagule pressure and a second one with increasing importance of adaptation is mirrored in numerous attempts of describing the courses of invasion Click here for additional data file.Figure S2Mean expected germination of Senecio vernalis of mixtures. Values are provided for diversity levels remote populations (RP) and proximate populations (PP) separately by experiment. Experiment 1 = seed addition, Experiment 2 = microsite limitation, with n = 6 for each diversity level and experiment. Different letters indicate significant differences between levels of main factors according to the Scheffé test. Statistical details following ANOVA. Diversity F1,20 = 6.41 p = 0.020; Experiment F1,20 = 0.506, p = 0.485¸ Diversity×Experiment F1,20 = 0.991, p = 0.331.(TIF)Click here for additional data file.Table S1Location and habitat type affiliation of sampled S. vernalis populations in Eastern Germany.(DOC)Click here for additional data file.Table S2Details on exact composition of seed mixtures of S. vernalis in the four diversity levels applied by population and individual identity in the two container experiments. Ind shows the identity of seed families, “-” indicates pooled samples of a population without exact knowledge of seed family identity. Pop indicates the identity of the population referred to in the mixtures. All mixtures were exactly replicated for application to different seed density levels (Experiment 1) and Festuca rupicola density levels (Experiment 2), respectively. Potential germination capacities give the expected germination for populations and seed families determined in a previous study . Potential germination capacities of a particular seed mixture applied were calculated as the mean germination capacities across the populations involved in the respective mixture.(DOC)Click here for additional data file.Table S3Experiment 1: Propagule pressure×genetic diversity. Repeated measures GLM for number of individuals Senecio vernalis with fixed factors diversity, seed density and time. N = 126. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold p values indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S4Experiment 1: Propagule pressure×genetic diversity. GLM for maximum abundance of Senecio vernalis, including expected germination as covariate in the model. N = 126. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S5Experiment 1: Propagule pressure×genetic diversity and population identity effects. GLM for response variables of Senecio vernalis testing for presence/absence effects of those populations  exclusively assigned to the highest diversity level (RP = remote populations) in this experiment only. Initial abundance reflects the number of Senecio individuals after 1 week. Establishment refers to the number of Senecio individuals after 15 weeks. Abundance and total biomass were log10(x+1)-transformed prior to analysis. Log size ratio gives the proportion of number of large individuals compared to those of small individuals as logarithm to the base 10. N = 126. The results of the Scheffé post hoc tests indicate the direction of significant differences between categories of each factor. 90, 60, 30 indicates seed density levels applied. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S6Experiment 2: Microsite availability×genetic diversity. GLM for number of individuals, including time as additional fixed factor in the model. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S7Experiment 2: Microsite availability×genetic diversity. GLM for maximum abundance of Senecio vernalis, including expected germination as covariate in the model. N = 168. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05). GLM for maximum abundance of Senecio vernalis, including expected germination as covariate in the model. N = 168. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.Table S8Experiment 2: Microsite availability×genetic diversity and population identity effects. GLM for response variables of Senecio vernalis testing for presence/absence effects of those populations  exclusively assigned to the highest diversity level (RP = remote populations) in this experiment only. Initial abundance reflects the number of Senecio individuals after 1 week. Establishment refers to the number of Senecio individuals after 12 weeks. Abundance and total biomass were log10(x+1)-transformed prior to analysis. Log size ratio gives the proportion of the number of large individuals compared to those of small individuals as logarithm to the base 10. N = 168. The results of the Scheffé post hoc tests indicate the direction of significant differences between categories of each factor. 15, 10, 5, 0 indicates numbers of Festuca individuals as density levels applied. Seed mixtures: RP = remote populations, PP = proximate populations, WP = within population, SF = seed family. The tests of fixed effects are based on type III SS, p values and degrees of freedom of numerator (df Num) and denominator (df Den) are shown. Bold numbers indicate significant effects (p<0.05).(DOC)Click here for additional data file.


Treated effluents from wastewater treatment works can comprise a large proportion of the flow of rivers in the developed world. Exposure to these effluents, or the steroidal estrogens they contain, feminizes wild male fish and can reduce their reproductive fitness. Long-term experimental exposures have resulted in skewed sex ratios, reproductive failures in breeding colonies, and population collapse. This suggests that environmental estrogens could threaten the sustainability of wild fish populations.Ne) of wild roach (Rutilus rutilus L.) living in English rivers contaminated with estrogenic effluents. Ne was estimated from DNA microsatellite genotypes using approximate Bayesian computation and sibling assignment methods. We found no significant negative correlation between Ne and the predicted estrogen exposure at 28 sample sites. Furthermore, examination of the population genetic structure of roach in the region showed that some populations have been confined to stretches of river with a high proportion of estrogenic effluent for multiple generations and have survived, apparently without reliance on immigration of fish from less polluted sites.Here we tested this hypothesis by examining population genetic structures and effective population sizes (Ne of up to 65% is still possible for the most contaminated sites because of the wide confidence intervals associated with the statistical model.These results demonstrate that roach populations living in some effluent-contaminated river stretches, where feminization is widespread, are self-sustaining. Although we found no evidence to suggest that exposure to estrogenic effluents is a significant driving factor in determining the size of roach breeding populations, a reduction in  Approximately two-thirds of the world’s freshwater is used to dilute wastewater discharges. The demand for freshwater is expected to rise by 70% by 2050  driving 2) and estrone (E1) from human excretion; 17 alpha-ethinylestradiol (EE2), a component of the female contraceptive pill [There is substantial evidence showing that experimental exposure of fish to WWTW effluents and the estrogens they contain can result in adverse health effects, including effects on reproductive development and breeding output. This has led to concerns that freshwater fish populations might also be affected with cascading consequences for freshwater ecosystems. Feminization of male fish is widespread in stretches of rivers downstream of WWTW outfalls as demonstrated in studies using wild ,4 and caive pill ; and a live pill , alterative pill , decreasive pill , alteredive pill  and chanive pill .Concern about estrogens in rivers in the United Kingdom drove a £40 M programme to evaluate the efficacy of various tertiary treatment processes in the removal of estrogens ,15. Impl2 (between 3 to 6 ng/L) in the aquatic environment could be a threat to the sustainability of fish populations. For example, a controlled exposure of an entire lake to EE2 in Canada resulted in the collapse of the fathead minnow  population within three years [Rutilus rutilus L.) to an undiluted WWTW effluent in large tanks resulted in an all-female population [2 at concentrations below 1 ng/L during the period of sexual development, have been shown to result in feminized gonads in roach [A critical question is whether chronic exposure to estrogenic effluents negatively impacts the viability of wild fish populations, but this has been difficult to address