Located in the center of the Mediterranean landscape and with an extensive coastal line, the territory of what is today Italy has played an important role in the history of human settlements and movements of Southern Europe and the Mediterranean Basin. Populated since Paleolithic times, the complexity of human movements during the Neolithic, the Metal Ages and the most recent history of the two last millennia  has shaped the pattern of the modern Italian genetic structure. With the aim of disentangling this pattern and understanding which processes more importantly shaped the distribution of diversity, we have analyzed the uniparentally-inherited markers in ∼900 individuals from an extensive sampling across the Italian peninsula, Sardinia and Sicily. Spatial PCAs and DAPCs revealed a sex-biased pattern indicating different demographic histories for males and females. Besides the genetic outlier position of Sardinians, a North West–South East Y-chromosome structure is found in continental Italy. Such structure is in agreement with recent archeological syntheses indicating two independent and parallel processes of Neolithisation. In addition, date estimates pinpoint the importance of the cultural and demographic events during the late Neolithic and Metal Ages. On the other hand, mitochondrial diversity is distributed more homogeneously in agreement with older population events that might be related to the presence of an Italian Refugium during the last glacial period in Europe. Due to its central position and to the extension of its coastal line , the modern Republic of Italy – e.g. the Italian Peninsula and the two major islands of Sicily and Sardinia – has been one of the focal points in the settlement history of Southern Europe and the Mediterranean Basin.Populated by early modern humans since approximately 30,000–40,000 years before present (YBP) Subsequently Italy has received the passage of multiple human groups in prehistoric and historic times, acting both as a step point and an area of expansion during the different major migratory events following the early Paleolithic colonization.The most recent archaeological syntheses Italian Late Neolithic and the Metal Ages revealed to be a complicated tapestry of different cultural strata, potentially associated with population movements. During the first millennium BC, Italy hosted a vast set of different peoples whose origins in some cases remain unknown , while in other cases are the result of specific migration processes  In addition, independent and/or intersecting subsequent historic events  also contributed to the present genetic composition of Italy. Unlikely to have completely deleted precedent genetic structures, such migrations may have resulted in partially overlapping patterns of diffusion within Italy.At present, only few studies addressed the reconstruction of the genetic structure and history of Italian populations. Barbujani and colleagues (1995), in a study based on mtDNA variability On the paternal perspective, Di Giacomo et al. (2003) carried out an investigation of Y-chromosome diversity in continental Italy Although a common north-south cline has been described for maternal and paternal lineages in Italy, recent data on the Neolithisation of southern Europe The present research aims to update our knowledge about Italian population genetic history, by increasing the specificity of sampling strategy and the resolution power of uniparental molecular markers. For the first time, we present an extensive study of both mitochondrial DNA and Y-chromosomal variation in the Italian Peninsula, Sicily and Sardinia. Almost 900 individuals from eight sampling macro-areas have been deeply typed for 136 SNPs and 19 STRs of Y-chromosome, as well as for the whole control region and 39 coding SNPs of mtDNA. We use this detailed and complete dataset to address the following issues. First, we seek to describe the genetic structure of Italy and compare it with the patterns obtained before, in order to distinguish between a clinal and a discontinuous pattern of genetic variation. Second, we want to investigate whether the structure observed is sex-biased and which factors could account for any differential contributes from paternal and maternal lineages. Third, we seek to identify which population movements mostly could be in the origin of the current genetic diversity of the Italian populations.For all subjects, a written informed consent was obtained, and Ethics Committees at the Universitat Pompeu Fabra of Barcelona (Spain), and at the Azienda Ospedaliero-Universitaria Policlinico S.Orsola-Malpighi of Bologna , approved all procedures.A total of 884 unrelated individuals from continental Italy, Sicily and Sardinia were collected according to the following sampling strategy. Firstly, based on the results of a precedent reconstruction of the surname structure of Italy DNA was extracted from fresh blood by a Salting Out modified protocol http://isogg.org/tree/ISOGG_YDNA_SNP_Index09.html). For simplicity reasons, we will use asterisks (*) to indicate those chromosomes that are derived at a certain SNP, but ancestral at all the tested downstream SNPs.A total of 884 samples were successfully typed for Y-chromosome markers. 121 SNPs in the non-recombining region of the Y chromosome were genotyped using the OpenArray® Real-Time PCR System (Applied Biosystems) as described previously All individuals were additionally typed for a set of 19 STRs: 17 using the Yfiler kit (Applied Biosystems) and two  included in the Multiplex2. As the Yfiler kit amplifies DYS385a/b simultaneously avoiding the determination of each of the two alleles (a or b), DYS385a/b were excluded from all the analyses performed. DYS389b was obtained by subtracting DYS389I from DYS389II 865 samples were successfully sequenced for the whole control region as in Behar et al. (2007) Haplogroup frequencies were estimated by direct counting. Standard diversity parameters  were calculated with Arlequin 3.5 The relationships between geographical distances and genetic diversity were investigated by using several spatial analyses. The correlation between geographical distances and genetic distances (Reynolds distance), based on haplogroup frequencies, was evaluated by means of a Mantel test . In order to distinguish any clinal pattern (Isolation-by-Distance pattern) from any discontinuous genetic structure (both of them can result in significant correlations with geography), geographical distances were plotted against genetic ones. A 2-dimensional kernel density estimation layer adegenetTo further explore spatial patterns of variation a spatial principal component analysis (sPCA) based on haplogroup frequencies was performed using the R software package adegenet package http://www.fluxus-engineering.com, To further test the significance of the structure found with the sPCA analysis, we carried out a series of hierarchical analyses of molecular variance (AMOVA) pooling populations according to the sPCA results. We used haplogroup frequencies (both Y-chromosome and mtDNA), RST distances (Y-STRs) and number of pairwise differences (HVRI-HVRII mtDNA sequences). In order to explore genetic variability within the most frequent haplogroups, and in particular within those identified by sPCA loadings, we applied a Discriminant Analysis of Principal Components (DAPC) to Y-STR haplotypes and mtDNA sequences  was estimated with BATWING The age of Y-chromosome DAPC clusters exhibiting peaks of frequency higher than 70% in any of the sPCA-identified populations  – with the exception of haplogroup G2a due to its particular relevance in our populations (see Results) – and composed by at least ten individuals, as well as the age of the entire haplogroups, were estimated with the standard deviation (SD) estimator TMRCA for the most common mtDNA haplogroups was estimated by means of the ρ (rho) statistic with the calculator proposed by Soares et al. (2009) However, results have to be taken with caution, given that molecular date estimates with ρ can be affected by past demography. Simulations show that error rates tend to increase with effective size, bottleneck and growth effects A total of 884 unrelated individuals from 23 Italian locations  were such), STR diversity (nπ) and mean number of pairwise differences (π) of the population samples are listed in h) are observed in Sardinia, while the Italian peninsula is characterized by a negative correlation between haplogroup diversity and latitude, resulting in a south-north decreasing pattern of variation . The most frequent haplogroups in Italy are R-U152* (12.1%), G-P15 (11.1%), E-V13 (7.8%) and J-M410* (7.6%). They are followed by three R1b-lineages , whose frequencies ranged from 6.9% to 5.7%; and finally from I-M26, which embraced more than the 4% of total variability. On the whole these haplogroups encompass ∼62% of Y-chromosomes lineages, while the remaining 38 haplogroups show frequencies lower or equal to 3.3%. Haplogroups distribution in the considered eight sampling areas is detailed in The haplotype and haplogroup diversity , even after removing Sardinian samples . However, a non-homogeneous distribution of points is apparent when plotting geographical distances against genetic ones , indicatThese general spatial patterns were further explored by means of sPCA based on haplogroup frequencies. The analysis showed that the Italian genetic structure is characterised by two significant global components  with similar variance values, being sPC1 characterized by a higher spatial autocorrelation (Moran's I) . These oGeographical patterns of sPC1 and sPC2 are plotted in When we tested the reliability of these three groups , by means of AMOVA based both on haplogroup frequencies and STR variability, the proportion of variation between groups .In order to quantify the contribution of each haplogroup to the genetic structure detected, the loadings values of the sPC1 and sPC2 were calculated and plotted in DAPC was performed within the most frequent haplogroups . Results  show howIt is noteworthy the structure shown by haplogroup G2a-P15 , which iDAPC comparisons with additional samples  suggest BATWING was used to estimate the age of split between the Italian regions identified by the first sPCA . BATWING modelled population growth starting at 12,890 YBP , with a rate of 0.00429 (95% CI: 0.00254–0.01219) per year. Our results suggest that the split happened around 5,490 YBP . Since BATWING does not consider migration, admixture between NWI and SEI would likely bias the split time estimate towards more recent dates.Concerning Y-chromosome lineages, STR variation within the 13 clusters