Batrachoseps attenuatus) inhabiting the six major islands of San Francisco Bay, formed following the rising of sea level around 9,000 years ago.Island populations are excellent model systems for studies of phenotypic, ecological and molecular evolution. In this study, molecular markers of mitochondrial and nuclear derivation were used to investigate the evolution, structure and origin of populations of the California slender salamander  where the evidence suggests that salamander populations are wholly or in part, the result of anthropogenic introductions.Comparison of the molecular data and the interpretations drawn therefrom with an earlier morphological study of many of the same salamander populations show some of the same evolutionary trends.B. attenuatus in the San Francisco Bay Area and its islands. With the two noted exceptions, it is clear that most island populations were established prior to the 9,000 years since the formation of the Bay. Results of coalescence-based analyses suggest that for most island populations the mtDNA lineages from which they were derived date from the Pleistocene.In spite of marked differences between the evolutionary rates of the two kinds of molecular markers, both indicate distinctive and similar patterns of population structure for It can be said that, based on observed values of genetic diversity, the last 9,000 years of evolution on these islands have been characterized by relative stability, with the occasional extinction of some haplotypes or alleles that were formerly shared between island and mainland populations but overall maintaining high levels of variation (with the exception of Alcatraz). In contrast, there is some evidence for rapid morphological changes between populations in some islands and their closest mainland counterparts. This pattern of rapid morphological divergence  is similar to that observed in other studies about recent colonization of island habitats. Islands have long been the subjects of both theoretical and empirical studies in Evolutionary Biology. They are often regarded as "natural laboratories" for the study of speciation because geographic isolation promotes morphological, ecological and genetic divergence of newly established populations with respect to their mainland counterparts and thus they offer an excellent opportunity to identify key factors in species formation. In more recent times, the use of molecular markers has added impetus to the analysis of the complex processes involved in differentiation of island populations. The study of colonization patterns and the effects of isolation on divergence may allow identification of potential source populations either from the mainland or other islands in a stepping stone or sweepstake model of colonization. Characterization of patterns of gene flow, with the addition of a temporal component, especially in cases when geological information is available allows dating with some precision the amount of time passed since populations have been isolated.Uroctonus mordax may have prevented the establishment of terrestrial isopods. However, on the remaining islands where these isopods form a substantial part of the slender salamander diet (pers. obs.) their presence is likely to have had a positive effect on salamander populations.The islands of the San Francisco Bay  constitute an interesting system from an evolutionary perspective because their origin is relatively recent (Holocene) and thus their study might provide clues to understanding the consequences of population isolation after the interruption of gene flow following the rising of the sea level, especially in a short time scale . The islands have been studied from different perspectives, including detailed geological, botanical and faunistic surveys . We noteAmong the faunal surveys of the San Francisco Bay islands, the salamanders in particular have been the subjects of thorough studies, including the description of morphological variation and the collection of extensive demographic data on these island populations . This stBatrachoseps attenuatus). Dispersal is very rare in this salamander, which has some of the lowest reported home ranges in vertebrates and exhibits strong phylopatry, promoting isolation by distance. Anderson [Of the two salamander species present on the islands of the San Francisco Bay area, the most abundant is the California slender salamander (Anderson  advanced1) Populations were continuously distributed before sea levels rose 9,000 years ago and were subsequently isolated as hills or mountaintops became islands. In this case, we would expect to find haplotypes that are exclusively found on islands and similar values of genetic diversity on mainland and island populations.2) Island populations were established more recently, after sea levels rose, by rafting or by anthropogenic introduction. In this case, haplotypes found on islands would include a subset of those found in nearby mainland populations and levels of genetic diversity would be lower on islands due to founder effects.Of course, each island constitutes a different "experiment", and test results may favor one or the other hypothesis, or combinations of both in different islands. For instance, a third possibility would be that populations were present before sea levels rose and were thereafter augmented by occasional immigrants by rafting. However, with the data at hand it is problematic to discriminate between cases where we find a mixture of exclusive (island) and widespread (mainland) haplotypes on islands because of incomplete sorting of polymorphisms  and cases where the same pattern is caused by a long history of isolation followed by occasional immigration introducing mainland genetic variants in island populations.B. attenuatus [B. attenuatus in the study region, including their most plausible evolutionary origin. This phylogeographic study indicated that populations are geographically structured and exhibit a significant pattern of isolation by distance, with populations in geographic proximity generally exhibiting greater genetic similarity, except in areas of narrow parapatry where some of the five well-differentiated mtDNA lineages identified in that study come into secondary contact. Several of these mtDNA lineages within B. attenuatus coexist in the vicinity of the San Francisco Bay, making more detailed examination necessary to understand the origin and evolutionary affinities of the island populations. In this paper, we use mitochondrial DNA (mtDNA) and allozyme data (genetic data of nuclear origin) to test hypotheses about the origin of island populations and to reconstruct their evolutionary history. These results are then used to discuss the patterns of morphological differentiation between islands and between island and mainland populations described by Anderson [A previous detailed phylogeographic study of tenuatus  providesAnderson .B. attenuatus on the islands of San Francisco Bay. Both of them include samples from all islands studied originally by Anderson [We compiled two independent, complementary datasets to investigate the evolution of populations of Anderson  Fig. 1)B. attenuAnderson . For Wesb gene were used to analyze patterns of genetic variation at different spatial scales in B. attenuatus. Details of DNA extraction, amplification and sequencing procedures are reported in a previous paper [DQ348971–DQ349064). The remaining 439 sequences were generated for this study and are also deposited in GenBank (Accession Numbers: FJ417408–FJ417846).Partial sequences of the mitochondrial cytochrome us paper . The finStandard estimates of genetic variability in selected island and mainland populations, including number of segregating