experimentally as it requires controlled experiments extending over periods of several years. Limited studies suggest that high concentrations of EEee years . Likewisee years -20 and apulation . It is npulation . Althougpulation , exposurin roach  and decrin roach . Evidencin roach  and sevein roach .Ne) – related to the breeding population of fish – may be decreased without necessarily impacting on population sizes [Ne can be several orders of magnitude smaller than census population sizes. One study found two populations of the exploited New Zealand snapper (Pagrus auratus) to have values of Ne less than 1,000 despite adult census population sizes in the millions [Morone saxatilis), a freshwater fish species, found cohorts to consist of a few, full sib families, despite an adult census size of over 300,000 [Ne influences long-term sustainability as it determines the rate at which genetic diversity is lost from a population through genetic drift [Ne, however, may act to increase the chances of losing some lethal or sub-lethal mutations through genetic purging.While these studies suggest that exposure to high concentrations of effluent could threaten the viability of fish populations, aggregates of cyprinid fish, including roach, are often found in effluent contaminated rivers. However, numbers alone may provide a misleading assessment of population sustainability as these could be sink populations maintained by substantial immigration from less contaminated locations where successful reproduction still occurs. Likewise, effective population sizes , a fish species in which feminization is widespread, in southern England. Southern England has some of the highest proportions of WWTW effluent in rivers known globally, and numerous weirs and locks which potentially confine fishes to heavily polluted stretches of river. We have used this system to evaluate whether stretches of river highly contaminated with estrogenic effluents have impaired breeding populations of roach. To do this we undertook analysis of population genetic structures of roach in the region using DNA microsatellite analysis. Microsatellite data were also used to calculate Ne and estimate levels of gene flow to determine the extent to which these populations are maintained through immigration of fish from less contaminated stretches of river.Understanding the impact of estrogenic effluents on the sustainability of fish populations is, therefore, paramount, but has been limited to date by the logistical challenges involved in undertaking long-term exposures to realistic effluent concentrations, and understanding the demographic history of wild fish populations at highly contaminated sites. In this study, we examine evidence for population impacts on wild roach (AR) ranged from 6.8 to 8.9, and expected heterozygosity (He) ranged from 0.69 to 0.75 , F = 2.1398, P = 0.00014) and observed heterozygosity (Ho)  = 1.8677, P = 0.0017) among samples were detected. Roach sampled at two relatively unpolluted sites (LamSha and LeeHUS’95) exhibited comparatively low AR and LeeHyd’95 exhibited comparatively relatively low Ho [see Additional file He were found. Additionally, there was evidence for genetic bottlenecks at two relatively unpolluted sites sampled within the rivers Arun (AruHUS) and Lee (LeeHUS), and at a polluted site in the Lee (LeeWhe)  from 32 different geographic locations in England were genotyped  indicated the majority of variation was partitioned among individuals within river locations, with river location accounting for a small 2.27%, but highly significant proportion of the genetic variation  for the study as a whole, while 262 of the 325 pairwise FST comparisons in the catchment were highly significant. There was a weak, but significant, relationship between genetic and geographic distance  within the catchment, indicating a tendency for individuals to produce offspring with fish from nearby populations rather than distant populations [see Additional file Despite Thames catchment roach appearing not to constitute a single genetic unit, distinct from roach in other regions, the study did find evidence for significant genetic structuring in roach populations within the Thames catchment. This suggests the existence of local subpopulations exchanging a limited number of effective migrants  rather than panmixia . For example, average Despite the proximity of some populations in the PCA and tree, we also found significant genetic differentiation between samples from some neighbouring stretches of the same river, sometimes occurring over small distances of separation (<10 km), for example, within the upper Lee (see below), between the Lee and the Stort, between the Blackwater and main Thames, between the Lambourn and the Kennet and within the Anglian Blackwater. In other cases, despite the separation of sampling locations by in-river impoundments such as weirs, we found no significant genetic differentiation between sites, for example, within the Stort, the main Thames, the Kennet, the Arun, the Nene and the Trent. Thus, patterns of within-river genetic structure differed between river stretches. For some other fish species analysis of genome-wide SNP data has provided greater resolution in population structure than that achievable using microsatellite data , and it Ne calculated from the microsatellite data using the approximate Bayesian computation method (Ne(ABC)), ranged from 54 to 301 for each sample, with higher precision for small Ne estimates  and predicted E2 equivalents (E2Eq), a measure of total estrogenicity of the river water due to contamination by sewage effluent , F = 0.7468, P = 0.40) or for an interaction between sample site and estrogen exposure  = 1.9954, P = 0.14) across the 28 sample sites where no recent restocking had occurred and had sufficient sample sizes for robust Ne calculation. However, the 95% confidence intervals (CI) for the model coefficient indicated Ne(ABC) could decrease by a maximum of 5.6% for each incremental increase in exposure of 1 ng/L E2Eq, or 65% at 11.6 ng/L E2Eq, equivalent to the most polluted river stretch included in this study. The inclusion of roach density as an additional covariate within the model also produced a non-significant result  = 1.3966, P = 0.26), albeit for a reduced number of sites (19). Similarly, there was no significant correlation between the other variables included in the statistical analyses  with Ne(ABC). This analysis makes the assumption that immigration of fish from remote sites is limited and this is discussed below.Estimates of Ne in roach sampled from more contaminated stretches of river compared to those sampled from less contaminated sites; all estimates above 6 ng/L E2Eq were below 100, whereas there was greater variation (54 to 301) where E2Eq was below 6 ng/L  ranged from 36 to 145 and also showed no relationship with E2Eq , and between these samples and two from its tributary, the Stort (FST ≥0.015) shown in Table A2 [Ne(ABC) estimates for these polluted sites in the upper Lee ranged from 70 to 84 (95% CI: 50 to 127) compared to only 54 (95% CI: 42 to 82) for the upstream uncontaminated location, suggesting no substantial impact of the effluent on the effective population size of these roach.The average proportion of effluent in the upper Lee downstream from Harpenden and East Hyde WWTWs ranges from 28% to 70% in different stretches. Significant genetic differentiation was detected between fish sampled from four of the five locations in this stretch of river  could not be ruled out, due to the wide 95% confidence intervals associated with the statistical model. Moreover, our analysis included relatively few samples from rivers in the highest risk category, largely because these sites are rare.In this study, analyses of population genetic structure bilities ,26. Bothbilities ), with pNe(ABC)[FST <0.002, Table As with any modelling exercise, this analysis makes assumptions that may affect the interpretation of the results. One of these assumptions is that migration between sites with different pollution profiles is limited over two to three generations, the time frame likely to have the greatest influence on Ne(ABC). This waNe(ABC). HistoloNe(ABC). This deNe(ABC) was relatively minor. Approximately 500,000 hatchery-reared roach just over one-year-old  have been introduced into the Thames catchment since 2000; broodstock for hatchery fish originate from the river Trent. The influence of restocking activities on Ne(ABC), however, is likely to be relatively minor, as