mentioned above suggests that most of them date back to relatively recent times . In factThe maternal genetic ancestry of Italian populations was explored by characterizing coding region SNPs and control region sequences from 865 individuals, which yielded to 79 distinct mtDNA haplogroups (including sublineages). Haplogroup frequencies and within-population diversity parameters are shown in The haplogroup distribution in Italy reflects the typical pattern of mtDNA variability of Western Europe. As described for other European and Italian populations In contrast to paternal lineages, correlation between geographical and genetic distances was non-significant . These results point to a strong homogeneity within the Italian Peninsula for the mtDNA gene pool composition. In order to extract further insights into the distribution of mtDNA lineages, a sPCA was performed using haplogroup frequencies. The highest absolute eigenvalues  correspoScores of the sPC1 and sPC2 are plotted in DAPC was performed within the eight most frequent haplogroups . They disaggregate in 24 haplotype clusters , rangingComparisons with other European samples  confirm TMRCA estimates for the most frequent haplogroups  could beAdditionally, we calculated TMRCA for the two DAPC clusters within HV haplogroup (2 and 3), given that they show a clear spatial polarity within continental Italy and Sicily. Their ages fall between the time estimate for the whole haplogroup  and the LGM, suggesting that their differentiation happened during this time frame .Previous reconstructions of the genetic structure of Italy agreed on two points: the peculiarity of the population of Sardinia – due to a distinct background and a high degree of isolation The discontinuous Y-chromosomal structure of continental Italy is also confirmed by the distribution of DAPC haplotype clusters identified for the most frequent haplogroups . HaplogrIn contrast to the results obtained for Y-chromosome, the mtDNA diversity in Italy is characterised by a high degree of homogeneity: the only exception  is due to significant differentiation found in the Sardinian samples compared to continental Italy and Sicily  and the Tyrrhenian (Western) coasts. Furthermore, archaeological sites from NWI are characterized by a deeper continuity with earlier Mesolithic cultures and a higher degree of local variability than SEI, while this last area, besides being culturally more homogeneous, shows clear links with the Southern Balkans For instance, the dates of several DAPC clusters fall within the range of the Metal Ages . During It is worth noting the older age estimate obtained for Y-haplogroup G2-P15  that, coupled with its high frequency (11.09%), makes it the most probable candidate for a continuity with Italian Mesolithic populations , North-Western Italy (NWI) and South-Eastern Italy (SEI). Furthermore, we observed that NWI and SEI are not separated according to latitude but following a longitudinal line. Such discontinuity may date at the Neolithic revolution in Italy, which was characterised by (at least) two independent diffusion processes involving the Western and Eastern coasts, respectively. Mitochondrial DNA, despite showing some correspondence with Y-chromosome results, depicts a substantially homogeneous genetic landscape for the Italian peninsula. Significantly different ages were estimated for mtDNA and Y-chromosome systems. mtDNA variability dates back to Paleolithic and supports the existence of an Italian human Refugium during the last glacial maximum whereas Y-chromosome points to the importance that the demographic events happened during the Neolithic and the Metal Ages had in the male Italian patterns of diversity and distribution.Figure S1Map showing the geographical location of populations sampled in the present study. Colors indicate the eight clusters of homogeneous Italian provinces (sampling macro-areas) identified after a preliminary surname-based analysis (TIF)Click here for additional data file.Figure S2Plot of geographical distances against genetic distances (based on frequencies of Y-chromosome haplogroups). A 2-dimensional kernel density estimation layer (Venables and Ripley 2002) was added to the plot. The analysis was performed including (a) and excluding (b) the Sardinian samples.(TIF)Click here for additional data file.Figure S3Eigenvalues of Y-chromosome-based sPCA analysis (A) with their decomposition in spatial and variance components (B). Eigenvalues are obtained maximizing the product of variance and spatial autocorrelation (Moran's I index). They are both positive and negative depending from Moran's I positive or negative values. Large positive components correspond to global structures (cline-like structures); large negative components correspond to local structures (marked genetic differentiation among neighbours).(TIF)Click here for additional data file.Figure S4Loadings of the most informative components . These values identify Y-chromosome haplogroups that mostly affect the genetic structure of Italian populations.(TIF)Click here for additional data file.Figure S5DAPC analysis of STRs variation for the most frequent Italian Y-chromosome haplogroups . Samples are grouped according to their affiliation to sPCA-identified areas . For each plot, the number of different haplotypes per cluster and their geographic distribution in the above areas are shown in the enclosed table. The DAPC eigenvalues are depicted in the enclosed barplot. Haplogroup I-M26, including two clusters only, is represented by a single discriminant function .(TIF)Click here for additional data file.Figure S6Median joining network for Italian G2a-P15 haplotypes. Individuals have been assigned and colored according to the correspondent DAPC-based clusters (clusters .(TIF)Click here for additional data file.Figure S7DAPC analysis of STRs variation for the most frequent Y-chromosome haplogroups. Results are based on Italian data and additional comparison samples ; CAU: Caucasus; WAN: Western Anatolia; symbols in the legend of each plot). For each plot, the number of different haplotypes per cluster and their geographical distribution are shown in the enclosed table. The DAPC eigenvalues are depicted in the enclosed barplot.(TIF)Click here for additional data file.Figure S8Eigenvalues of mtDNA-based sPCA analysis (A) with their decomposition in spatial and variance components (B). Eigenvalues are obtained maximizing the product of variance and spatial autocorrelation (Moran's I index), and are both positive and negative, depending from Moran's I positive or negative values. Large positive components correspond to global structures; large negative components correspond to local structures (marked genetic differentiation among neighbours).(TIF)Click here for additional data file.Figure S9Loadings of the most informative components . These values identify mtDNA haplogroups that mostly influence the genetic structure of Italian populations.(TIF)Click here for additional data file.Figure S10DAPC analysis of HVS variation for the most frequent mtDNA haplogroups  in the Italian data set. Results have been grouped geographically using the same categories as for Y-Chromosome ; “0” codes were attributed to those populations for which Y-chromosome information was not available and whose geographical position lies along the boundary between NWI and SEI . For each plot, the number of different haplotypes per cluster and their geographical distribution are shown in the enclosed table. The DAPC eigenvalues are depicted in the enclosed barplot. Haplogroup K1a, including two clusters only, is represented by a single discriminant function .(TIF)Click here for additional data file.Figure S11DAPC analysis of HVS variation for the most frequent mtDNAhaplogroups. Results are based on Italian data and comparison European populations ; AUST: Central Europe (Austria); MAC: Macedonians; ROM: Romanians; BALK: Balkan Peninsula; symbols in the legend of each plot). For each plot, the number of different haplotypes per cluster and their geographical distribution are shown in the enclosed table. The DAPC eigenvalues are depicted in the enclosed barplot.(TIF)Click here for additional data file.Table S1Frequencies of Y-chromosome haplogroups. Absolute values are reported for the whole Italian data set, while the frequencies within the eight sampling areas (from I to VIII) are expressed in percentage (%).(XLS)Click here for additional data file.Table S2Y-Chromosome STRs haplotypes in the 884 Italian samples of the present study.(XLS)Click here for additional data file.Table S3Diversity indices computed for the different Italian sampling points. Standard diversity parameters were calculated for both Y-chromosome and mtDNA based on haplotype/sequence data and haplogroup frequencies.(XLS)Click here for additional data file.Table S4Analyses of the molecular variance (AMOVA). Apportionment of the variance in %. Samples were grouped according to the geographic clusters (eight macro-areas) and to the sPCA results.(XLS)Click here for additional data file.Table S5DAPC membership probabilities to the SPCA-identified groups.(XLS)Click here for additional data file.Table S6Frequencies of Y-Chromosome DAPC clusters based on Italian data and comparison to other populations. The absolute number of haplotypes and individuals are shown for each population ; CAU: Caucasus; WAN: Western Anatolia).(XLS)Click here for additional data file.Table S7Frequencies of mtDNA haplogroups. Absolute values are reported for the whole Italian data set, while the frequencies within the eight sampling areas (from I to VIII) are expressed in percentage (%).(XLS)Click here for additional data file.Table S8Frequencies of mtDNA DAPC clusters in Italy. Values were calculated both grouping according to the geographical clusters identified with Y-Chromosome sPCA  as well as considering the continental Italy (including Sicily) altogether (ITA). The absolute number of haplotypes and individuals are shown for each DAPC-cluster, and the maximum frequency for each cluster is expressed in percentage (max%).(XLS)Click here for additional data file.Table S9Frequencies of mtDNA DAPC clusters based on Italian data and comparison to other populations. The absolute number of haplotypes and individuals are shown for each population ; AUST: Central Europe (Austria); MAC: Macedonians; ROM: Romanians; BALK: Balkan Peninsula).(XLS)Click here for additional data file.Methods S1Spatial Principal Component Analysis (sPCA). Discriminant Analysis of Principal Components. Batwing analysis. “Jackknife-like” procedure for outliers identification.(DOC)Click here for additional data file.