sites, haplotypic diversity, nucleotide diversity and average number of nucleotide differences within populations were estimated with the software package DNASP .B. attenuatus and to investigate the phylogenetic affinities of island populations  prior to phylogenetic analyses, which were based on Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian inference as implemented in the software packages PAUP, GARLI and MrBayes v3.1.2 [At the broadest spatial scale, phylogenetic analyses were used to identify the main mtDNA lineages within us study ). Sequens v3.1.2 -8.Because haplotypes found on islands did not form monophyletic groups in phylogenetic analyses, we further investigated population history by inferring population (rather than gene) trees in the Southern_N clade, the most widespread in the study area. We used the "minimize deep coalescences" method  as impleb to reflect a priori uncertainty in this parameter based on the values reported by Mueller [Batrachoseps dating from 5.3 million years ago have been recovered in the vicinities of the San Francisco Bay Area [Finally, in order to estimate the temporal scale associated with the evolutionary history of island populations, we estimated the time to the most recent common ancestor (TMRCA) of selected groups of haplotypes. These groups include the five main mtDNA lineages identified in a previous study , referen Mueller . We alsoBay Area , thus pr2 and 0.01 M Tris adjusted to neutrality with hydrochloric acid. Tissue homogenates were stored frozen until needed for electrophoretic assay.Approximately 1 to 2 mm of tissue excised from the tail tip of the salamanders was mechanically homogenized in an estimated five volumes of grinding buffer composed of 0.25 M sucrose containing 10 mM mercaptoethanol, 0.001 mM EDTA, 0.1 M MgClFor electrophoresis we used a standard horizontal starch-gel system -19. The O and HE), number of private alleles, and allelic richness per population were calculated with the software package Biosys-1 [A total of 1148 individuals from 14 populations were scored at 21 loci. Estimates of observed and expected heterozygosity  with a highest posterior probability without taking into account prior information on the number of sampling localities. We used an admixture model with correlated allele frequencies for values of K ranging from one (panmixia) to eight clusters, and performed five runs for each value of K, with a "burn-in" period of 500,000 iterations and posterior searches of 1,000,000 MCMC iterations. Individuals with 10% or more missing data were excluded from the analyses, which included 949 individuals from the 14 populations sampled.We tested for genetic structure in the allozyme dataset with three methods. First, we estimated pairwise FT values  and theiT values . AdditioT values  with BioT values  using thT values . FinallyT values  to inferet al. (2007) [B. attenuatus where salamanders of two of the haplotype clades have been found in sympatry. Finally, samples from Brooks Island grouped with Martínez-Solano et al.'s (2007) [In the islands of San Francisco Bay we found mtDNA haplotypes corresponding to three of the five mtDNA lineages identified in Martínez-Solano . (2007) . The rems (2007)  Eastern In general, levels of mtDNA variability are high in all islands studied, with unique haplotypes found on each island with the exception of Alcatraz, where only one haplotype was found. This haplotype also occurs in the Angel Island population. With this exception, all other haplotypes found on Angel Island and all from Red Rock are unique to these two islands, whereas in the remaining islands we found a mixture of exclusive island haplotypes and those shared with mainland populations Table . For exaPhylogenetic analyses performed on datasets composed of all haplotypes for each mtDNA clade plus an outgroup chosen from its sister lineage did not in general resolve well. Some nodes, however, were strongly supported in all analyses Figure . In the The program "Mesquite" found 11 population trees that minimized the number of deep coalescences in the best ML gene tree for the pruned Southern_N dataset, including all island as well as reference mainland samples. Figure vs Tiburon and Brooks vs Albany Hill, see Table Results of BEAST analyses are summarized in Table Levels of polymorphism were also high in the allozyme dataset, with values of mean number of alleles per locus ranging from 1.4 in Alcatraz to 2.3 in Presidio and Marin Headlands and percentages of polymorphic loci of 20–45%  North Bay populations  [et al. (2007) [et al. (2007) [The results of "Structure" analyses suggest that l., 2007 ); 2) Easl., 2007 ); and 3). (2007) . Assignm. (2007) : Brooks . (2007) , althougA feature of the allozyme data that is in contrast to that of the mtDNA findings for the island populations is the infrequent occurrence of unique characters. Whereas each island has one or more unique mtDNA haplotypes (Alcatraz Island excepted), private alleles, that is, alleles that occur on a single island at very low frequency, are less common than in the mainland samples Table . There aBatrachoseps attenuatus, we have collected them at the high tide mark in both East Marin and Red Rock Islands, indicating some degree of salt tolerance in this species  that rafting was a common occurrence, distinguishing between the two hypotheses could become problematic because the impact of founder effects would be diminished.Seawater is generally considered to be an effective barrier retarding dispersal in amphibians. However, recent studies have shown that rafting is the only possible explanation for the presence of certain frog species on oceanic islands far removed from any mainland source . In the ies . These findings are consistent with a recent introduction involving individuals from Angel Island, in keeping with the proposal of previous studies [ST values , with high assignment probabilities in all cases, also highlighting the genetic distinctiveness of these populations among the total. This is in agreement with Anderson's [With respect to the evolutionary history of salamander populations in the study region, data derived from both mitochondrial and nuclear genes support the existence of three distinct evolutionary units, although there are discordances with respect to their individual limits. The two major barriers identified by both mtDNA and allozymes seem to be associated with the Colma and Raccoon Straits Figs. . Higher Morphometric variables studied by Anderson (1960)  includedB. attenuatus is in fact a complex of cryptic species. However, values of DNei between the Eastern populations from Brooks Island and Albany Hill and the remaining populations range from 0.079–0.142 , suggesting that this contact zone was established naturally rather than being related to anthropogenic introductions. However, the allozyme data for the Yerba Buena population suggest a complex history with discordant pictures derived from the mitochondrial and nuclear data. This population stands out from others as genetically distinct, and although "Structure" analyses suggest that this is clearly an admixed population with assignment probabilities to inferred clusters always <70%, some analyses, including the preferred structuring into three clusters, indicate a significant genetic input from populations in the Eastern clade Table . This isAnderson  considerBatrachoseps populations may have been small enough to have had their gene pools affected by detectable genetic drift. East Marin and West Marin Islands are in close proximity, the channel separating them being only 170 m. wide and of shallow depth. It seems probable that prior to their isolation from each other, and notwithstanding the