the sites sampled in this study were separated by major physical barriers from sites where these introductions had occurred. Moreover, introductions prior to 2000 are unlikely to have had a large influence on Ne(ABC) as this is primarily affected by the size and variance in reproductive success of the parental generation, which would have spawned between 2004 and 2007 for most of the samples in this study. However, we cannot exclude some influence of introductions prior to 2000 as some of the summary statistics used to calculate Ne(ABC) are known to be affected by demographic processes over a longer time period [A second assumption is that no restocking of the rivers sampled had occurred or that the effect of restocking activities on e period . The effe period . Using oNe of fish populations, it suggests that roach populations can be self-sustaining despite exposure to estrogens over several generations. These findings are consistent with the fact that the prevalence of male fish with moderate to severely feminized gonads  is generally less than 10% in English rivers [Ne by reducing the reproductive dominance of large older males in estrogen-contaminated rivers. While the effects on females are less well studied, female roach exposed to an undiluted effluent for three years in large tanks were able to breed, despite the fact that this exposure caused complete gonadal feminization of males [While this study does not exclude the possibility that estrogenic effluents reduce h rivers ,25,26,46of males ; similar2/L [2 has been measured up to approximately 4 to 8 ng/L in English WWTW effluents [2 was detected in only 21 of 135 water samples from the Lee, although occasionally reaching 4 ng/L [2Eq and would be below 21 ng/L for 90% of the time. Only 1% to 3% of 10,313 individual river reaches in the UK receiving WWTW effluent were predicted to have average E2Eq >10 ng/L, and, of these, many are ditches composed almost entirely of sewage effluent [2 is approximately 10 times less potent than EE2 in inducing gonadal feminization in fish [et al. [The results of this study on wild roach populations seem to contrast with studies that have assessed population risk through long-term exposures to estrogens, where exposure to concentrations between 3 and 6 ng EE2/L -20 or to2/L  resulted2/L . The mos2/L . While Effluents ,48, for ffluents ,49, and g 4 ng/L . Consideeffluent . As E2 i in fish ,50, it i [et al.  recentlyThe population genetic structure of roach in southern England observed in this study may have been influenced by historical biogeography, migratory behaviour, human translocations and in-river barriers. Roach can be highly mobile and can migrate over 10 km, particularly in the spawning period April to June, if migration is not obstructed ; additioNe of roach populations and predicted exposure to estrogens, although because of the wide confidence intervals, a reduction in Ne of up to 65% is still possible at the most contaminated sites.Despite the widespread feminization of male roach in effluent-contaminated rivers of southern England, using nuclear DNA microsatellites we were able to identify some populations that have been confined to stretches of river with moderate to high exposure to estrogenic effluents over multiple generations. We also found no evidence of a correlation between the Southern England, particularly the region within the Thames catchment, was chosen for this study for four reasons. Firstly, it is a densely populated region with relatively low rainfall and, therefore, includes some river stretches with some of the highest concentrations of WWTW effluents in the United Kingdom . SecondlRoach was selected as the study species because it is native and widely distributed in the United Kingdom, including in rivers polluted with WWTW effluents. Additionally, widespread feminization has been reported in wild populations and with a proven association with exposure to estrogenic effluents ,19,21,57HO) and expected heterozygosity (He) using GenAlEx 6 [AR) was calculated using Fstat v2.9.3 [FST, calculated using Arlequin 3.5 [D, Dest[FST estimates was assessed based on 10,000 permutations. AMOVA was performed using Arlequin. In order to test whether fish are more likely to produce offspring with local mates, compared to mates in geographically distant locations within the Thames catchment, isolation by distance analysis was performed using the Mantel test [A2 [FST values . See Additional file To understand the extent to which roach populations are restricted to various stretches of river, several approaches were used to investigate population genetic structure. We analysed microsatellite loci variation in 1,769 fish sampled between 1995 and 2011. Each fish was genotyped at between 14 to 19 microsatellite loci. Microsatellite genotypes are provided in Additional file enAlEx 6 ; allelict v2.9.3  – see Adt v2.9.3 ,61 was uquin 3.5  and Jost [D, Dest, calcula [D, Dest. The sigtel test  in GenAltel test . Genetictel test , PCA in tel test  and a Batel test . Finallytest [A2 ,68 was uNe), which relate to the number of breeding fish and skews in breeding success, were estimated using the microsatellite genotypes. We compared Ne from sites ranging from little/no upstream WWTW effluent inputs to those where the majority of the flow can comprise WWTW effluent. Two single sample (generation) methods, that use different aspects of the microsatellite data, were used to estimate Ne for each population; the Approximate Bayesian Computation (ABC) method using ONeSAMP 1.2 [Ne(ABC); and the sibling assignment method (SA), Ne(SA)[Ne, which are calculated from the change in allele frequencies between generations, were also estimated for sites where fish had been sampled more than once using TempoFs [To test whether WWTW effluents substantially reduce the size of breeding populations, effective population sizes , Ne(SA). Tempora TempoFs  and NeEs TempoFs . For furNe(ABC). For further details see Additional file GLM were used to examine the relationship between predicted exposure to estrogenic effluents and AMOVA: analysis of molecular variance; ANOVA: analysis of variance; AR: allelic richness; CI: confidence interval; E1: Estrone; E2: Estradiol; EE2: 17 alpha-ethinylestradiol; GLM: Generalized Linear Models; He: expected heterozygosity; HWE: Hardy-Weinberg equilibrium; MCMC: Markov chain Monte Carlo; MEGA: Molecular Evolutionary Genetic Analysis; Nb: effective number of breeders; Ne: effective population size; Ne(SA): effective population size estimate calculated using the sibling assignment method; Ne(ABC): effective population size estimate calculated using the approximate Bayesian computation method; PCA: principal component analysis; SNP: single nucleotide polymorphism; WWTW: Waste Water Treatment Work (WWTW).The authors declare that they have no competing interests.PH, EN, JS, SJ, JS, JS and CT participated in the design of the research. RW modeled estrogen exposure in rivers. PH, EB and EN participated in data collection, analysis of microsatellite data and statistical analysis. PH, EN, EB, RW, JS, SJ, JS and CT wrote the paper. All authors read and approved the final manuscript.PH, the corresponding author, is a molecular ecologist and evolutionary biologist, and is a Research Fellow at the Biosciences Department at the University of Exeter.Details of microsatellite genotyping methods, population-genetic analysis and statistical analysis.Click here for fileGenetic diversity estimates for each locus in each population.Click here for fileGraph showing statistical differences in genetic diversity among roach Rutilus rutilus populations.Click here for filePairwise FST and Dest among roach population samples.Click here for fileMultidimensional scaling plots of pairwise DA distances [.stances .Click here for fileStructure analyses plots using the locprior model and analysis of optimum number of genetic units in Structure Harvester [.rvester .Click here for fileCorrelation between genetic distance and geographic distance (km) between pairs of sites for 24 population samples from the Thames catchment.Click here for fileMicrosatellite genotypes for 1,769 roach genotyped in this study.Click here for file