Metacrangonyx longipes, is endemic to the Balearic islands of Mallorca and Menorca (W Mediterranean). It has been suggested that the origin and distribution of thalassoid crustaceans could be explained by one of two alternative hypotheses: (1) active colonization of inland freshwater aquifers by a marine ancestor, followed by an adaptative shift; or (2) passive colonization by stranding of ancestral marine populations in coastal aquifers during marine regressions. A comparison of phylogenies, phylogeographic patterns and age estimations of clades should discriminate in favour of one of these two proposals.Metacrangonyctidae  is an enigmatic continental subterranean water family of marine origin . One of the species in the genus, M. longipes based on three mitochondrial DNA (mtDNA) and one nuclear marker revealed five genetically divergent and geographically structured clades. Analyses of cytochrome oxidase subunit 1 (cox1) mtDNA data showed the occurrence of a high geographic population subdivision in both islands, with current gene flow occurring exclusively between sites located in close proximity. Molecular-clock estimations dated the origin of M. longipes previous to about 6 Ma, whereas major cladogenetic events within the species took place between 4.2 and 2.0 Ma.Phylogenetic relationships within M. longipes displayed a surprisingly old and highly fragmented population structure, with major episodes of cladogenesis within the species roughly correlating with some of the major marine transgression-regression episodes that affected the region during the last 6 Ma. Eustatic changes (vicariant events) -not active range expansion of marine littoral ancestors colonizing desalinated habitats-explain the phylogeographic pattern observed in M. longipes. Subterranean fauna provides unique opportunities for the study of evolutionary mechanisms and speciation processes . In receGeological and hydrological processes, in particular shifts in water tables, can lead to the isolation or connection of aquifers, with consequent effects on gene flow between populations of subterranean aquatic organisms . In the Among the obligate dwellers of subterranean waters (stygobionts), a high number belong to so-called thalassoid lineages, organisms that are derived directly from marine ancestors . ThalassMetacrangonyx longipes Chevreux, 1909, a euryhaline stygobiont amphipod crustacean that is endemic to Mallorca and Menorca . On Mallorca, it occurs in various types of groundwater habitats, from coastal anchialine caves  of raiss , cytochrome b (cob) and 16S rRNA (rrnL)) and one nuclear (Histone H3A)-with a total sequence length of about 1.7 Kb were sequenced from 34 Metacrangonyx longipes specimens and the outgroups Metacrangonyx ilvanus, M. remyi and M. sp  analyses of the combined mitochondrial and nuclear data set yielded a similar topology, in which five divergent monophyletic lineages of Cox1 uncorrected distances between collection sites ranged from a minimum of 0.5% between those located close to each other  to a maximum of 8.9% (corrected to 9.8% using a GTR model) between populations from the two islands  or between some Mallorcan populations. As deduced from the phylogenetic analyses, clade A was the most divergent (7.8-8% mean uncorrected genetic distance with respect to the remaining clades), whereas the distance between the other clades fell between 6.3-7.5%. The distance between subclades B1 and B2 averaged 5.5%.cox1 haplotypes were identified in the sampled specimens from the 31 populations analysed (EMBL accession numbers FR729731-FR729892) (Table cox1. Six populations included only one haplotype (h = 0), although in three of them only two specimens (the only ones collected) were analysed. In sharp contrast, three populations showed maximum diversity indices, as every individual bore a different haplotype (h = 1). The rest of populations attained low-to-moderate h values, in the range of 0.25-0.86. Diversity was much lower in the two Menorcan populations  compared with the Mallorcan populations . Nucleotide diversity (mean number of pair-wise differences π) were low at most locations (π < 0.5%); population 28 alone exhibited a π value > 1% because of the presence of an individual bearing a divergent haplotype. Neutrality tests were non-significant in all cases, with the exception of populations 7  and 8 .Fifty different r = 0.467, P < 0.001) between genetic and geographic distances. In addition, cox1 pair-wise population FST values were generally high (FST = 0.5-0.99) and highly significant, with the exception of between neighbouring populations . Accordingly, SAMOVA showed that the optimum number of population groups necessary to maximize FCT values was K = 19-20 (FCT = 0.961-0.963), whereas the corresponding FSC values were the lowest in the K series tested, as expected [K values was explored using AMOVA, as this method allows recognition of population groups with any number of K. This analysis yielded a similar geographical setting, but identified wells 16, 19 and 26 as different populations; AMOVA showed that FCT values reached a plateau at 0.96 at K = 19-27, with the highest value attained at K = 23.The Mantel test revealed a moderately significant correlation  [M. longipes established far inland in completely fresh waters and those of the coastal anchialine medium, and among anchialine population themselves.The thalassoid condition of pletely) -19. If tM. longipes clades were linked to particular or neighbouring hydrographic catchments and were found nowhere else , populations do not appear necessarily clustered according to palaeodrainage channel. This pattern could result from the occurrence of gene flow or range expansion between palaeodrainages in the past, before populations became isolated in particular calcretes. The ulterior isolation of populations could be associated with a major period of aridification that affected the region between 10 and 4 Ma [In a study on hyalid and crangonyctoid stygobiont amphipods from W Australian calcrete aquifers, Cooper et al.  showed tand 4 Ma .M. longipes can be related to the succession of past sea-level changes in the Mediterranean (with the caveat of the limitations and errors associated with molecular-clock estimations). During the Tortonian (11.3 Ma), Mallorca and Menorca were invaded by an epicontinental sea that reduced the former to a cluster of small islands roughly corresponding to its current uplands, whereas the southern half of Menorca was probably completely submerged  may have induced the repeated range expansion, contraction and fragmentation of populations of l scales . Our stul scales .M. longipes were collected from seven anchialine and one freshwater cave, and from 23 freshwater wells spanning the entire geographic range of the species  and at the type locality located 1280 m above sea-level in the High Atlas, respectively. M. ilvanus Stoch, 1997 was collected in a well at Elba Island .One hundred and sixty-two specimens of cox1 gene using the primers described in [rrnL) gene were amplified using the specific primers 16SmetaF (5'- RGTATTTTGACCGTGCTAAGG-3') and 16SmetaR (5'- TGTAAAAATTAAARGTTGAACAAAC-3'), 360 bp of the cytochrome b (cob) gene were amplified using the primers described in [M. longipes individuals and outgroup species for rrnL, cob and Histone H3A are FR846024-FR846060, FR846061-FR846096 and FR846097-FR846133, respectively.Genomic DNA was isolated from whole specimens using the DNeasy Tissue Kit , according to the manufacturer's recommendations. PCR was used to amplify a fragment of ~650 bp of the mitochondrial ribed in  or, in sribed in . EMBL achttp://www.ebi.ac.uk/Tools/mafft/index.html, with default parameters.PCR was performed on a PTC-100 thermocycler (MJ Research) using a reaction volume of 25 μl and amplification conditions consisted of one cycle at 94°C for 2 min and 40 cycles of 94°C for 30 s, 47-55°C for 30 s and 72°C for 1 min, followed by a final incubation step at 72°C for 10 min. Amplified products were purified with Invitek columns , according to the manufacturer's instructions. The fragments were sequenced in both directions using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit v. 2.0 and electrophoresed and detected on an ABI 3100 automated sequencer . Alignments were performed using MAFFT rrnL and Histone H3A, respectively. Competing partition strategies were compared using Bayesian Information Criterion [cox1 and cob, third codon positions of cox1 and cob, rrnL and Histone H3A as separate partitions), whereas two partitions were selected in the case of cox1-only data sets (first + second vs third codon positions). Two independent runs were performed for each Bayesian search with default prior values, random trees and three heated and one cold Markov chains running for five million generations and sampled at intervals of 1000 generations. All parameters were unlinked and rates were allowed to vary freely over partitions. The burn-in and convergence of runs were assessed by examining the plot of generations against likelihood scores using the sump command in MrBayes. The convergence of all parameters in the two independent runs was also assessed using the Tracer program, v. 1.4 [sumt command in MrBayes, and the frequencies of the nodes in a majority rule tree were taken as a posteriori probabilities [M. longipes habitat character state at ancestral nodes (inland fresh vs. brackish groundwaters) using parsimony. In this analysis, we used the cox1 phylogenetic tree (as it represents a full population sampling) and the observed habitat distribution among populations to minimize the number of steps of habitat change.Partition Bremer Support values were estimated using TreeRot v. 3  and PAUPriterion . In the , v. 1.4 . Trees rbilities . Maximumbilities . Finallybilities  to reconcox1) and geographic distances of populations using the ZT program [cox1 data set, such as number of haplotypes, haplotype and nucleotide diversity, and pair-wise FST distances and their significance based on 10,000 permutations were obtained using ARLEQUIN v. 3.01 [SC). The method calculates F statistics  using AMOVA [K from K = 2 to K = 20. Neutrality tests were performed for individual populations calculating Fu's FS [2 [2 and FS are better at detecting population growth compared with other tests, the former being superior for small sample sizes [A Mantel test was performed on genetic  and to posterior probability values > 0.95 (second number).Click here for filecox1 mtDNA treeAncestral habitat tracing on the Bayesian . Parsimonious reconstruction of M. longipes habitat at ancestral nodes. Inland fresh groundwater and brackish groundwater populations are indicated in blue and yellow, respectively.Click here for file