low vagility of Batrachoseps, the two populations would have constituted a single deme. This conjecture is bolstered by analyses of the allozyme data, where values for pairwise Nei's genetic distances and FST are, excepting those separating Brooks Island and Albany Hill, the lowest in the dataset . In contrast, there is some evidence for rapid morphological changes between populations in some islands and their closest mainland counterparts  [In summary, our study provides a general picture of the results of 9,000 years of independent evolution of salamander populations on at least four of the islands in San Francisco Bay: East Marin, Red Rock, Angel and Brooks Island. Additionally, signal of older historical events that predate island formation is also prominent in our dataset; genetic and morphometric data delineate well-differentiated lineages that diverged from a common ancestor in the Miocene . These ern, see . In addiichmond) . This paichmond) -43.IMS collected samples, compiled the mtDNA dataset and analyzed both datasets. RL collected samples and compiled the allozyme dataset. Both authors wrote the manuscript, read and approved the final version of the manuscript.Appendix 1. Sampling localities, including mtDNA clade, latitude and longitude coordinates, sample sizes and voucher numbers.Click here for fileAppendix 2. List of enzymes surveyed. EC = Enzyme Commission.Click here for file


Anopheles nili is a widespread efficient vector of human malaria parasites in the humid savannas and forested areas of sub-Saharan Africa. Understanding An. nili population structure and gene flow patterns could be useful for the development of locally-adapted vector control measures.An. nili from Senegal, Ivory Coast, Burkina Faso, Nigeria, Cameroon and the Democratic Republic of Congo (DRC).Polymorphism at eleven recently developed microsatelitte markers, and sequence variation in four genes within the 28s rDNA subunit (ITS2 and D3) and mtDNA (COII and ND4) were assessed to explore the level of genetic variability and differentiation among nine populations of All microsatellite loci successfully amplified in all populations, showing high and very similar levels of genetic diversity in populations from West Africa and Cameroon  and much lower diversity in the Kenge population from DRC . Bayesian clustering analysis of microsatellite allelic frequencies revealed two main genetic clusters in the dataset. The first one included only the Kenge population and the second grouped together all other populations. High Fst estimates based on microsatellites  were observed in all comparisons between Kenge and all other populations. By contrast, low Fst estimates  were observed between populations within the second cluster. The correlation between genetic and geographic distances was weak and possibly obscured by demographic instability. Sequence variation in mtDNA genes matched these results, whereas low polymorphism in rDNA genes prevented detection of any population substructure at this geographical scale.An. nili gene pool was found across its distribution range in West and Central Africa, although demographic events probably resulted in a higher level of genetic isolation in the marginal population of Kenge (DRC). The role of the equatorial forest block as a barrier to gene flow and the implication of such findings for vector control are discussed.Overall, high genetic homogeneity of the  Anopheles gambiae s.l. and, to a lesser extent, Anopheles funestus whereas research on other important vectors has critically lagged behind. Here, the first results of a large-scale population genetics analysis of the level and distribution of  genetic diversity in the mosquito Anopheles nili are presented.The recent shift in strategic emphasis from malaria control to elimination and eradication has highlighted major gaps in knowledge that need to be addressed before such achievement is contemplated -3. VectoAnopheles nili is a widespread efficient vector of Plasmodium parasites in the humid savannas and forested areas of sub-Saharan Africa . Liksly used . Moreovesly used ,56. NoneAn. nili population from DRC observed with both mtDNA genes. Apparent segregation of different haplotypes between these two genetic clusters in An. nili prompts for further investigation to increase sample sizes and the geographic span of sampling eastwards and southwards of the present study area.Genetic homogeneity within the rDNA genes is not surprising, given the particular evolutionary dynamics of the rDNA operon subject to concerted evolution [e.g. ], renderet al  togetheThe authors declare that they have no competing interests.CN, CAN, IM and FS designed the study and monitored its implementation. CN, CAN, PAA participated to field sampling. CN conducted microsatellite genotyping and gene sequencing, under guidance of PK and IM. CN, PK, DA, AC and FS participated to data analysis. CN, CAN and FS wrote the manuscript which was critically revised by AC, DA, PN and DF. DC was involved in the design of the maps contained in this paper. All authors read and approved the final manuscript.Supplementary table S1. Estimates of null alleles frequencies per locus and per geographical population of An. nili.Click here for file


Genetic ancestry is known to impact outcomes of genotype-phenotype studies that are designed to identify risk for common diseases in human populations. Failure to control for population stratification due to genetic ancestry can significantly confound results of disease association studies. Moreover, ancestry is a critical factor in assessing lifetime risk of disease, and can play an important role in optimizing treatment. As modern medicine moves towards using personal genetic information for clinical applications, it is important to determine genetic ancestry in an accurate, cost-effective and efficient manner. Self-identified race is a common method used to track and control for population stratification; however, social constructs of race are not necessarily informative for genetic applications. The use of ancestry informative markers (AIMs) is a more accurate method for determining genetic ancestry for the purposes of population stratification.st), allele frequencies and their suitability for efficient genotyping. Genotype analysis using CoAIMs along with a Bayesian clustering method (STRUCTURE) is able to discern continental origins including Europe/Middle East (Caucasians), East Asia, Africa, Native America, and Oceania. In addition to determining continental ancestry for individuals without significant admixture, we applied CoAIMs to ascertain admixture proportions of individuals of self declared race.Here we introduce a novel panel of 36 microsatellite (MSAT) AIMs that determines continental admixture proportions. This panel, which we have named Continental Ancestry Informative Markers or CoAIMs, consists of MSAT AIMs that were chosen based upon their measure of genetic variance (FCoAIMs can be used to efficiently and effectively determine continental admixture proportions in a sample set. The CoAIMs panel is a valuable resource for genetic researchers performing case-control genetic association studies, as it can control for the confounding effects of population stratification. The MSAT-based approach used here has potential for broad applicability as a cost effective tool toward determining admixture proportions. Population stratification refers to the subdivision of a population into different ancestral groups having different allele frequencies and different disease prevalence. In contemporary populations there has been recent admixture between individuals from different populations resulting in variable ancestry, e.g. in African American