We present theoretical arguments that suggest plankton species may respond rapidly to selection on mildly beneficial mutations due to exceptionally large population size, and consider the circumstantial evidence that supports our inference that selection may be particularly important for these species. We also review all primary population genetic studies of open ocean zooplankton and show that genetic isolation can be achieved at the scale of gyre systems in open ocean habitats (100s to 1000s of km). Furthermore, population genetic structure often varies across planktonic taxa, and appears to be linked to the particular ecological requirements of the organism. In combination, these characteristics should facilitate adaptive evolution to distinct oceanographic habitats in the plankton. We conclude that marine zooplankton may be capable of rapid evolutionary as well as ecological responses to changing ocean conditions, and discuss the implications of this view. We further suggest two priority areas for future research to test our hypothesis of high evolutionary potential in open ocean zooplankton, which will require (1) assessing how pervasive selection is in driving population divergence and (2) rigorously quantifying the spatial and temporal scales of population differentiation in the open ocean.Open ocean zooplankton often have been viewed as slowly evolving species that have limited capacity to respond adaptively to changing ocean conditions. Hence, attention has focused on the ecological responses of zooplankton to current global change, including range shifts and changing phenology. Here, we argue that zooplankton also are well poised for Recent attention has focused on the ecological responses of open ocean zooplankton to current global change, including range shifts and changing phenology. Here, we argue that marine zooplankton also are well poised for evolutionary responses to global change. The oceans are changing on a global scale and, in some cases, at rates greatly exceeding those observed in the historical and recent geological record  can quickly drive beneficial mutations to fixation at population sizes typical of marine zooplankton , whereas genetic variation in Homo sapiens and Arabidopsis  conformed to a background selection model, with a large number of segregating deleterious polymorphisms . In these cases (and others), barriers to dispersal in the open sea occur at oceanographic regions with sharp transitions in both biotic and abiotic properties , while the most oceanic lineage entirely lacked population genetic structure across the same sampling sites. Similar ecophysiological mechanisms likely control dispersal among populations of open ocean zooplankton, though there are no studies that have examined this for truly oceanic taxa. What constitutes optimal habitat will vary across species, with the nature of dispersal barriers highly dependent on the ecological niche of the organism. Therefore, it is more appropriate to consider population genetic structure a species-specific trait that varies across holoplanktonic taxa, as does any other ecological or life history trait, than to view the zooplankton as monotypically ‘high dispersal’ species.Third and finally, increasingly it appears that population genetic structure is species-specific in marine zooplankton, with closely related species often showing very different patterns of differentiation across the same pelagic habitats Goetze . These rThis emerging view of the complexity of dispersal in marine zooplankton extends a number of observations that have been made in other marine species to the pelagic habitat. Panmixia was once expected for all marine species with large population size and a planktonic phase of the life history , we hypothesize that open ocean zooplankton have large adaptive potential and may be capable of strong evolutionary responses (genetic adaptation) to changing ocean conditions. These responses could be rapid, occurring on ecological time scales  technologies have made possible the rapid generation of large-scale sequence data from nonmodel organisms at reasonable cost. We expect that these technologies . One cost-effective approach to examining adaptive variation in natural populations is to study functionally important traits related to, for instance, morphology or life history. Differentiation at these quantitative traits is typically expressed as In order to rigorously quantify the spatial and temporal scales over which differentiation occurs in the open ocean, we need to substantially increase the types and amounts of data that are collected. First, it is critically important to be able to compare genetic results across marker types in order to obtain rigorous estimates of dispersal through Bayesian, Maximum Likelihood, or coalescent analytical techniques (e.g., Pritchard et al. Finally, a wide range of questions on the fundamental basis of evolutionary change in marine zooplankton remain to be addressed. We hope that the ideas outlined here, in combination with the suite of high-throughput genomics techniques now available for development of genome-wide markers in nonmodel species will catalyze research in this field.


S. s. semitorques and S. s. cinereicapillus but not detected among geographic populations of S. s. semitorques. Divergence time of the two subspecies was dated back to 87 Kyr which is congruent with the interglacial MIS 5. A weak phylogeographic structure due to strong gene flow among geographic populations was identified in this species, suggesting complex topography of South China has not formed barriers for this species.Recent phylogeographical studies indicated that glacial oscillations played a key role on the phylogeographic pattern of extant species. As most studies have previously been carried out on heavily ice-covered regions, such as in European and North American regions, potential effects of climatic oscillations on species that are distributed on ice-free regions are less known. To address this, we investigated the phylogeographic pattern of an avian species endemic to South China, which was not glaciated during the Pleistocene glaciations. By using 2142 bp mitochondrial DNA, we identified 89 haplotypes defined by 39 polymorphic sites. A combination of high haplotype diversity (0.786–1.00) and low nucleotide diversity (0.00132–0.00252) was detected among geographic populations. Explicit genetic divergence was observed between  It is widely accepted that glacial oscillations during Pleistocene shaped the geographic distribution and genetic pattern of many plant and animal species on previously glaciated regions –3. For sIn contrast, the influence of recent climatic oscillations due to glacial cycles on the phylogeographic patterns of species in unglaciated regions, especially in East Asia, remain poorly understood ,6,7. RecEast Asia harbors one of the most diverse temperate fauna as it was never heavily covered by ice-sheets ,10. DuriSpizixos semitorques). The Collared Finchbill is endemic to south China, with two described subspecies: S. s. semitorques and S. s. cinereicapillus. S. s. semitorques cover a large area of South Mainland China, while S. s. cinereicapillus is only found in Taiwan. The Collared Finchbill distributes mainly in shrubbery, bamboo forests, and grassy areas on hilly and flat terrain below 1000 m, and its distribution is roughly between 20° N and 30° N in latitude, and 105° E and 120° E in longitude [S. semitorques might enrich our understandings about the role of climatic changes during the Pleistocene glaciations on the phylogeographical formation of birds endemic to South China.To address this, we used two fragments of mitochondrial DNA to study the phylogeographical structure of a passerine species, the Collared Finchbill  and partial control region and tRNA-Glu (1165 bp) was obtained. Sequences aligned unambiguously have no gaps and there were no nonsense codons in protein-coding regions. Sixty-nine variable sites define 89 haplotypes. Most haplotypes are singletons, while there are only a few common haplotypes (10 see Attachment 1) shared by several geographical populations . The hapD values detected negative values for all populations, but none are significant  and GSWX (<0.01) populations. Fu and Li’s D tests showed negative values for SXFP (<0.05), HNYJ (<0.01) and GZGD (<0.01) populations. No population showed significant negative value in all three neutrality tests.Neutrality test was conducted on geographic populations which contained at least five individuals. Tajima’s nificant . Fu’s FsS. s. semitorques. However, AMOVA could not find a pattern with significant variation within any grouping. This indicates that there is no substantial genetic subdivision across subspecies on the mainland of China. Most of the variation is accounted for within-population variation, as