Bemisia tabaci Q, commonly known as B. tabaci biotype Q) in China, which was introduced in approximately 2003. A total of 619 B. tabaci Q individuals in 20 provinces throughout China were collected and analyzed using five microsatellite loci.Even though introductions of exotic species provide ready-made experiments of rapid evolution, few studies have examined the genetic structure of an exotic species shortly after its initial introduction and subsequent spread. To determine the genetic structure of its populations during the initial introduction, we investigated the invasive sweet potato whitefly (B. tabaci Q in China represent eight genetic clusters with different geographic distributions. The populations in Yunnan Province, where B. tabaci Q was first detected, are genetically different from the other populations in China.The introduced populations of B. tabaci Q in China have high spatial genetic heterogeneity. Additional research is required to determine whether the heterogeneity results from multiple introductions, rapid evolution following one or few introductions, or some combination of multiple introductions and rapid evolution. The heterogeneity, however, is inconsistent with a single introduction at Yunnan Province, where B. tabaci Q was first detected, followed by spread.The introduced populations of  The introduction of invasive species can cause substantial economic loss and ecological damage in the introduced regions Although many empirical studies on the genetic structure of invasive species e.g.,  have revBemisia tabaci, is an important agricultural pest that is distributed on all continents except Antarctica Bemisia tabaci has been regarded as a species complex that includes many morphologically indistinguishable biotypes that differ in host range, virus transmission, insecticide resistance, and other traits B. tabaci Q (commonly known as biotype Q) has been introduced into many non-Mediterranean countries from its native Mediterranean regions over the past decade B. tabaci populations has been analyzed on a large geographic scale (Asia-Pacific regions) B. tabaci has been analyzed only rarely.The sweet potato whitefly, B. tabaci Q populations in China. Bemisia tabaci Q, a cryptic species in the B. tabaci species complex B. tabaci Q was present in Yunnan Province, Beijing, and Henan and that B. tabaci Q represented only a small percentage of the B. tabaci individuals detected B. tabaci Q was detected in 19 of 22 whitefly populations in 15 surveyed provinces, and the percentage of B. tabaci Q individuals within 10 populations was higher than 50% B. tabaci Q was detected in 11 of 14 whitefly populations in eight surveyed provinces; of the 11 populations, nine were pure B. tabaci Q populations, and two were mixed populations of B. tabaci Q and B (commonly known as biotype B) B. tabaci Q was detected in 12 of 16 surveyed provinces B. tabaci Q B. tabaci Q spread across China and became the dominant whitefly species in field ecosystems within approximately 4 years after its introduction. The well-documented invasion of B. tabaci Q in China or partial regions in China B. tabaci Q resulted from multiple introductions and subsequent spread rather than from a single introduction and subsequent spread.Here, we examined the genetic structure of B. tabaci Q across the entire country of China, and this was accomplished with microsatellite markers, which have been widely used in the analysis of invasive species B. tabaci Q in China resulted from a single introduction  and subsequent spread vs. multiple introductions and subsequent spread.The first objective of the present study was to determine the genetic structure of the introduced populations of The research complies with all laws of the country (China) in which it was performed and was approved by the ‘Department of Scientific Management of Chinese Academy of Agricultural Sciences, China’ (permit number: 20090112).B. tabaci were collected from a variety of field crops  and weeds  in 25 provinces of China in 2011–2012 (B. tabaci Q in 22 of 25 surveyed provinces (unpublished data), and the specimens of B. tabaci Q from 20 provinces were used in the present study. At least 100 living specimens were collected from the major host plants at each site  and L2-N-3014 (5′-TCCAATGCACTAATC TGCCATATTA-3′) and then sequenced et al. B. tabaci Q if their divergence from the consensus sequence of B. tabaci Q  and stored at −20°C. The mtCOI gene sequence was used to determine the species of B. tabaci Q individuals as template et al. PCR primers were used to amplify the microsatellite DNA loci  using the DNA of B. tabaci Q, the average number of alleles per locus (Na), the effective number of alleles (Ne), the observed heterozygosity (Ho), the expected heterozygosity (He), and Nei's expected heterozygosity (Nei) were calculated using POPGENE v.1.31 For each of the 20 populations of FisThe Wahlund effect within each population was quantified by calculating the Weir and Cockerham's Fst values. Weir and Cockerham's estimator of the fixation index FstWe used a traditional population differentiation approach based on B. tabaci Q populations in China and to estimate individual coefficients of ancestry with regard to the detected clusters. When estimating individual ancestry coefficients via admixture analyses, we used the recommended values: the number of iterations used to estimate the admixture coefficients for the individuals was 100; the number of reference individuals from each population was 200; and the number of iterations used to estimate the admixture coefficients for the reference individuals was 20.The program BAPS v.4.14 To estimate the variance among clusters, among populations within clusters, and within clusters, a hierarchical analysis of molecular variance (AMOVA) was performed with ARLEQUIN v.3.5 Na) ranged from 3.20 to 5.80, and the effective number of alleles (Ne) ranged from 1.9330 to 3.0546. The expected heterozygosity (He) ranged from 0.3977 to 0.6004 while the observed heterozygosity (Ho) ranged from 0.2158 to 0.4698. The He in each population was higher than the value of Ho. Nei's expected heterozygosity (Nei) ranged from 0.3901 to 0.5937. The levels of genetic diversity indices were similar but not always consistent. For instance, population YN had the lowest level of genetic diversity according to Na (3.20), Ne (1.9330), He (0.3977), and Nei (0.3901) but not according to Ho (0.3846).Values of genetic diversity indices of the Chinese populations are given in Fis, demonstrated the presence of sub-structure within all populations (P<0.05) in only three populations , which account for 15% of the Chinese populations. The significant heterozygosity deficit for the three populations, however, may result not from demographic expansion but rather from within-population substructure Fis) in these populations  was detected in only four populations   , indicat and AH) .Fst values were associated with a significant exact test (Fst in comparisons of the population from Yunnan (YN) and other populations ranged from 0.3622 to 0.4937, which were much higher than other Fst values (<0.2366). The main results from Fst computations were confirmed by Bayesian analyses. Analyses using BAPS identified eight genetic clusters overall  act test . The val overall : one cluB. tabaci Q was observed at three hierarchical levels   and the variation among clusters  were small but significant.Significant genetic structure of lusters) . Most ofB. tabaci Q in China at the national level. Our results show that all Chinese populations have sub-structure even though B. tabaci Q was only recently introduced into the country. Most microsatellites fit a two-phase model of mutation (TPM) better than a strict stepwise mutation model (SMM) or infinite alleles model (IAM) To the best of our knowledge, this is the first study to report the genetic structure of Fst data and analyses using BAPS show that the introduced populations are genetically different. There are many possible explanations for the genetic heterogeneity of B. tabaci Q populations in China. One possible explanation is that there have been multiple introductions of exotic B. tabaci Q in China, with diversity in the genotypes among introductions. The second possibility is rapid evolution following only one or a few introductions. A third possibility would include both multiple introductions and rapid evolution. With regard to rapid evolution, both genetic drift and natural selection (from biotic interactions and abiotic factors in the new environment) may result in genetic heterogeneity of the introduced populations The B. tabaci Q in China because only four of the 20 populations exhibited significant heterozygosity excess (a signature of a bottleneck) under the TPM model, and none exhibited heterozygosity excess under either the IAM or SMM model. The effects of a bottleneck on heterozygosity are transient and observable only for a few generations B. tabaci generally has 11–15 generations each year (http://www.ipm.ioz.ac.cn/them_gefeng/fenshi/yuce.asp). Thus, approximately 88–120 generations have occurred since the introduction of Q in about 2003 B. tabaci Q populations have been mitigated. The nuclear genetic diversity of introduced populations of B. tabaci Q in China (He range: 0.3977–0.6004) in this study is similar to that of B. tabaci Q in native regions (He range: 0.3594–0.6124) as reported by Chu et al. Our study suggests (but does not prove) that bottleneck effects did not play an important role during the genetic differentiation of B. tabaci Q, especially those in northern China, have spread to nearby provinces by natural expansion or human activity. For example, the populations in cluster I (except SH) are mainly composed of the populations in northern China including populations in nine neighboring provinces   . ClusterShaanxi) . ClusterShaanxi) . AlthougB. tabaci Q was first detected in the USA B. tabaci Q in field and greenhouse systems showed that it cannot establish self-sustaining populations in the field in the USA B. tabaci Q have limited invasiveness and that the genetic composition of B. tabaci Q populations differs in the USA and China. It seems, therefore, that the B. tabaci Q populations in China were not derived from those in the USA.B. tabaci Q populations in other provinces of China are not derived from populations in Yunnan Province, where the pest was first detected, even though infested ornamental plants and flowers are likely to be transported from Yunnan Province to the other provinces (http://www.accci.com.au/keycity/yunnan.htm). The introduced populations in Yunnan Province may not have acted as bridgehead population Fst value indicates that the Yunnan population is highly differentiated from the other populations.Our data on genetic diversity and differentiation also suggest that B. tabaci Q in China, which demonstrates that the introduced populations of B. tabaci Q in China have high spatial genetic heterogeneity. The heterogeneity may have resulted from a combination of multiple introductions of diverse populations and rapid evolution following introduction. Determining the relative contributions of these two sources of heterogeneity will require additional research.Although introductions of exotic species have provided excellent opportunities to investigate rapid evolution, few studies have examined the genetic structure of an exotic species shortly after its initial introduction and subsequent spread. In this study, we found eight genetic clusters among the introduced populations of Table S1Summary statistics for five microsatellite loci screened for Bemisia tabaci Q. Abbreviations are as follows: sample size (N), number of alleles observed per locus (Na), observed heterozygosities (Ho) and expected heterozygosities (He), Wright's fixation index (Fis). Fis values in bold indicate significant departures from Hardy–Weinberg proportions.(DOC)Click here for additional data file.