and Hispanic populations. Population stratification can act as a confounding factor in genetic studies, such as genome-wide association studies (GWAS), where the presence of uncontrolled population structure can lead to false-positive or false-negative findings Reliably detecting population structure can be difficult. Perhaps the simplest approach to control for population stratification is to use the self-reported race/ethnicity of the study participants. While in some populations and for some studies this information may be sufficient, it is inadequate when considerable admixture is present, such as exists in North America An alternative and more accurate approach to detect population stratification is to determine genetic ancestry using AIMs. AIMs are polymorphic markers that exhibit high allele frequency differences among parental populations  and can be used to accurately estimate individual admixture and identify population structure We have developed CoAIMs, a comprehensive panel of 36 MSATs suitable for contemporary genetic research with the potential future clinical applications. This set of AIMs (1) differentiates among continental groups including Europe/Middle East (Caucasians), East Asia, Africa, Native America, and Oceania, (2) accurately measures individual ancestry proportions in admixed populations, and (3) is efficient and cost-effective.Table S1. All samples are available from Coriell Cell Repositories at the Coriell Institute for Medical Research .DNA samples used as reference material for parental populations were generously provided by the National Institute of General Medical Sciences (NIGMS) and the National Human Genome Research Institute (NHGRI). These include 234 samples from the NIGMS Human Population Collection as follows: Oceania ; East Asia ; Native American, also referred to as Americas ; Africans of sub-Saharan ancestry ; Caucasian, Europe ; Caucasian, Middle East . The population controls also included 476 HapMap samples from NHGRI as follows: East Asia ; Africans of sub-Saharan ancestry ; Caucasian, Europe . 385 samples from the National Institute for Neurological Disorders and Stroke (NINDS) Repository generously provided 385 samples with self-declared ancestry: 92 Caucasian from North America (NDPT020), 92 African American (NDPT111), 20 Asian American, 92 Caucasian Hispanic from North America (NDPT112), 20 non-Caucasian Hispanic from North American, 25 American Indian, and 14 Pacific Islander and 30 of undeclared or mixed race. Genomic DNA derived from either peripheral whole blood or lymphoblastoid cell lines, were utilized in this study. No human subjects were recruited for this study; de-identified samples were obtained from the NIGMS, NHGRI and NINDS Repositories at Coriell. A list of samples used can be found in http://rosenberglab.bioinformatics.med.umich.edu/). The dataset included genotype results for 783 MSATS on 1048 samples from 7 distinct worldwide geographical locations. This panel of samples has been used in multiple population genetic studies Genotype data from the Human Genome Diversity Panel http://ccr.coriell.org/Sections/Search/MSK.aspx?Ref=MSK&PgId=202) Table S2 for a list of the MSATs).Population controls from the NIGMS and NHGRI collections were initially genotyped using the ABI Identifiler panel  comprised of the 15 Combined DNA Index System (CODIS) markers, and the Coriell Identity Mapping kit (6-plex) (Table S3). The groupings were based upon MSAT base pair size ranges. One primer from each pair was fluorescently-end labeled with PET, VIC, 6FAM, and NED . Each PCR was setup with 30 ng of genomic DNA, 0.4 µl AmpliTaq Gold (5 U/µl), 5.0 µl primer mix, 2.0 µl dNTPs (2.5 mM), 2.0 µl MgCl2 (25mM), 2.5 µl 10× PCR buffer, and deionized H2O to 25 µl and the PCR cycling conditions are in Table S4. The multiplex PCR products were analyzed by capillary electrophoresis using an Applied Biosystems 3730 DNA Analyzer. The NIGMS and NHGRI parental population samples were genotyped to set marker bins to generate the expected genotype calls using the ABI GeneMapper v3.5 software as well to analyze the fragment sizes, colors and intensities of the PCR products.The CoAIMs panel was optimized to be genotyped in three multiplex PCR reactions, each containing 12 primer pairs (Distruct program Population structure was inferred using a Bayesian clustering approach implemented using STRUCTURE v2.3 To determine the best estimate or “fit” of the correct number of population clusters (K), all sets of markers tested were run with varying numbers of markers with K ranging from K = 2 to K = 12, and five replicates performed at each value of K. To statistically determine the correct number (K) of clusters for a given dataset, we used StructureSum, an R script Principal component analysis (PCA) was performed to test further the number of population clusters observed with STRUCTURE. This method can be used to infer population structure by clustering samples into groups based upon ancestral groups st) is a measure of surplus of homozygotes within subpopulations, and is also used to examine the overall genetic divergence among subpopulations. Fst values range from 0 to 1; markers with the highest values are most informative for ancestry determination st values for each genetic marker tested, and was also used to perform pairwise Fst calculations. Pairwise Fst values provide a measure of the inter-population genetic variance as compared to intra-population genetic variance.F-statistic (FTable S2). Parental population samples from 6 continental regions (Table S2) were genotyped using the combined marker panel and the results were analyzed using both STRUCTURE We determined if combining two MSAT panels used at Coriell as part of routine quality control and using current software tools would be useful for determining population structure. The markers in both the ABI Identifiler panel and the Coriell Identity Mapping Kit (6-plex) were developed to identify unique individuals Figure S1). These results further that this marker panel is insufficient for determining genetic ancestry, and indicate that a more informative set of MSAT AIMs is required to adequately address genetic ancestry.The genotype data generated with the 19 marker panel were analyzed using PCA. Individual specific Principal Component (PC) values, when plotted, can be interpreted according to geographic origins in silico analysis of publically available genotype data. Following the identification of a set of markers, the second step is to independently confirm the ability of these markers to distinguish among population groups in a separate set of population samples. Finally, the ability of the markers to assess continental admixture proportions in samples of self-declared ancestry will be tested.To identify an informative set of MSAT AIMs to determine genetic ancestry, a two step approach was undertaken. The first was to identify a minimal panel of markers through the In Silico identification of a panel of markers involved the analysis of downloaded genotype data from the HGDP (see st) values were determined for all 783 MSATs genotyped on this sample panel. Of the 783 MSATs, 78 of these markers displayed values ≥0.1, and were selected for further analyses. Pairwise Fst values of these 78 markers .Both PCA and STRUCTURE demonstrated that the 78 markers identified via Froups , , demonstThe 3 smaller groupings of markers  were tested further using STRUCTURE and PCA. Analyses of the STRUCTURE data for the 48 and 36 MSAT panel indicated that five population groups (K = 5) are the best fit for both datasets, which effectively resolve the five major continental population clusters Figure 2Figure S2). Taken together, the results provide