could be expected given the large number of singletons. There is no correlation between Fst genetic distance and geographic distance  in S. s. semitorques . The majority of the variations , which is widespread in southern China and often flocks in the medium and understory of tropical rain forests and subtropical broadleaf evergreen forests [S. semitorques usually inhabits the canopy and edge of shrub land and has a larger body size [S. semitorques to have vagility and adaptability superior to A. morrisonia. The phylogeographical structure of A. morrisonia showed a deep lineage divergence while S. semitorques only have a shallow differentiation among geographical populations. Palynological and palaeoclimatic data suggest that the vegetation in East Asia in 35 Kyr ago similar to observed today might be ascribed to high precipitation, especially at middle or low latitudes [S. semitorques might stay relatively stable in certain areas of southern China.Unlike  forests ,25, S. sody size  which miatitudes –28. The S. semitorques from then on.East Asia has experienced several cyclical changes due to the sea level rising and falling. These sea level fluctuations started in the Early Miocene (24 Myr BP), and became more frequent after 10 Myr, and particularly during the Quaternary. Such changes of sea level led to occasional connection/disconnection between mainland and Taiwan. Divergence time of the two subspecies dated back to 87 Kyr BP while the TMRCA was estimated to 0.44 Myr BP. This result implied that the rising sea level did not effectively block the two subspecies for a long time until one of the warmest interglacial marine isotope stage 5 MIS 5:130–74 Kyr) . The sea–74 Kyr .A total of 120 samples of Collared Finchbill were collected from 14 localities from 2003 to 2009, covering most of its distribution range in southern China . Liver/mS. s. semitorques and one location for S. s. cinereicapillus  network by program Network4.6 . Mantel H (=2Nel), M (=Nem = number of migrants between populations per generation), and T . Each analysis used an infinite-site model and 3000,000 generations of Markov chain Monte Carlo run and the first 500,000 generations was discarded as burn-in. Priors of maximum of M and T were set to 5 and 10 respectively. The divergence time between subspecies was estimated using the Formula tdivergence time = Tpop × (Theta/2 μk).MDIV  was emplMolecular clock is widely used for the liable of sequence information. But the constancy of clocks have been controversial, particularly if they clash with estimates taken from more traditional sources such as the fossil record. Songbirds (Passeriformes) have a very limited fossil record in spite of making up more than one-half of the world’s 10,000 living species of birds. There has been no fossil reported for Pycnonotidae birds. Different species have different molecular clock due to life cycles and metabolic rates. So for a specific species, a useful clock should be close to calibrated relatives. We used conventional 2% –46 molecM) in the assumption of both populations and migration are at equilibrium with the MCMC chain approach implemented in the program LAMARC 2.1.3 [We estimated effective population size (Θ) and migration rate (RC 2.1.3 . All anaRC 2.1.3 . Two simRC 2.1.3 .S. semitorques. Approximate random gene flow indicated there might be no distinctive geographic barriers for this species in south China during late Pleistocene, which differs from the other three species of previous studies. Shallow differentiation due to considerable gene flow and lack of barriers might lead to high haplotype diversity and relative low nucleotide diversity. The mtDNA data showed a short evolutionary history of S. semitorques and undistinguished effects of the LGM on its historical demography.Our study showed undistinguished genetic structure among geographical populations of


Many species have been heavily exploited by man leading to local extirpations, yet few studies have attempted to unravel subsequent recolonization histories. This has led to a significant gap in our knowledge of the long-term effects of exploitation on the amount and structure of contemporary genetic variation, with important implications for conservation. The Antarctic fur seal provides an interesting case in point, having been virtually exterminated in the nineteenth century but subsequently staged a dramatic recovery to recolonize much of its original range. Consequently, we evaluated the hypothesis that South Georgia (SG), where a few million seals currently breed, was the main source of immigrants to other locations including Livingston Island (LI), by genotyping 366 individuals from these two populations at 17 microsatellite loci and sequencing a 263 bp fragment of the mitochondrial hypervariable region 1. Contrary to expectations, we found highly significant genetic differences at both types of marker, with 51% of LI individuals carrying haplotypes that were not observed in 246 animals from SG. Moreover, the youngest of three sequentially founded colonies at LI showed greater similarity to SG at mitochondrial DNA than microsatellites, implying temporal and sex-specific variation in recolonization. Our findings emphasize the importance of relict populations and provide insights into the mechanisms by which severely depleted populations can recover while maintaining surprisingly high levels of genetic diversity. Recently established populations may experience rapid genetic divergence, a process often attributed to founder effects  gazella, is a typical pinniped species, being highly polygynous .The Antarctic fur seal, Arctocephalus genus, Antarctic fur seals were subject to uncontrolled exploitation for their fur and oil during the early nineteenth century. At South Georgia (SG), sealing began in 1786 and it was estimated that by 1822, up to 1.2 million seals had been taken  in the South Shetland archipelago, with intense sealing activities from 1822 to 1825 leading to local extirpation (McCann and Doidge ST) between SG and the South Shetland Islands. Wynen et al. (n ≤ 20 per population), the authors interpreted the absence of certain haplotypes from SG as meaning that at least some contemporary fur seal populations may have been founded from more than one source. This merits further exploration, as being able to reliably exclude SG as the main source of fur seal immigrants to these populations would have important implications for understanding modes of recolonization that allow long-lived and highly mobile species to maintain high levels of genetic diversity despite dramatic historical reductions in population size.Despite the Antarctic fur seal having experienced drastic population reductions, only a single genetic study has so far examined this species' global population structure  and genotyped at 17 highly polymorphic microsatellite loci. We also added a fine-scale perspective by sampling three populations at LI that were successively established during the late twentieth century. Our aims were to evaluate support for the hypothesis that fur seal colonies at LI were mainly established by individuals from SG, and to test for genetic differences among the three colonies at LI that could be indicative of subtle differences in their recolonization histories.SG (35°47′–38°01′W and 53°58′–54°53′S) is a sub-Antarctic island situated approximately 1000 km southeast of the Falkland Islands . AntarctCape Shirreff fur seal pups were sampled at three sites  for DNA used in genotyping.Tissue samples were preserved in either 20% dimethylsulfoxide (DMSO) saturated with salt (NaCl), or 95% ethanol (ETOH) stored at −20°C. Total genomic DNA was subsequently extracted from LI tissue samples using a NaCl precipitation method  amplified using the primers Thr/Pro (5′-TCCCTAAGACTCAAGGAAGAG-3′) and Cent (5′-GAGC GAGAAGAGGTACACTTT-3′) as detailed by Wynen et al.  diversity, the number of polymorphic sites (S), nucleotide diversity (π), and the average number of nucleotide differences (k) were assessed using DNAsp v. 5.10.01 (Librado and Rozas n sampled and unsampled) given the number of observations and number of haplotypes sampled in a population. In order to obtain accurate estimates of variance, the analysis was set to increase n until its probability dropped below a 1/1010 proportion of the highest probability.Lastly, the total number of haplotypes at SG and LI was estimated to assess potential biases caused by incomplete haplotype sampling. We employed Dixon's method Dixon , which urxy) for all