Atrina pectinata) is a popular food source with a high commercial value in a number of Asian Pacific areas. The natural A. pectinata population has been declining continuously over the past several decades. Microsatellite DNA markers are a useful DNA-based tool for monitoring the genetic variation of pen shell populations. In this study, 20 polymorphic microsatellite (MS) DNA markers were identified from a partial genomic pen shell DNA library enriched in CA repeats, and used to compare allelic variation between wild and hatchery pen shell populations in Korea. A total of 438 alleles were detected at the 20 MS loci in the two populations. All loci were easily amplified and demonstrated allelic variability, with the number of alleles ranging from 5 to 35 in the wild population and from 5 to 22 in the farmed population. The average observed and expected heterozygosities were 0.69 and 0.82, respectively, in the hatchery samples and 0.69 and 0.83, respectively, in the wild samples. Statistical analysis of fixation index (FST) and analysis of molecular variance (AMOVA) showed minor, but significant, genetic differences between the wild and hatchery populations . These microsatellite loci may be valuable for future aquaculture and population genetic studies for developing conservation and management plans. Further studies with additional pen shell samples are needed to conclusively determine the genetic diversity between the wild and hatchery populations.Pen shell ( Atrina pectinata (Linnaeus 1767), belongs to the Pinnidae family and is a large (shell length up to 30 cm) suspension-feeding bivalve common along the coasts of Korea, Japan, and China [A. pectinata is an infaunal bivalve found in habitats ranging from muddy to sandy sediment and from tidal flats to shallow subtidal environments up to 20 m in depth [Marine shells of the Pinnidae family are a popular food source with a high commercial value in a number of Asian Pacific areas. The pen shell, Linnaeus 767, beloin depth ,5. Thus,With the rapid development of pen shell aquaculture and breeding projects, molecular markers for studying the genetic variation can help elucidate the genetic differences among wild populations, assess genetic variation within captive stocks, and determine the genetic impacts of aquaculture on wild populations, thereby promoting sustainable aquaculture. Because of their high degree of variability, microsatellite (MS) DNA markers or simple sequence repeats (SSRs) are molecular markers that are suitable tools for monitoring changes in the genetic variation of farmed stocks, assigning parentage, and evaluating the genetic diversity and structure of various marine species for the improvement of fisheries and resource conservation –10. Despn-enriched genomic DNA library, 150 of which were screened by PCR for the presence of a repeat-containing insert. Sequencing of the inserts from these 150 colonies revealed 90 loci containing MS arrays with a minimum of five repeats, corresponding to an enrichment efficiency of 22.5%. These were primarily 2-bp repeat motifs, some of which were combined with other 2-bp repeat motifs. Primers were designed and tested for 43 loci that exhibited adequately long (>20 bp) and unique sequence regions flanking the MS array. Forty-seven loci were discarded because the MS sequences were so close to the linker sequence that primer sequences could not be designed for amplification. After initial PCR assays, only 21 primer sets successfully yielded variable profiles. The remaining 22 primer sets gave either inconsistent or no PCR products, despite adjusting the dNTP concentrations and using an annealing temperature gradient. An initial evaluation of the polymorphic status of each locus was done by genotyping in 16 individuals randomly selected from the wild population. With the exception of KAp25, which had one allele, all loci were polymorphic and showed different degrees of variability. The primer sequences, repeat motifs, annealing temperatures, fluorescent labels, and GenBank accession numbers for the 21 newly identified MS loci are summarized in In total, more than 400 white colonies were obtained from the transformation with the Korean pen shell (CA)et al. [Pinctada maxima (25%) [Haliotis midae (47%) [Mercenaria mercenaria (59.4%) [Generally, MS DNA loci isolation by screening of genomic libraries using repetitive probes is tedious but can result in the acquisition of numerous MS DNA loci. In case of magnetic bead-based enrichment, the types and ratios of biotin-labeled probes and the positive clone selection strategy can affect the success of cloning and the efficiency of enrichment. In this study, we created MS libraries enriched for CA repeat sequences by following the protocol of Hamilton et al.  with modet al. ,15. Of tma (25%) , but lowae (47%)  and Merc (59.4%) . Except  (59.4%) .A. pectinata collected from around Jangheung, Korea, were screened for variation at the 20 new polymorphic MS loci. The 20 primer sets yielded variable profiles; reruns were conducted for 20% of the samples to ensure that the allele scoring was reproducible. No differences were observed, indicating that genotyping errors did not affect allele scoring. Samples that failed to amplify after the rerun were not included, which made it unlikely that poor DNA quality affected our results.Samples of 63 wild and 50 hatchery-bred FST value was estimated with and without these loci. With KAp12, KAp18, KAp26, and KAp32, however, our data indicated that these loci were affected by null alleles in only one sample; thus, they were included in further analyses.The MICRO-CHECKER analysis showed that some loci may have been influenced by one or more null alleles in both the wild and hatchery samples. Our data showed that the loci, KAp12, KAp21, KAp23, KAp32, KAp33, KAp40, and KAp43, in the farmed samples and the loci, KAp18, KAp21, KAp23, KAp26, KAp33, KAp40, and KAp43, in the wild population were affected. The KAp21, KAp23, KAp33, KAp40, and KAp43 loci appeared to be influenced in both the wild and hatchery samples, indicating that the use of these particular loci for population genetics analyses that assume HWE may be problematic. Thus, a global multi-locus AR) was employed to compare different populations independent of sample size. Overall allelic richness varied from 4.59 to 32.11 (AR = 17.41) than the hatchery population (AR = 17.05) based on minimal sample size of 50. The wild population had a higher number of alleles and a higher allelic richness than the hatchery-bred population, but the difference was not significant . The mean observed and expected heterozygosities were respectively 0.689 and 0.825 in the wild samples while the corresponding parameters were 0.684 and 0.822 in the hatchery samples, respectively. There were not significant differences of expected and observed heterozygosity between the wild and hatchery populations .All 20 MS loci were found to be highly polymorphic in both populations. A total of 438 different alleles were observed and the average number of alleles per locus was 21.9. The number of alleles varied from five at the KAp30 and KAp42 loci to 35 at the KAp21 locus . Not allto 32.11 . A higheIn this study, a high level of genetic diversity  in the wild population was detected. A similarly high genetic diversity has been reported in many other marine bivalves such as oyster , clam 2, and scaFIS) varied among markers from −0.144 (KAp30) to 0.883 (KAp21) in the hatchery samples and from −0.023 (KAp39) to 0.574 (KAp23) in the wild samples. The average FIS, including all markers, was 0.170 in the hatchery samples and 0.166 in the wild samples (Inbreeding coefficients ( samples .P < 0.003). The allele frequencies of the eight microsatellites in the wild and hatchery samples are shown in n = 42), and the allele frequencies at this locus were clearly different between the wild and hatchery populations. Distinct differences in the allele frequencies between the wild and hatchery populations were also observed at the loci, KAp12, KAp17, KAp20, KAp26, KAp30, KAp33, and KAp40. More unique alleles were observed in the wild population (96) than in the hatchery population (64). Allele frequency distributions indicated the presence of 217 rare alleles (frequency <5%) out of a total of 341 alleles over all loci  in the hatchery sample, whereas 263 rare alleles out of a total of 374 alleles  were observed in the wild sample. Rare alleles were detected at most loci in both populations. Examination of pair-wise linkage disequilibrium revealed that all of the 20 microsatellite loci were in linkage equilibrium (P > 0.003).P > 0.05) through the sign test, standardized differences test and Wilcoxon sign rank test, suggesting that both the wild and hatchery populations have not experienced a recent bottleneck.In testing the departure from mutation-drift equilibrium based on heterozygosity excess or deficiency for both populations, bottleneck analysis was conducted using Bottleneck software under the two-phased model of microsatellites. No population displayed significant heterozygosity excess (P < 0.003) were found at seven loci  in the hatchery samples and seven loci  in the wild samples, indicating that deviations from HWE detected at nine of the 20 microsatellite loci were due to heterozygote deficiency.Significant departures from HWE after Bonferroni correction . When the KAp21, KAp23, KAp33, KAp40, and KAp43 loci were excluded, the global multi-locus FST was estimated to be 0.0106 (P < 0.01).Single-locus P = 0.000), 5.67% (P = 0.000), and 1.06% (P = 0.006), respectively  of the 15 microsatellites yielded results similar to those from the FSTAT analysis; variation within individuals, among individuals within populations, and among populations was 