evidence that a minimal panel of 36 MSATs can be used to distinguish human ancestries from five major continental regions with the same efficiency as a larger panel of 78 MSATs. The panel of 36 MSATs that comprise this set of markers, listed in Table S3, has been termed CoAIMs, for Continental Ancestry Informative Markers.Similarly, PCA analyses Figure 3in silico analyses of datasets representing previously genotyped samples of the HGDP. To confirm further that CoAIMs can distinguish among the continental population groups, the 36 MSATs were optimized to be genotyped in three multiplex PCR reactions consisting of 12 markers each . Moreover, pairwise Fst analyses further demonstrate high intercontinental Fst values. Similar to the results obtained with HGDP data, the two closely related Caucasian populations from Europe and the Middle East displayed small pairwise Fst values  assumed ranging from two to twelve (K = 2 to K = 12). As the number of population clusters assumed, K, is increased from K = 2 to K = 5, so increases the number of distinguishable population groups Figure 4). Taken together, results obtained using 710 NIGMS and NHGRI ancestral population samples allowed independent verification of the results obtained via in silico experiments using a publically available HGDP genotype dataset.PCA analyses Figure 5DNA samples from the NINDS Human Genetics DNA and Cell Line Repository were examined with CoAIMs to determine continental ancestral proportions. Currently, more than 29,000 samples from diverse ethnic groups have been banked and more than 4,000 have been used in GWAS of Parkinson's disease, ALS, and other disorders  and  and Based on previous studies of self-declared ancestry Hispanic populations are known to have Caucasian, African, and Native American ancestries The current study was performed to develop a set of AIMs for efficiently and reliably discerning among continental population groups. The results presented here demonstrate the development and utility of CoAIMs, a 36-MSAT panel that measures genetic ancestry. This MSAT-based approach uses a routine and cost-effective genotyping methodology. CoAIMs can be used to determine continental ancestry and admixture, as well as to cluster individuals from a cohort into discrete ancestry groups to control for the confounding effects of population stratification in genetic studies. The clustering patterns observed for the five major continental ancestries studied here are similar to those obtained in other studies using larger sets of markers st values, and STRUCTURE and PCA results of genotyping parental population samples confirm that these markers can distinguish five discrete population groups: Caucasian (European and Middle Eastern), African, East Asian, Native American and Pacific Islander. Interestingly, our primary decision in marker choice was based upon high informativeness value (Fst), and not the specific repeat structure of the MSAT (i.e. dinucleotide vs. tetranucleotide). Previous studies have shown that dinucleotide repeats are more stable and are more suitable for determining population structure Both SNPs and MSATs have been used to determine genetic ancestry. SNP AIM panels that require large numbers of markers (>100) to determine continental ancestry have been described We hypothesized that CoAIMs can be used to measure continental admixture proportions in subjects of admixed descent. To test this hypothesis we examined individuals from heterogeneous population groups, utilizing DNA samples banked in the NINDS Repository. Analyses of African American individuals demonstrated admixture proportions (African ancestry 0.81±0.20 and 0.14±0.20 Caucasian ancestry) similar to those observed using SNP AIMs panels consisting of a large numbers of markers In addition to African Americans, individuals from two separate Hispanic populations were examined representing those self-declared as Caucasian, and those self-declared as non-Caucasian. Genetic studies of Hispanic populations have displayed a trihybrid ancestral population structure between Caucasian, Native American and African populations. The proportions of these three ancestral population groups varied greatly Measurements of genetic continental ancestral proportions in samples of self-declared ancestry by CoAIMs is made possible by the inclusion of ancestral population groups. In the analyses performed here, individuals from the NIGMS and NHGRI Repositories were used as parental population reference groups. As expected, NIGMS and NHGRI samples from the Caucasian, African and East Asian populations separated into highly discrete population clusters. This is in contrast to the Native American ancestral population group which displayed individuals with measurable proportions of European ancestry. These individuals were identified as Mexican and Mexican Indian descent from the NIGMS Human Population Collection. Previous genetic studies of these population groups have shown them to have European admixture The complex genetic heterogeneity that exists within admixed populations often confounds genetic association studies, a major application of AIMs is to control for these adverse effects of population stratification in GWAS. CoAIMs can be readily applied to GWAS as an efficient method to adjust for the differences in continental ancestry between cases and controls. This panel can be used to include or exclude subjects from a study cohort based upon continental ancestral proportions. This would be particularly effective for studies involving Hispanic or African American populations that contain wide ranges of admixture among ancestral populations. Additionally, applying CoAIMs prior to performing whole-genome genotyping can eliminate the expense of high throughput SNP genotyping of extraneous samples. Ultimately, the use of CoAIMs can help facilitate a better understanding of the significance of existing GWAS data as well as future genetic studies in both Caucasian and non-Caucasian populations.Finally, it is becoming increasingly evident that many health-related traits are influenced by an individual's genetic ancestry. For example, increasing proportions of Native American ancestry have been associated with milder asthma among Mexican Americans Figure S1PCA Plots of the Top Three PCs of the set of 19 MSATs (ABI identifiler and Coriell 6-plex). Plot of PC1 vs. PC3 (A) which captures 56.1% and 7.2% of the total variation of the data. Plot of PC2 vs. PC3 (B) which captures 22.9% and 7.2% of the total variation of the data.(0.19 MB TIF)Click here for additional data file.Figure S2PCA Plots From Analysis of the HGDP MSATs. (A) PC1 vs. PC3 and (B) PC2 vs. PC3 for the 78 MSAT marker set. (C) PC1 vs. PC3 and (D) PC2 vs. PC3 for the 48 MSAT marker set. (E) PC1 vs. PC3 and (F) PC2 vs. PC3 for the36 MSAT marker set. (G) PC1 vs. PC3 and (H) PC2 vs. PC3 for the 24 MSAT marker set. In all cases, the addition of the third PC does not allow for further separation of population cluster.(0.16 MB TIF)Click here for additional data file.Figure S3PCA Plots of CoAIMs with the NIGMS and NHGRI Population Samples. Plot of PC1 vs. PC3 (A) and PC2 vs. PC3 (B) of the CoAIMs panel with the NIGMS and NHGRI population samples.(0.17 MB TIF)Click here for additional data file.Table S1Samples from the NIGMS, NHGRI and NINDS Cell Repositories used.(0.08 MB DOCX)Click here for additional data file.Table S2The 19 MSATs of the ABI Identifiler and Coriell Identity Mapping kit and their Fst values.(0.04 MB DOC)Click here for additional data file.Table S3MSATs of the CoAIM panel.(0.02 MB DOCX)Click here for additional data file.Table S4PCR conditions of the 3 multiplex PCRs for CoAIMs.(0.01 MB DOCX)Click here for additional data file.