individuals within SG and LI using COANCESTRY v. 1.0 (Wang The microsatellite data set was tested for deviations from Hardy–Weinberg equilibrium (heterozygote deficit) and linkage disequilibrium using 100,000 dememorizations and 10,000 iterations per batch within GENEPOP v. 4.0  were calculated according to Nei (FSTAT v.2.9.3.2 Goudet  was nextg to Nei  within FK) based solely on the log probability of data (Ln[Pr(x|K)]) is not always straightforward within STRUCTURE, particularly where population structure is weak or follows an isolation-by-distance pattern. Consequently, we applied the ad hoc statistical method of Evanno et al.  indicates the uppermost hierarchical number of clusters present in the data set. We initially ran STRUCTURE without a priori sampling location information, but later repeated the same analyses incorporating location information by setting LOCPRIOR to 1. All analyses were conducted using the following parameters: admixture, allele frequencies correlated, 10,000 burn-in period and 100,000 Markov chain Monte Carlo (MCMC) repetitions . We conducted five independent runs for K = 1–10 and used STRUCTURE HARVESTER web core  for each of three alternative migration model priors .Maximum likelihood methods as implemented in the program MIGRATE can be powerful for exploring migration rates among populations or subpopulations. However, these approaches can be strongly affected by unsampled or “ghost” populations Slatkin . Having n = 246 individuals), five of which were represented by more than one individual. Fifteen haplotypes were unique to LI , 10 of which were sampled more than once. Remarkably, unique regional haplotypes were found in 51% of the individuals sampled at LI, with the highest incidence being observed at the oldest colony , the lowest at the youngest colony , and an intermediate proportion at the colony of intermediary age .A total of 52 polymorphic sites and 41 haplotypes were observed among the 365 HVR1 mtDNA sequences. Thirteen haplotypes were only observed in SG , whereas the remaining 5% was largely partitioned between SG and LI . A negligible proportion of the total variance was explained by sampling sites within these two regions . Consistent with this pattern, most of the significant pairwise ΦST values  and three unresolved links (loops) despite attempts to reduce its complexity using postprocessing calculations within the program NETWORK. Nevertheless, P(n) = 0.99) were estimated for SG , which corresponds to the exact number of observed haplotypes. At LI, the number of estimated and sampled haplotypes was equivalent (n = 28), but this number had a lower probability (P(n) = 0.62) and higher variance  suggesting a greater probability of missed haplotypes.Analyses conducted to estimate the total number of haplotypes revealed that our sampling thoroughly encompassed haplotype diversity at both study areas, particularly SG. A total of 26 haplotypes (HE = 0.81) and the proportion of missing data was low at 1.6%. There was no clear indication of null alleles, allelic dropout, or linkage disequilibrium (rxy) distribution centered tightly around a mean of zero at both SG and LI. Only two of 30,135 pairwise comparisons at SG and four of 7140 pairwise comparisons at LI yielded rxy values ≥0.50, suggesting a negligible effect of sampling kin.Our microsatellite panel was highly informative  for the microsatellite data set was 0.014 . Pairwise FST values among sampling sites were mostly significant in comparisons involving SG and LI . At LI, a similar overall result was obtained  with only comparisons involving the youngest colony (LI-E) reaching statistical significance . Allelic richness and mean observed (Ho) and expected (Hs) heterozygosities did not differ significantly between SG and LI .The global K = 2) based on the approach of Evanno et al. . Two of these were assigned to SG with >99.5% probability, whereas the third individual was not confidently assigned to either SG or LI, suggesting that it could have originated from another, unsampled location. The program STRUCTURE identified one of the same migrants using a migration prior of 0.01 and confirmed the second migrant with a higher migration prior of 0.05, while assignment probabilities to the population of origin (LI) were 0.004 (P < 0.0001) and 0.391 (P < 0.01), respectively.The program GENECLASS2 detected three pups with migrant ancestry via exclusion tests within LI-N . Repeating the analysis after randomly selecting the same number of individuals from LI-W and LI-N, the genetic differences within LI became no longer significant. However, the latter analysis is highly conservative and it would be worthwhile collecting more samples from these three colonies in the future to explore this phenomenon in greater detail.As initially reported for SG (Hoffman et al. We also found evidence for at least two pups from LI having immigrant ancestry from SG within the last two generations. Although we were not able to formally estimate migration rates within a maximum likelihood framework due to incomplete population sampling, this provides evidence in support of some level of contemporary gene flow between SG and LI, primarily directed toward the more recently founded LI-E colony. This makes sense because the SG population reached carrying capacity fairly recently and may thus be spilling over into relatively nearby, lower density sites. In fact, Boyd  suggesteOur results are interesting in a broader context, partly because very few genetic studies have explored the impacts of historical exploitation on long-lived vertebrate species, but also because those that have done so have reported little or no population structure. For example, Australian and Northern fur seals were both found to be panmictic despite these species having also been historically harvested (Dickerson et al. In conclusion, our findings strongly support the hypothesis that LI was not simply recolonized from SG and instead point toward a more complex recolonization history in which the genetic contribution of SG may have varied both temporally and by sex. Our results also highlight the importance of relict populations, which although demographically less significant, can harbor unexpectedly high levels of genetic diversity. Such populations could become increasingly important for maintaining the diversity of polar species that are facing mounting threats from rapid environmental change.


We evaluated the performance of GBLUP including dominance genetic effect (GBLUP-D) by estimating variances and predicting genetic merits in a computer simulation and 2 actual traits (T4 and T5) in pigs. In simulation data, GBLUP-D explained more than 50% of dominance genetic variance. Moreover, GBLUP-D yielded estimated total genetic effects over 1.2% more accurate than those yielded by GBLUP. In particular, when the dominance genetic variance was large, the accuracy could be substantially improved by increasing the number of markers. The dominance genetic variances in T4 and T5 accounted for 9.6% and 6.3% of the phenotypic variances, respectively. Estimates of such small dominance genetic variances contributed little to the improvement of the accuracies of estimated total genetic effects. In both simulation and pig data, there were nearly no differences in the estimates of additive genetic effects or their variance between GBLUP-D and GBLUP. Therefore, we conclude GBLUP-D is a feasible approach to improve genetic performance in crossbred populations with large dominance genetic variation and identify mating systems with good combining ability. Genomic selection refers to the use of genome-wide dense single nucleotide polymorphism (SNP) markers to predict breeding values and subsequently select individuals http://animalgene.umn.edu/) Most published models only include additive genetic effects The present study aimed to evaluate the performance of GBLUP including dominance genetic effect by estimating variance components and predicting genetic effects for both simulation and actual pig data.