93.32%  and analysis of molecular variance (AMOVA) showed minor, but significant, genetic differentiation between the wild and hatchery populations. This is confirmed by the allele frequency distributions and the numbers of unique allele and rare allele in each MS locus between the wild and hatchery populations. In pen shell populations in Jangheung, Korea, the progeny produced for stock abundance had a different genetic composition although no significant reductions were found in the diversity compared with the wild population . DNA fragments in the range of 300–800 bp were isolated and purified using the QIAquick Gel Extraction Kit . The selected fragments were ligated to an adaptor (SNX/SNX rev linker sequences), and the linker-ligated DNA was amplified using SNX as a linker-specific primer for PCR. For enrichment, the DNA was denatured and biotin-labeled repeat sequences ((CA)12GCTTGA) [A partial genomic library enriched for CA repeats was constructed using a slightly modified enrichment procedure with pre-hybridization polymerase chain reaction (PCR) amplification, as described previously . The ext2GCTTGA)  were hybEscherichia coli DH5 competent cells. White colonies were screened for the presence of a repeat insert by PCR using the universal M13 primer and non-biotin-labeled dinucleotide primers. PCR products were examined on 2% agarose gels, and inserts producing two or more bands were considered to contain a microsatellite locus. Positive clones were cultured and purified. Plasmids from insert-containing colonies were recovered using the QIAprep Spin Miniprep Kit  and sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit  and an automated sequencer .The purified PCR products were digested with NheI, cloned using an XbaI-digested pUC18 vector , and transformed into Ex Taq DNA polymerase , 1× PCR buffer, 0.2 mM dNTP mix, 100 ng of template DNA, and 10 pmol of each primer, where the forward primer from each pair was 5′-end-labeled with 6-FAM, NED, and HEX dyes (Applied Biosystems). The PCR reaction ran for 11 min at 95 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at the annealing temperature . Newly designed PCR primer pairs were tested to optimize the annealing temperatures; a gradient PCR with a 50–60 °C range was performed on a sample set from eight pen shells captured from Jangheung, Korea. The PCR amplification was performed using a PTC 200 DNA Engine  in a 10-μL reaction containing 0.25 U of perature , and 1 mA), size of alleles in base pairs (S), frequency of the most common allele (F), and number of unique alleles (U) were determined for each local sample at each locus using the program GENEPOP . This was also used to identify deviation from Hardy-Weinberg equilibrium  and the observed and expected heterozygosities, indicating an excess or deficiency of heterozygotes. FSTAT  was used to calculate the inbreeding coefficient (FIS) [AR) [FST; 1,000 permutations) [Samples were screened for variation at the newly developed MS loci. MICRO-CHECKER 2.2.3  was usednt (FIS)  per locuIS) [AR) , suitablIS) [AR) , componeIS) [AR) . The sigIS) [AR)  and to ctations) . Signifitations) . As hatctations)  was emplIn conclusion, genetic studies on pen shell with microsatellite DNA markers are very rare with only two existing reports on the development of new microsatellites ,12. No d


Anopheles funestus is one of the major malaria vectors in tropical Africa. Because of several cycles of drought events that occurred during the 1970s, this species had disappeared from many parts of sahelian Africa, including the Senegal River basin. However, this zone has been re-colonized during the last decade by An. funestus, following the implementation of two dams on the Senegal River. Previous studies in that area revealed heterogeneity at the biological and chromosomal level among these recent populations.An. funestus population located in the sudanian domain.Here, we studied the genetic structure of the newly established mosquito populations using eleven microsatellite markers in four villages of the Senegal River basin and compared it to another An. funestus population, suggesting a situation of panmixia. Moreover, no signal from bottleneck or population expansion was detected across populations. The tests of genetic differentiation between sites revealed a slight but significant division into three distinct genetic entities. Genetic distance between populations from the Senegal River basin and sudanian domain was correlated to geographical distance. In contrast, sub-division into the Senegal River basin was not correlated to geographic distance, rather to local adaptation.Our results presume Hardy Weinberg equilibrium in each An. funestus endured.The high genetic diversity among populations from Senegal River basin coupled with no evidence of bottleneck and with a gene flow with southern population suggests that the re-colonization was likely carried out by a massive and repeated stepping-stone dispersion starting from the neighboring areas where  Anopheles funestus Giles, 1900 is one of the major malaria vectors in tropical Africa with An. gambiae sensu stricto and An. arabiensis. Each ru results. The estPr (X|K).Groups of populations were defined according to STRUCTURE results and the hierarchical genetic variation existing within groups of populations and within populations was analyzed by Analysis of Molecular Variance (AMOVA) using Arlequin version 3.5.FST/(1 – FST) on the logarithm (ln) of geographical distance[2)[The correlation between genetic and geographical distances was assessed by the regression of distance, and tesance[2).Kruskal-Wallis test was used to compare the average number of alleles and the average proportions of heterozygosity between populations using XLSAT Pro 2006®.The Bonferroni correction procedure was applAn. funestus females were scored at 11 microsatellite loci. These microsatellite loci were highly polymorphic with a number of distinct alleles per locus ranging from 6  to 15  in our five populations (Table P = 0.83). Mean observed heterozygosity across all loci ranged from 0.68 to 0.72 and was not significantly different among populations (P = 0.89). Heterozygosity tests were performed to explore demographic stability in An. funestus populations and compliance to mutation-drift equilibrium. Significant deviations from mutation-drift equilibrium, as a result of heterozygote excess, was found in a population of Loboudou, when the studies were performed under TPM 70% and TPM 90% model. In Dielmo, significant deviations associated with strong heterozygote deficiencies were detected under SMM model. For the populations of Gankette, Diaminar and Mbilor, no deviation from MDE was found  of 11 loci showed significant deviations from Hardy-Weinberg equilibrium due to significant heterozygote deficiency (Table AFUB11 and AFND19 respectively in populations of Mbilor and Diaminar after the Bonferroni correction was applied. These deviations from Hardy-Weinberg expectations were associated with heterozygote deficiency (Table P > 0.05).When the pooled samples were analyzed as a single population, 3  to 0.0519 (Dielmo-Mbilor). The highest FST estimates were obtained between the most distant populations. FST estimates were highly significant (P <10-4) between Dielmo and the populations of the Senegal River basin and between Mbilor and the populations around Guiers Lake area . After all, only three comparisons were not significant after Bonferroni correction: Diaminar-Loboudou (P > 0.005), Diaminar-Gankette (P > 0.005), and Gankette-Loboudou (P > 0.005).The levels of genetic differentiation between pairs of populations were estimated by Structure v 2.2. provided consistent results over 5 replicated runs tested for each K. The probability of the data (LnPr(X|K)) greatly increased from K = 1 to K = 2, and then reached a maximum value at K = 3, after which the values decreased gradually  of all the eleven microsatellites then confirmed the differentiation and structure analysis with the variation within individuals, among individuals within populations, among populations within groups and among groups being 92.73%, 4.21%, 0.08%, and 2.97% respectively  correlation between FST/(1- FST) and the logarithm of distance, when considering the five villages (Dσ2 = 7.23)  (Dσ2 = 20.94).Isolation by distance was tested and showed a positive and significant [An. funestus populations may occur each year at the rainy season, when breeding sites are numerous and close enough to allow mosquito dispersal among populations.The observed genetic stability of . gambiae. The absilibrium. For insseasons),47. On tseasons). TherefoAn. funestus and our estimates are consistent with previous studies on genetic structure of An. funestus populations in Senegal and other parts of Africa using microsatellites[An. funestus populations from the Senegal River basin and the sudanian population (252 km apart), revealed by our analysis, indicate the existence of continuous populations of this malaria mosquito are inter-connected. Such observations were already reported in the other genetic studies in the populations of An. funestus[An. gambiae s.l[An. funestus in the Senegal River basin was probably carried out by a step by step dispersion starting from the neighbouring areas where An. funestus had not disappeared.Our results showed low but significant levels of genetic differentiation between populations of the Senegal River basin and those from the sudanian area. High levels of gene flow have been repeatedly reported on tellites,20,49,50 funestus,49,51,52mbiae s.l-56. TherAnopheles[Anopheles funestus[An. funestus populations from the Senegal River basin may be subdivided into two distinct genetic entities: populations around Guiers lake area  and populations