Fundulus heteroclitus as well as closely related Fundulus species .By targeting SNPs contained in both coding and non-coding areas of the genome, we are able to identify genetic differences and characterize genome-wide patterns of variation among individuals, populations and species. We investigated the utility of 454 sequencing and MassARRAY genotyping for population genetics in natural populations of the teleost, Fundulus. Specifically, pyrosequencing identified 96 putative SNPs across coding and non-coding regions of the F. heteroclitus genome: 88.8% were verified as true SNPs with MassARRAY. Additionally, putative SNPs identified in F. heteroclitus EST sequences were verified in most (86.5%) F. heteroclitus individuals; fewer were genotyped in F. grandis (74.4%), F. majalis (72.9%), and F. similis (60.7%) individuals. SNPs were polymorphic and showed latitudinal clinal variation separating northern and southern populations and established isolation by distance in F. heteroclitus populations. In F. grandis, SNPs were less polymorphic but still established isolation by distance. Markers differentiated species and populations.We used 454 pyrosequencing and MassARRAY genotyping technology to identify and type 458 genome-wide SNPs and determine genetic differentiation within and between populations and species of Fundulus genome and provide markers for population genetic studies.In total, these approaches were used to quickly determine differences within the  High throughput sequencing and genotyping has become increasingly faster, less expensive and more accurate. In recent years this has lead to the establishment of myriad data sets ranging from increased coverage of variation in the human genome at the individual level -5 to theFundulus, includes closely related species that range in physiology, environmental and habitat preference, and geographic locales; Fundulus heteroclitus and Fundulus majalis inhabit the Atlantic coast, and Fundulus grandis and Fundulus similis inhabit the Gulf Coast. Many Fundulus species and/or populations have extensive euryhaline capabilities, respond well to varying ranges of hypoxia [F. heteroclitus [One non-model genus,  hypoxia -15, live hypoxia . A varieroclitus -27 much Fundulus. Once markers are established they can be further studied to look for signatures of selection to any number of evolutionary forces . A few studies have established genetic differences between populations of F. heteroclitus mainly with respect to phylogeographic constraints [Establishing a set of genetic markers, which can be used to assess regions of the genome involved in local adaptation and in speciation is important to understand fundamental similarities and differences between populations and species of straints ,29 or sestraints -38. ThesFundulus populations and species.SNPs have been used to establish differences between individuals , populatF. heteroclitus were collected using minnow traps during the spring of 2005. Spleen and testes were sampled from 20 individuals from each of ten collection sites along the East coast of the United States  and Corpus Christi, Texas .s Figure . F. gran9 Figure . Fin cliet al. [Genomic DNA from spleen and testes was extracted by phenol and chloroform as described in Wirgin et al. , and DNAet al.  and DNA F. heteroclitus genomic DNAs (500 ng) from eight individuals in each of ten collection sites  and 2 μL of diluted product was amplified with primers  method. For the Bayesian method, 10ion rate . At the Prior = 1 × 10-4 represents the mutation rate; N represents the total number of unique mapping loci with multiple allelic types; Ai and ai represent, respectively, the major and minor alleles at locus i; Ni represents the total number of alleles observed for locus i, and Yj is the type of the jth allele copy among these Ni alleles where j = 0 ⋯ Ni; finally, ej is the probability of error of the jth allele where the error probability is computed as Q is the corresponding quality score after rescaling.For the Bayesian model, for each contig, ith locus being homozygous or heterozygous is:The posterior probability for the Based on the posterior probabilities from above, we classified each of these N loci as homozygous or heterozygous exclusively. If a locus was classified as heterozygous, it was further tested using a likelihood ratio test (LRT) as follows:i on the contig:For a particular locus Xj stands for the true allele that we should have observed. For each Yj, we have an error probability of ej associated with it.where Then we have:Therefore we have:andI was computed as:Based on all of the above, the likelihood of locus Ij = 1 if Yj = Ai; and Ij = 0 if Yj = aiWhere HO: p = 0.5 versus Ha: p >0.5 and -2 × LRT ~ χ2(1).The LRT was performed with the hypothesis of Ai and randomly simulated Ni number of alleles copies to be Ai or any of the other three allele types from a uniform distribution with probability (1 - ej) and ej respectively. We repeated this process 10,000 times and recorded the different numbers of second alleles found in the simulation. The critical value was chosen as the number of second alleles with a right-side p-value of 0.001.In order to call SNPs at the population level, we simulated the error model for each locus with multiple allelic types; we assumed that a particular locus was homozygous with major allele F. heteroclitus individuals, 90 F. grandis individuals, 23 F. majalis individuals, and 21 F. similis individuals were attempted using the Sequenom MassARRAY technology. These consisted of 81 putative SNPs identified by the F. heteroclitus pyrosequencing, 350 putative SNPs previously identified in F. heteroclitus ESTs [e.g., K = G or T). Reaction conditions were performed by iPLEX chemistry as recommended by Sequenom across 13 plates at the University of Minnesota's BioMedical Genomics Center. SNP genotypes were called using the Sequenom System Typer 4.0 Analysis package. This software uses a three-parameter model to calculate the significance of each putative genotype. Based on the relative significance, a final genotype is called and assigned a particular name . Non-calls also were noted .Multiplex assays targeting 458 SNPs in 250 tus ESTs , and 27 tus ESTs , lactatetus ESTs , and theF. heteroclitus and F. grandis) by calculating the percentage of polymorphic SNPs (PO), observed (HO) and expected heterozygosity (HE), and the within-population fixation index (F) [F. heteroclitus populations within North and South regions. For F. grandis, the AMOVA was performed to calculate the distribution of variance within populations as well as between populations longitudinally along the Gulf of Mexico. Since SNPs were initially identified from F. heteroclitus sequence data, a maximum of 5% missing data was used as a parameter for calculations involving F. heteroclitus and 10% for all others.Arlequin v.3.11 was used to calculate genetic diversity among populations (of ndex (F) . FixatioF. heteroclitus and F. grandis.A Mantel test was performed to assess the assumption of isolation by distance using XLSTAT 2009 for F. heteroclitus, F. grandis, F. majalis and F. similis along both the Western Atlantic and the Gulf of Mexico and to assign individuals to these populations. The Monte Carlo Markov Chain was run for 105 iterations following a burn-in period of 105 iterations for K = 1 to 14 using the correlated allele frequencies model and assumed admixture. Distruct v. 1.1 [STRUCTURE v.2.2 ,68 was ut v. 1.1  was usedA total of 111,001 reads were obtained in one run of the GS FLX instrument producing 5,346,445 total bases of sequence (average read length of 218 bases) with 99.98% of bases having a quality score of 20 or greater. Across the eight regions of the plate, there were on average 1,982 reads per individual. The third barcode produced many less reads per region  amongst all regions. All other barcodes performed very similarly with respect to the number of reads per individual across regions. Only 46% of the number of expected reads  were obtained from sequencing. Prior to sequencing, the amplification success of loci on the beads was checked for quality using a Bioanalyzer 2100, and all samples passed. However, three of the eight regions produced half the expected number of reads and a fourth region produced only 15% the expected number of reads. This indicated local problems in sequencing with respect to particular regions and the samples in those regions rather than the plate as a whole. All control beads passed the filter control with an average percentage of 90% across all regions, whereas the percentage of samples passing the filter control varied between regions and averaged 36%: regions