−4 per locus per generation. Recombinations were sampled from a Poisson distribution with a mean of 1 per Morgan and were then randomly placed along the chromosome. The historical population evolved over 2,000 generations of random mating and random selection with a population size of 100  to reach mutation–drift balance.A historical population was simulated to establish mutation drift equilibrium. The simulated genome comprised one chromosome 1 Morgan long that contained 6,000 SNP markers and 300 randomly spaced biallelic quantitative trait loci (QTL). In the first generation of the historical population, the initial allele frequencies of all markers and QTL were assumed to be 0.5. The recurrent mutation process was applied, and the mutation rate of markers and QTL was 5.0×10After 2,000 historical generations, a base population (G0) and the subsequent 6 generations (G1 to G6) were generated as a recent population. The population size of G0 increased to 300 . In G1 to G6, 30 sires were randomly selected and mated to 150 dams in each generation. Each dam had 1 son and 1 daughter; thus, each sire had 5 sons and 5 daughters.jth QTL. The genetic values are then given by jth animal was calculated by summing all QTL genotypic values, and its variance . The total number of individuals was 3,512, and all have phenotypes for 2 traits (T4 and T5) and genotypes available from the PorcineSNP60 chip . These phenotypes were already adjusted for environmental fixed effects such as sex, farm, and year of birth Publicly available data including pedigree, genotypic, and phenotypic information on a single Pig Improvement Company (PIC) nucleus pig line were used  In T4 and T5 from the pig data, dominance genetic variance accounted for 9.6% and 6.3% of the phenotypic variance, respectively . The estIn T4 and T5 from the pig data, there were nearly no differences between GBLUP and GBLUP-D with respect to . In T5, The GBLUP-D method captured the substantial ratios of the dominance genetic variances and estimated the individual dominance genetic effects although there were nearly no differences in estimates of additive genetic variance and narrow-sense heritability between GBLUP and GBLUP-D. Our result indicates that GBLUP-D is expected to improve performance of the crossbreds, in particular when degree of dominance is large.In the present study, the simulated genome comprised one chromosome 1 Morgan long. However, the whole genome sizes of livestock are larger. Here, another simulation data were constructed to evaluate the effect of the genome size on predictive ability. This simulation data comprised five chromosomes of 1 Morgan. The numbers of markers and QTL set to be 5,000 and 250 to obtain the same distances between markers and QTL as the initial simulation. The degrees of estimated dominance genetic variance in T4 and T5 were nearly equal to those in simulation data with a GBLUP-D has two practical uses. First, selection on the basis of GBLUP-D in crossbreds is useful for commercial production. In swine and poultry, crossbreds are the end product. The marker information from a purebred and its crossbred relatives enables the selection of candidate purebreds for the performance of their crossbred offspring jth locus are In the presence of dominance genetic effects, the breeding values of ith individual at the jth marker is calculated asA GBLUP method including dominance genetic effects was also proposed in a previous study This element corresponds to the heterozygosity coefficient but not the dominance genetic effect. To compare the performance of GBLUP-D in the present study and the model in the previous study Assuming linkage equilibrium and uncorrelated marker effects, the dominance genetic variances in the present study (In addition, the estimated dominance genetic effects in the present study (+ cells in publicly available mouse data were almost 0 Increasing knowledge about biological pathways and gene networks highlights the importance of gene–gene interactions, i.e., epistasis; some authors argue that much of the genetic variance in a population is due to such interactions


Aedes albopictus  (Diptera: Culicidae), a mosquito native to Asia, has recently invaded all five continents. In Central Africa it was first reported in the early 2000s, and has since been implicated in the emergence of arboviruses such as dengue and chikungunya in this region. Recent genetic studies of invasive species have shown that multiple introductions are a key factor for successful expansion in new areas. As a result, phenotypic characters such as vector competence and insecticide susceptibility may vary within invasive pest species, potentially affecting vector efficiency and pest management. Here we assessed the genetic variability and population genetics of Ae. albopictus isolates in Cameroon , thereby deducing their likely geographic origin.STF = 0.068, P<0.0001). Analysis of mtDNA sequences revealed four haplotypes each for the COI and ND5 genes, with a dominant haplotype shared by all Cameroonian samples. The weak genetic variation estimated from the mtDNA genes is consistent with the recent arrival of Ae. albopictus in Cameroon. Phylogeographic analysis based on COI polymorphism indicated that Ae. albopictus populations from Cameroon are related to tropical rather than temperate or subtropical outgroups.Mosquitoes were sampled in 2007 in 12 localities in southern Cameroon and analyzed for polymorphism at six microsatellite loci and in two mitochondrial DNA regions (ND5 and COI). All the microsatellite markers were successfully amplified and were polymorphic, showing moderate genetic structureamong geographic populations (Ae. albopictus isolates is in keeping with recent introduction and spread in this country. The genetic structure of natural populations points to multiple introductions from tropical regions.The moderate genetic diversity observed among Cameroonian  Aedes (Stegomyia) albopictus  (Diptera: Culicidae), is a major vector of dengue  and chikungunya  viruses in tropical areas Aedes albopictus was first reported in South Africa in 1989, where it was immediately controlled Ae. albopictus was first recorded in Cameroon in 2000 Aedes albopictus is now the dominant species at the periphery of urban centers such as Yaoundé and Douala Aedes aegypti .The Asian “tiger” mosquito, Ae. albopictus, numerous DENV and CHIKV epidemics occurred in Cameroon Ae. albopictus was incriminated as the main vector of both viruses during a 2007 dual outbreak in Libreville, Gabon Ae. albopictus isolates in Cameroon proved to be orally susceptible to DEN-2 virus and highly competent for CHIKV Coinciding with the spread of Aedes albopictus exhibits strong physiological and ecological plasticity, allowing it to thrive in a wide range of climates and habitats. Known as a sylvatic species in Asia Ae. albopictus has now adapted to human environments and is preferentially found in suburban areas, although it has also been recorded in densely populated urban areas such as Rome, Italy Ae. albopictus is extremely broad and includes natural water collections  and artificial containers  Ae. albopictus vary with the geographic origin of local mosquito populations Ae. albopictus to feed on different animal species enhances not only its biological traits  but also its capacity to propagate zoonotic pathogens across animal species and to humans Ae. albopictus populations. An early study based on isoenzymatic markers suggested that North and South American populations (USA and Brazil) were closely related to Japanese populations Ae. albopictus varies according to its geographic origin Ae. albopictus are genetically determined, and as these traits may influence virus transmission in newly colonized areas, it is important to determine the geographic origin of invading populations.Several phylogeographic studies have been undertaken to determine the origin of invasive Ae. albopictus in Africa, we undertook a genetic study of Ae. albopictus in Cameroon.As little is known of the geographic origin or genetics of An institutional clearance for this study, including the sampling of mosquito, was granted by Health Ministry of Cameroon.Aedes albopictus mosquitoes were sampled as larvae or pupae in 12 localities in South Cameroon during April 2007. The geographic locations and main characteristics of the sampling sites are summarized in Ae. albopictus and were then stored in individual tubes containing a desiccant at −20°C until molecular analysis.Total DNA was extracted from whole mosquito bodies, as described by Morlais et al. 2 , 0.2 mM each dNTP , 10 pmol of each primer, and 0.5 U of Taq polymerase . The 5′ end of the forward primer was labeled with a fluorescent dye . Cycling conditions were as follows: 5 min at 94°C, followed by 35 cycles of 45 s at 94°C, 30 s at the annealing temperature (TA), 45 s at 72°C, and 30 min at 72°C. PCR products were diluted in water  and pooled with other compatible products according to allele size range and fluorescent dye. Pools were