from the low valley of Senegal River (Mbilor). The existence of these three genetic entities  was confirmed by different genetic approaches (i.e. Structure and AMOVA). When Dielmo was excluded from the isolation by distance analysis, geographical distance did not explain the genetic differentiation observed between Mbilor and populations around Guiers Lake area. Thereby, other factors rather than geographical distance should play a key role into the population structuration of An. funestus populations in the Senegal River basin. Chromosomal differentiation detected between An. funestus populations of the low valley of Senegal River and the Guiers Lake area (difference of frequency for the inversion 3La)[An. funestus populations of Burkina Faso[An. funestus populations of the low valley of Senegal River and the Guiers Lake area[The genetic analysis clearly distinguished between sahelian and sudanian populations. The genetic tests of isolation by distance suggested that genetic differences observed between domains are linked to the geographical distance. This is a common pattern in Anopheles,57,58 an funestus,20,50,51ion 3La) may be iina Faso. Becauseake area could beAn. funestus: populations around Guiers Lake area, populations from the low valley of Senegal River, and populations from Dielmo. The high genetic diversity among populations from the Senegal River basin coupled with no evidence of bottleneck and with a gene flow with the southern population suggested that the re-colonization was likely carried out by massive and repeated stepping-stone dispersions starting from the neighbouring areas where An. funestus endured. Geographical distance is not the only factor involved in shaping of the genetic structure observed between the An. funestus populations from the low valley of the Senegal River and The Guiers Lake area and we hypothesize that the different breeding sites created by human activities may have shaped chromosomal structuration and may explain the restricted but still occurring gene flow. Our study is therefore indicative of adaptation of malaria vectors to the environment modified by humans.Our study showed the existence of three genetically different subpopulations of The authors declare that they have no competing interests.ID, LK and DF designed and supervised the study. BS, ID and LK performed field activities. BS performed laboratory work under guidance of AC. BS, DA, AC analyzed the data. BS, DA, ID and AC drafted and revised the manuscript. All authors approved the final version of the manuscript.


Gambusia holbrooki) that had invaded Spanish watersheds, along with the American locations close to the suspected potential source populations. Mosquitofish populations from the Spanish streams that were studied had similar levels of genetic diversity to the American samples; therefore, these populations did not appear to have undergone substantial losses of genetic diversity during the invasion process. Population structure analyses indicated that the Spanish populations fell into four main clusters, which were primarily associated with hydrography. Dispersal patterns indicated that local populations were highly connected upstream and downstream through active dispersal, with an average of 21.5% fish from other locations in each population. After initially introducing fish to one location in a given basin, such dispersal potential might contribute to the spread and colonization of suitable habitats throughout the entire river basin. The two-dimension isolation-by-distance pattern here obtained, indicated that the human-mediated translocation of mosquitofish among the three study basins is a regular occurrence. Overall, both phenomena, high natural dispersal and human translocation, favor gene flow among river basins and the retention of high genetic diversity, which might help retain the invasive potential of mosquitofish populations.Genetic analyses contribute to studies of biological invasions by mapping the origin and dispersal patterns of invasive species occupying new territories. Using microsatellite loci, we assessed the genetic diversity and spatial population structure of mosquitofish ( Pseudorasbora parva) began accidentally when humans introduced it during the establishment of new cultured stocks of the common carp (Cyprinus carpio), and was followed by further natural dispersal of short distances Biological invasions are a central component of global change, and a major threat to the biodiversity of freshwater ecosystems Gambusia holbrooki, is one of the most commonly introduced freshwater species G. holbrooki has caused a decline in the feeding rates and reproductive success of two Iberian endemic fish species, Valencia hispanica and Aphanius iberusG. holbrooki were able to competitively displace A. iberus populations. Mosquitofish introductions were originally supported by governmental health agencies to control mosquito populations, which are vectors of various diseases, such as malaria G. holbrooki were initially introduced into a pond in southern Spain in 1921 G. holbrooki throughout the Mediterranean basin G. holbrooki have probably contributed to its successful integration into Iberian fish communities The eastern mosquitofish, Ne), with reduced effective sizes intensifying the loss of diversity due to genetic drift Temporal fluctuations in population size reduce the average effective population size  provided by the Agriculture, Fisheries, Food and Environment Department of the Autonomous Community of Catalonia. All work was performed in compliance with and approved by the Ethics Committee of the University of Girona and meets the requirements stated by the Spanish (RD53/2013) and Catalonian (D214/1997) laws of animal care, and experimentation.G. holbrooki were collected from 15 sites along three watersheds  in northeastern Spain. The largest of these rivers is the Ter, with a basin area of 2955 km2, with its headwaters in the Pyrenees and its upper course being partially snow-fed. The Fluvià (974 km2) and Muga (758 km2) are typical Mediterranean streams with smaller watersheds, and have their headwaters located in mountainous areas. All three rivers are subject to a Mediterranean climate, with severe summer droughts and autumn floods Phragmites australis). We also analyzed 36 individuals from the Potomac River (Washington) and 16 individuals from Brunswick (North Carolina), because it has been suggested that these populations are the closest to the main American source of G. holbrooki individuals that were introduced to Europe A total of 556 Gambusia holbrooki specimens from Iberian rivers were collected from the riverbank using dip nets. All samples were collected from July to August 2010 and, to minimize any seasonal effects on population demography, only adult individuals born during the spring of the same year were selected by discarding females with a standard length of less than 2.5 cm and more than 3.5 cm, and males with a body length of less than 2.0 cm in situ, and then preserved in 96% ethanol until DNA was extracted at the laboratory.Pooc-G49, Mf13, and Gafμ3, Gafμ5, Gafμ6, Gafμ7, Gaaf7, Gaaf9, Gaaf10, Gaaf13, and Gaaf15), with two optimized multiplex PCR as described in Diez-del-Molino et al. (submitted). Both multiplex PCR were conducted under the same conditions: 30 µl of reaction volume containing 5–15 ng genomic DNA, 0.34 µM of each primer, 200 µM dNTPs, 1.5 mM MgCl2, and 0.75 units of Taq polymerase. The PCR cycling conditions were as follows: initial denaturation at 94°C for 3 min, followed by 35 cycles of 30 s at 94°C, 90 s at 60°C, 90 s at 72°C, and ending with a final extension of 10 min at 72°C. Forward primers were fluorescently labeled, and genotype peaks were resolved on a 3130 Genetic Analyzer and using GeneMapper 4.0 software .For each fish that was collected, genomic DNA was isolated from the caudal muscle using the Realpure Genomic DNA extraction toolkit  following the manufacturer’s instructions. Genomic DNA was stored at –20°C until further use in Polymerase Chain Reactions (PCRs). Variation was analyzed at 11 loci (OH) and the number of alleles per locus (A). Genetic diversity was also measured using the estimated expected heterozygosity (EH) and allelic richness (r) from allele frequencies using FSTAT 2.9.3 Genetic diversity within each study site was estimated from direct counts as the mean observed heterozygosity (STF) and significance values were calculated using FSTAT software. To assess the relevance of stepwise mutations on population differentiation (STR), an allele permutation test was performed with 1000 randomizations that simulated the distribution of allele sizes and STR values using SPAGEDI version 1.1 EH and STF) within basins were compared among basins using permutation tests in FSTAT (1000 permutations). Non-parametric Wilcoxon signed-rank tests were used to compare allele richness and gene diversity (EH) between upstream and downstream collections within each study basin.Pairwise population differentiation (STF/(1-STF) and geographical distance was log-transformed for these analyses Nm) between populations pairs (Nm =  (1 – STF)/4STF) and their geographical distances (both variables log-transformed). Negative relationships indicate IBD, and the slope (b) of the linear regression (Log(Nm)  = a + bLog(d), where d equals geographical distance) is –1 for one-dimensional stepping stones models and –0.5 for the two-dimensional models Isolation-by-distance (IBD) within and among watersheds was estimated from the correlation between genetic and geographical distance matrices among sampling sites. We used geographical distances, rather than hydrographical distances; because natural dispersal within linear river basins or human-mediated translocation