with fewer than expected reads had fewer samples passing the filter control. Two regions had very high failure rates due to mixed samples, indicating more than one amplicon per bead.2 = 0.01).Upon alignment 1,464 contigs were obtained with an average length of 213 base pairs. The average coverage across all loci was 22 reads per contig  were polymorphic, 8 loci (9.8%) were monomorphic, and one locus did not amplify. Among all other putative SNPs genotyped with MassARRAY, 83% were successfully amplified. However, 13.5% of all loci in F. heteroclitus, 25.6% in F. grandis, 27.1% in F. majalis and 39.3% in F. similis did not amplify  were not carried out for these two species.On average, 80% (SD = ± 7.4%) of the putative SNPs identified with 454 pyrosequencing were amplified with MassARRAY in y Figure . Many nos Figure . Due to F. heteroclitus. These SNPs may represent segmental duplication where the two duplicate regions are identical, except that a SNP has been driven to high frequency or become fixed in one of the duplicates.SNPs which were identified by Sequenom software as low probability in greater than 50% of all individuals were removed . An additional 20 SNPs were excluded from analyses due to their excessive heterozygosity across individuals and populations of PO) ranged from 3.7% to 67% (Table F. heteroclitus where levels decreased in populations further north and east (p = 0.035). Among F. grandis populations, the percentages of polymorphic SNPs did not significantly differ along latitude (p = 0.143) or longitude (p = 0.415). Among populations, most loci were in Hardy-Weinberg equilibrium (Table HO) among all populations ranged from 0.016 to 0.17 with a mean of 0.10 (Table F. heteroclitus in comparison to southern populations (p = 0.04) and did not differ along latitude (p = 0.72) or longitude (p = 0.33) in F. grandis. Average expected heterozygosity (HE) ranged from 0.019 to 0.20 with a mean of 0.11 .SNPs identified F. heteroclitus had a frequency greater than 0.10 and were considered common SNPs  in F. heteroclitus clustered north to south and F. grandis as its own separate cluster. Among F. heteroclitus, individuals from Maine and Georgia, the most northern and southern collection sites, formed their own distinct clusters. Individuals from sites between Maine and Georgia clustered with others from geographically similar sites. F. majalis and F. similis clustered together and away from the other two species. Similarly, in the PCA analysis, which does not rely on modeling the data, northern and southern F. heteroclitus stratified by latitude and were distinct from each other ) identified species and population differences in all samples Figure . STRUCTUr Figure  and eachr Figure . F. grans Figure .F. heteroclitus, AMOVA showed that most of the variation was distributed within populations (59.05%), but another large proportion of variation (31.1%) was distributed among northern and southern regions. The remaining 9.85% of variation was explained by differences among populations within regions. In F. grandis, most of the variation was distributed within populations (82.4%), and a smaller proportion (17.6%) of variation was distributed longitudinally between populations across the Gulf of Mexico.In F. heteroclitus populations (p < 0.001) and F. grandis populations (p = 0.032).A Mantel test showed significant isolation by distance among Fundulus genus. Genotype data sharply differentiated northern and southern populations of F. heteroclitus as well as other species in this genus . Within the species where SNPs were originally annotated, most can be successfully verified and used to study population structure as well as the role and outcome of selection forces on a genome-wide scale.We used high throughput sequencing and genotyping technology to identify and verify SNP markers in four non-model species within the Eucalyptus ESTs identified 0.05% of contigs containing SNPs [Using the 454 FLX pyrosequencing system, we observed 111,001 reads yielding an average of 22× coverage across 1,464 contigs. Read lengths and quality scores were similar to many other studies using the 454 FLX system to sequence uncharacterized genomes ,70, but ing SNPs  and pyroF. heteroclitus. Thus, we attempted to perform genome reduction with selective PCR reactions to approximately 200 loci that could be sequenced in 10 populations of 8 individuals. With 30,000 reads per one-eighth of a 454 sequencing plate, each region would have 15× coverage per individual or 980× coverage across all populations, enabling accurate genotype calls for most individuals. However, preselective amplication was not perfect, and many more than 200 loci were sequenced; most amplified only a single time in a single individual (these singlets therefore were not useful for variant detection). Furthermore, we obtained only 46% of the expected number of reads. In the end, these problems led to the inability to directly call individual genotypes. We were hoping to both identify SNPs and genotype individuals in a single step, but a more successful approach  is to mF. heteroclitus cDNAs using the MassARRAY system. Similar percentages of 454 pyrosequencing derived SNPs and SNPs identified from ESTs were verified . Of the 458 putative SNPs, 379 (82.75%) were polymorphic, but only 264 had a greater than 90% successful call rate among all individuals. Among F. heteroclitus, most SNPs amplified  indicating that differences in amplification rate between species led to the lower overall call rate. In white spruce, 91% of SNPs verified with the Illumina SNP bead array platform [F. heteroclitus, 70% of SNPs in spruce were called in greater than 95% of individuals [To increase our ability to measure population genetic parameters within and among populations, we verified SNPs identified through 454 sequencing and additional SNPs annotated from platform ,72 were ividuals . OverallF. heteroclitus, F. grandis and F. majalis and similis as well as population structure within F. heteroclitus . PCA and STRUCTURE did not differentiate sister species, F. similis and F. majalis, from each other or establish population structure within these species. Small sample sizes , high levels of monomorphism , and the fact that only 10% of SNP alleles differed between these two species, decreased the power to detect such differences when analyzed in conjunction with F. heteroclitus and F. grandis. Population structure also was masked in F. grandis when data was analyzed with other species. However, when F. grandis individuals were analyzed separately, they also showed distinct population structure (data not shown). One other study has reported multiple fixed differences in mitochondrial sequences between F. heteroclitus and F. grandis [Species differentiation was demonstrated using principle component analysis (PCA) as well as STRUCTURE analysis. Both analyses showed separation between s Figure . These a grandis , but no F. heteroclitus and F. grandis species, within-population fixation indices  ranged from 0.09 to 0.32. Among F. heteroclitus, all populations had an overall significant deficiency of heterozygotes indicated by positive FIS values. In these populations, approximately 10% of loci had similarly very large FIS values (>0.5) across populations causing the skew in the average FIS value for each population. Within a population, these loci were predominately homozygous for one allele with a complete absence of the heterozygote and one or a few individuals homozygous for the alternative allele. The loci which presented this pattern were called conservatively at both alleles by Sequenom software across all individuals indicating that genotyping error was not the main reason for this pattern.Within F. grandis. Among F. grandis populations, most (70%) SNPs with high FIS values were different between populations. This is in contrast to F. heteroclitus populations where loci with high FIS values were shared across populations. Within any one F. grandis population, one allele was predominant as a homozygote with one or a few individuals with the alternative homozygote. The most parsimonious explanation is that there is undetected substructure.Furthermore, all northern populations were predominately homozygous for one allele and all southern populations were predominately homozygous for the alternative allele indicating strong demographic patterns in the data. The same demographic pattern was not found in F. heteroclitus. In F. grandis, SNPs were shown to lack polymorphism (7.18%). The higher percentage of monomorphic loci in F. grandis likely is due to ascertainment bias in SNP discovery caused by only using F. heteroclitus populations. Many of