prepared by adding 0.4 µl of GS 500 Liz internal size standard™  and HD formamide to 1 µl of each diluted amplification product, for a total volume of 20 µl. The mixture was heated at 95°C for 3 minutes before migration in an ABI Prism™ 3130 XL automatic sequencer (Applied Biosystems). Microsatellite alleles were scored with GeneMapper software (Applied Biosystems).DNA was amplified in a GeneAmp 9600 thermal cycler  in 25-µl reaction mixes containing 2 µl of 1/5-diluted total DNA, 2.5 µl of 10× reaction buffer , 0.5 mM MgClRseHISF and STF estimates were calculated as described in Genetic diversity per locus and per sample was characterized by estimating allele richness, ST/(1−FST) on the natural logarithm (Ln) of straight-line geographic distance Isolation by distance (IBD) was estimated with Mantel's test in GENEPOP4.0.7, using the correlation between genetic and geographic distances by the regression of pairwise FK) in the data set, without prior information of the sampling locations K (K = 1 to 12), with a burn-in period of 100 000 iterations and 400 000 replications. The method of Evanno et al. ad hoc quantity, ΔK, based on the second-order rate of change of the likelihood function between successive values of K.A Bayesian approach available in STRUCTURE 2.2 was used to infer the number of clusters (ND5 and COI. DNA samples were used as templates to amplify a 400-bp fragment of ND5 and a 550-bp fragment of COI. Two sets of primers were used: 5′TCCTTAGAATAAAATCCCGC-3′(FOR-ND5) and 5′-GTTTCTGCTTTAGTTCATTCTTC-3′ (REV-ND5) for ND5 5′-GGAGGATTTGGAAATTGATTAGTTC-3′ (FOR-COI) and 5′-CCCGGTAAAATTAAAATATAAACTTC-3′ (REV-COI) for COI 2 (for ND5), 0.2 mM dNTP, 10 pmol of each primer and 0.5 U of Taq polymerase (Qiagen). The thermal cycling conditions consisted of 3 min at 94°C, 35 cycles of 30 s at 94°C, 45 s at TA (52°C for ND5 and 54°C for COI), and 45 s at 72°C, followed by 10 min at 72°C. PCR products were detected by agarose gel electrophoresis in Tris-borate-EDTA buffer (TBE), stained with 0.5 µg/ml ethidium bromide in TBE, and visualized over UV light. PCR products were purified with the Ampure kit  and sequenced on both strands using ABI Prism BigDye Terminator version 3.1 (Applied Biosystems). Each 10-µl sequencing reaction mix contained 1 µl of Ready Reaction mix (Applied Biosystems), 1 µl of 5× sequencing buffer, 3.2 pmol/µl primer, and 0.5–1.0 µl of purified PCR product. The reaction consisted of an initial denaturation step of 1 min at 96°C, followed by 25 cycles of 10 s at 96°C, 5 s at 50°C, and 4 min at 60°C, with a final elongation step of 3 min at 72°C. Sequencing reaction products were purified with the Agencourt CleanSeq kit (Beckman Coulter) and analyzed on an ABI 3130XL automatic sequencer (Applied Biosystem).At least ten individuals per locality, selected among those previously analyzed for microsatellite markers, were used to explore sequence polymorphism in the mitochondrial genes ND5 and COI sequences were numbered according to the reference sequences . Basic sequence statistics, including the number of haplotypes per sample (Hp), the number of segregating sites (S), haplotype diversity (HpD), nucleotide diversity (π) and the average number of nucleotide differences were computed with DnaSP 4.10.9 Ae. albopictus from Asia, the Americas, the Indian Ocean and Europe were explored with Bayesian inference analysis. MrModeltest v2.2 COI and ND5 nucleotide sequence data (under Akaike's information criterion). Analyses were performed with MrBayes 3.1.2 Sequences were analyzed with SEQSCAPE software 2.5 (Applied Biosytems) and aligned with Clustal W Ae. albopictus specimens collected in 12 localities in Cameroon were successfully genotyped at all 6 microsatellite loci (Rs and He) were lowest in Bafoussam and highest in Buéa . Pairwise analysis of populations indicated significant genetic differentiation for most sample pairs (61/66), associated with FST estimates ranging from 0.006 (Bertoua-Abong-Mbang) to 0.176 (Buea-Yaounde) (P = 0.33). Bayesian cluster analysis identified 2 distinct genetic clusters in the dataset, although most of geographic populations appeared as a mixture of specimens from the two clusters except Buea and Garoua-Boulaï that appear made up of a single dominant cluster  . The UPGYaounde) . The topster see .Ae. albopictus specimens sampled at the 12 Cameroonian locations. Overall, the polymorphism in the ND5 region was low, with only three segregating sites defining four closely related haplotypes, resulting in low haplotype and nucleotide diversity indexes (Nucleotide sequences of mtDNA ND5 (400 bp) and COI (500 bp) were obtained for 153  indexes  and 5. T indexes  and 5. TAe. albopictus populations invading Cameroon, phylogenetic relationships between COI and ND5 sequences recorded in Cameroon and previously published sequences were assessed by using Bayesian inference. COI sequences segregated into two lineages .This is the first study of the genetic variation and differentiation of Ae. albopictus population from Indonesia Ae. albopictus in Africa.The set of microsatellite makers used here had already been tested on a single Ae. albopictus populations across most of its geographic range. The level of polymorphism found here within COI and ND5 sequences was low (four haplotypes for each gene) and consistent with previous studies of populations sampled in newly invaded areas Ae. albopictus in Cameroon was not related to the sample size. This low overall level of genetic diversity is consistent with recent introduction of Ae. albopictus in Cameroon.MtDNA markers have been extensively used to assess the genetic diversity of Ae. albopictus populations in Cameroon. No obvious relation between genetic and geographic distances was found, suggesting that the genetic structure has been shaped by additional biotic or abiotic factors. The Bayesian cluster analysis (i.e. existence of 2 genetic clusters co-occuring in most of cameroonian samples) supports a multiple introduction hypothesis. Recent studies have shown the important role of multiple introductions in successful colonization by invasive species Ae. albopictus colonization in Africa were recorded in the 1990s Ae. albopictus through accidental transportation of eggs, larvae or adults. Further analyses including samples from Nigeria would help to elucidate the relationships between Nigerian and Cameroonian populations of Ae. albopictus. Besides the effect of multiple introductions on the genetic structure of Ae. albopictus in Cameroon, the role of genetic drift must be considered. Indeed, the genetic variation observed in local populations of Ae. albopictus in Brazil The pattern of pairwise genetic differentiation estimated from the microsatellite data indicated a significant level of genetic structure among Ae. albopictus were related to specimens originating from tropical rather than temperate or subtropical areas. Unfortunately, we were unable to determine the geographic origin more precisely. Several studies have shown that Ae. albopictus populations that invaded Brazil Analyses of COI sequences revealed that Cameroonian Ae. albopictus populations, this study suggests that Ae. albopictus invasion of Cameroon involved multiple introductions from tropical sources. Further studies with more extensive sampling and a larger number of genetic markers, including other nuclear loci, are needed to confirm this hypothesis and to determine the precise geographic origin of Cameroonian Ae. albopictus.Despite the low genetic variation across the sampled Figure S1Microsatellite based Bayesian cluster analysis using STRUCTURE . (A). Estimates of Δ K, based on the second order rate of change of the likelihood function with respect to KK) in the data set. (B). Graphical representation of the data set for the most likely K = 2, where each color corresponds to a suggested cluster and each individual is represented by a vertical bar. The numbers in the x-axis correspond to a specific sample: 1) Abong-Mbang, 2) Bertoua, 3) Garoua-Boulai, 4) Ayos, 5) Bafia, 6) Yaoundé, 7) Buea, 8) Douala, 9) Pouma, 10) Bamenda, 11) Banganté and 12) Bafoussam.(TIF)Click here for additional data file.Table S1Estimates of null allele frequencies per locus and per sample of Ae. albopictus. Bolded: loci showing significant (P<0.05) heterozygote deficiency after correction for multiple testing.(DOC)Click here for additional data file.Table S2Accession numbers of COI and ND5 sequences of outgroup specimens used for phylogenetic analysis.(DOC)Click here for additional data file.