by road could be involved in the connectivity between locations, given their geographical proximity (less than 50 km on average) and anastomosed road network . The geoK) over sampled individuals was estimated using the MCMC method in STRUCTURE 2.3.3 K (1 to 15) were repeated 10 times. Each run used a burn-in of 40000 iterations, a run length of 100000 iterations, and the model of independent allele frequencies. The most likely value of K was selected following Evanno The minimum number of homogeneous units (STF). These analyses were conducted using the STF matrices from single-locus information corrected by the presence of null alleles (FREENA software). We identified the main barriers for each locus, and only retained those confirmed by at least six loci.Major genetic discontinuities in the study area were assessed using Monmonier’s algorithm in BARRIER 2.2 Analyses of molecular variance (AMOVA) were conducted in ARLEQUIN 3.5 6 iterations were performed until the MCMC chains reached stationarity . Migration parameters were estimated by sampling every 1000 iterations after a burn-in of 106 iterations. Delta values were adjusted following the BAYESASS manual recommendations. Five runs using different starting points were performed, and the results with the highest likelihood were retained.Contemporary migration rates among populations were estimated by using the Bayesian inference as implemented in BAYESASS 3.0 software Mf13, Gaaf15, and Gaaf9 loci) to nine (Gaaf13 locus). Average allelic richness (r) ranged from 2.45 in ES (Fluvià River) to 3.31 in CO (Ter River) (OH) ranged from 0.336 in OF (Fluvià River) to 0.475 in PP (Fluvià River), and the expected heterozygosity (EH) ranged from 0.345 in BA (Fluvià River) to 0.500 in CE (Muga River). At the studied American collections, diversity levels averaged 4.45 for allelic richness, 0.465 for OH, and 0.557 for EH. After adjusting for differences in sample sizes, FSTAT permutation tests demonstrated lower allele richness (P = 0.009) and EH (P = 0.010) in the invaded Spanish locations compared to the potential American sources. Non-significant STF differentiation was detected between males and females  . The obs females . SubsequISF values. Significant null allele frequency was estimated at Gafμ5 , Gafμ6 , Gafμ7 , Gaaf10 , and Gaaf15 . No significant pairwise linkage disequilibria were observed. According to the BOTTLENECK analyses, heterozygosity excess relative to mutation-drift equilibrium was observed at CE (Muga River), BL, and MF (Fluvià River), and VE, and OY (Ter River); however, the allele-shift model test reported additional signals for a bottleneck at VE only.Deviations from HWE were detected at seven Spanish locations after Bonferroni correction . AccordiSTF and STR estimates were observed (P = 0.345), indicating that local mutations have limited effects on population structure. Significant genetic differentiation was detected between almost all population pairs . The collection OY from the Onyar tributary in the Ter basin had the largest average pairwise STF (0.194).No significant differences between on pairs , except on pairs . EstimatP = 0.062) and Ter basins (P = 0.052). Overall, among-basin differences in genetic diversity  were non-significant between upstream and downstream locations. Marginal (P = 0.067) increased divergence among the upstream locations of the three basins was indicated (P = 0.042). Non-significant differences were obtained in the Muga River basin, and an unexpected higher richness was indicated at the upstream CL location compared to the downstream TV in the Ter River basin (P = 0.034). None of these changes in allele richness between upstream and downstream locations resulted in significant differences on the estimated amount of heterozygosity.Despite the different values on estimates of average allele richness, the average expected heterozygosity, and population differentiation observed in the Iberian rivers , FSTAT pndicated . Within-r = 0.326, P<0.001). Within watersheds, only the Fluvià River displayed a significant correlation . Significant negative regression of the log-transformed effective number of migrants and geographical distances were detected for the whole data set of the studied locations in Spain. Furthermore, as the slope of the regression (b = –0.629) was closer to –0.5 than to –1, a two-dimensional stepping stone model better explained the population relationships (P = 0.052) was obtained for the whole Ter River basin (b = –0.455); however, this significance disappeared when the analysis focused on the mainstem of the river .Genetic and geographical distance matrices were positively correlated across all three watersheds (ionships . Within a) and third (c) barriers reflected the isolation of the three watersheds, with the exception of the PP location in the Fluvià River basin, which was grouped with the Muga River collections. The second (b) and fourth (d) barriers reflected the distinct genetic composition of the OY and BY locations separated from the mainstem of the Ter River by river transects where mosquitofish were not detected during our surveys. Overall, these results mainly agreed with the population relationships depicted by the two principal axes of the PCA analysis . In the hydrographical model, the variance assigned to divergence among populations within basins (8.8%) was smaller compared to the variance among river basins (10.3%). This pattern reflected the above noted population divergence among and within drainages. In the AMOVA based on the four STRUCTURE clusters, the proportion of genetic variance explained within clusters decreased to 6.8%, while the variance among these clusters increased (12.3%), probably reflecting the distinctiveness of the OY collection from locations in the mainstem of the Ter River.Overall, contemporary dispersal rates indicated an average of 21.4% of immigrant individuals at each location  per generation during a founder effect depending on the effective number (Ne) of introduced individuals (see for instance STF = STR), all of the diversity present in the invaded range should be attributed to the population sources of the invasion. Based on the average diversity in the two American collections studied here . This observation indicates minimal, if any, loss of genetic diversity in the introduced populations of the Iberian Peninsula. Nevertheless, a significant reduction in allele richness was detected at the invaded Spanish locations, because this parameter is more sensitive to bottlenecks compared to average heterozygosity Poecilia reticulata) showed strong genetic bottlenecks in genetic diversity when measured with neutral markers; yet, these populations retained substantial additive variation In colonized territories, the level of genetic diversity of the invading species is expected to be reduced compared to original sources as a result of founder events G. holbrooki in northern American drainages indicated postglacial colonization from refuge populations in Georgia or Florida G. holbrooki populations in North Carolina and northward compared to populations that occurred to the south in South Carolina and Florida Lower neutral genetic variation is often detected in populations at the limit of the distribution range G. holbrooki was first introduced to southwestern Spain in 1921, it was absent from the study basins in 1942 when insecticides (DDT) replaced mosquitofish as the major agent against malaria vectors. Malaria was eradicated in 1964 from Spain; however, mosquito control, including mosquitofish introductions, continued (reviewed in TH = 0.532), with this diversity mainly being distributed within locations (EH = 0.523).Precise historical records are not available about the introduction of mosquitofish to the study basins. While 1/2Ne per generation of diversity losses), or more bottleneck-generations with larger Ne. In fact, signals of recent bottlenecks were detected at 30% of the locations, and affected populations from all river basins.Significant genetic divergence among study locations indicated the isolation of current mosquitofish populations both within and among the three studied basins. According to Smith et al. G. holbrooki populations exhibited similar patterns to that observed for the endemic killifish Aphanius iberus, in which increased gene flow was observed between populations during floods STF = 0.46) has been detected among remaining native populations of the three-spine stickleback (Gasterosteus aculeatus) In the lowlands, differentiation among BAYESASS indicated current relevant migration rates between some neighboring population pairs in all of the study basins. This phenomenon resulted in BAPS clustering the VM and CE locations in the Muga River, the BL and ES and the BA and OF in the Fluvià River basin, and the CL and VE in the Ter river basin. These location pairs were separated by a distance of 5.3 km on average (range 3.50–8.28), with significant dispersal occurring both downstream and upstream (BA to OF). High positive spatial autocorrelation of allele frequencies at hydrological distances of 6–150 km has been observed in American drainages  observed between the log-transformed effective number of migrants (Nm) and the geographical distances also supported a two-dimensional stepping stone model of gene flow. Thus, in addition to active linear and flood-mediated dispersal along river basins, human-mediated translocations between road-neighboring populations from separate basins have probably contributed to the spread of mosquitofish in the area (for instance to OY and BY in the Ter river basin). In Australia, unreported and unregulated human-mediated dispersal has led to the introduction of mosquitofish to areas outside of its first sites of introduction