the monomorphic loci (24%) represent fixed differences between F. heteroclitus and F. grandis. Thus, while SNP markers developed in F. heteroclitus are not necessarily polymorphic in other Fundulus species, they still can be used to differentiate F. heteroclitus from other species.SNPs in Hardy-Weinberg were shown to be moderately polymorphic (average of 60%) in F. heteroclitus populations, genotype data revealed strong latitudinal clines between the Northern and Southern F. heteroclitus populations. PCA, STRUCTURE, FST values, and the isolation by distance test identified that individuals from Northern populations (above 40-41°N) were distinct from Southern populations. This split is centered around the southern-most extent of the Atlantic coastal advancement during the late Pleistocene [F. heteroclitus in the south [Among istocene . Specifiistocene  numerousistocene -36,75 anistocene . The larhe south  would maST of 0.20 among northern populations in comparison to that of an average FST of 0.10 among southern populations. This statistic is also evident for the north and south split, where populations from respective regions had an extremely high FST value of 0.44 when compared against one another. Similar genetic divergence has been reported for F. heteroclitus using microsatellites . Simila Georgia  and mari Georgia  and blue Georgia , and, asF. grandis, and a Mantel test shows significant isolation by distance. However, there were no significant differences between either levels of polymorphism or observed heterozygosity along latitude or longitude. Williams et al., 2008 reported significant isolation by distance as well as decreased allelic richness with increasing latitude. In this 2008 study, microsatellites were used, and two additional sites southern to those used in our study were included. Since microsatellites have many more alleles than SNPs and two additional sites were found to have relatively higher allelic richness in comparison to all other sampling sites along the gulf, this may account for the differences found in levels of polymorphism.A similar latitudinal cline occurs between populations of Fundulus genome. Similar studies using high throughput methods to sequence SNP markers have been developed in Atlantic cod [Eucalyptus [Eucalyptus) or for use in breeding programs (swine)By targeting SNPs contained in both coding and non-coding areas of the genome, we are able to better understand how evolutionary forces are shaping the ntic cod , white sntic cod , Eucalypcalyptus , and swicalyptus . Like ouLMW designed experiments, carried out laboratory and statistical analyses, and drafted the manuscript. XM carried out SNP detection of 454 data. ARB carried out SNP detection of 454 data, assisted in statistical analyses of MassARRAY data, and provided comments on earlier versions of this manuscript. CDB assisted in designed experiments and help develop SNP detection software. MFO designed experiments, assisted on statistical analysis, and helped to draft the manuscript. All authors read and approved the final manuscriptSNP minor allele frequencies. Distributions of SNP minor allele frequencies (MAF) within F. heteroclitus and F. grandis populations.Click here for file


Alu insertions reveal different degrees of polymorphism in human populations, and a series of characteristics that make them particularly suitable genetic markers for Human Biology studies. This has led these polymorphisms to be used to analyse the origin and phylogenetic relationships between contemporary human groups. This study analyses twelve Alu sequences in a sample of 216 individuals from the autochthonous population of Galicia (NW Spain), with the aim of studying their genetic structure and phylogenetic position with respect to the populations of Western and Central Europe and North Africa, research that is of special interest in revealing European population dynamics, given the peculiarities of the Galician population due to its geographical situation in western Europe, and its historical vicissitudes.The most recent Alu elements analysed were within the variability range of European populations, while Yb8NBC125 proved to be the lowest so far recorded to date in Europe.The insertion frequencies of eleven of the Taking the twelve polymorphisms into account, the GD value for the Galician population was 0.268. The comparative analyses carried out using the MDS, NJ and AMOVA methods reveal the existence of spatial heterogeneity, and identify three population groups that correspond to the geographic areas of Western-Central Europe, Eastern Mediterranean Europe and North Africa. Galicia is shown to be included in the Western-Central European cluster, together with other Spanish populations. When only considering populations from Mediterranean Europe, the Galician population revealed a degree of genetic flow similar to that of the majority of the populations from this geographic area.The results of this study reveal that the Galician population, despite its geographic situation in the western edge of the European continent, occupies an intermediate position in relation to other European populations in general, and Iberian populations in particular. This confirms the important role that migratory movements have had in the European gene pool, at least since Neolithic times. In turn, the MDS and NJ analyses place Galicia within the group comprised of Western-Central European populations, which is justified by the influence of Germanic peoples on the Galician population during the Middle Ages. However, it should also be noted that some of the markers analysed have a certain degree of differentiation, possibly due to the region's position as a 'cul-de-sac' in terms of Iberian population dynamics. Alu sequences are a type of transposon from the SINEs group that are found in the genome of primates, into which they have inserted and expanded over the last 65 million years . Th. Th46]. Also, the conditions of anonymity and confidentiality established by the Ethics Committee of the University of Santiago de Compostela were strictly adhered to during the process of obtaining the samples.Alu insertions , typed using DNA isolated from peripheral venous blood extracted from voluntary healthy donors, without close biological parentage and with a level of Galician autochthony up to the third preceding generation.The genetic characterisation of the Galician population was carried out by analysing the polymorphism of twelve autosomal The extraction of genomic DNA from the leukocyte fraction of the blood samples was carried out using the GFX Genomic Blood DNA Purification kit from GE Healthcare. The extracted DNA was stored at -20°C.Alu insertions were typed by PCR amplification, using the protocols and primers previously established by other authors, then carrying out electrophoresis in 2% agar gel and staining using ethidium bromide (0.5 μl/ml); visualisation and documentation of the gels was carried out using the Gel Doc 2000 system (BioRad) for gel analysis and documentation.The twelve Alu loci typed were calculated by direct counting. The Hardy-Weinberg equilibrium test was carried out using the exact test to calculate the P values from the Markov chains in the Monte Carlo method [The allele frequencies of the twelve o method  using veo method . Intrapoo method .Alu insertion frequencies in populations from the selected areas that had been previously published in the scientific literature.In order to analyse the phylogenetic relationships of the Galician population with other populations in its geographic surroundings, a comparative analysis at two levels was carried out, using data on Alu insertions analysed in this study were typed, meaning that in the comparative analysis we omitted the Alu polymorphisms Sb19.3, Sb19.10, Sb19.12, Ya5NBC221, Yb8NBC120 and Yb8NBC125, and in order to use a number of markers that provides more reliable results, the three Alu polymorphisms were included  that had been previously studied in Galicia by this team [The first level was carried out including populations from Central and Southern Europe, as well as Northern Africa, together with the Galician population in order to make the comparison Figure . It shouhis team . This mehis team ,41,51-53In the second level, using the same markers, a comparison was made exclusively between the Galician population and European Mediterranean populations, with the aim of analysing the model of the genetic structure in the populations in this geographic area.The relationships of affinity between the Galician population and other European and North African populations were analysed using non-metric multidimensional scaling (MDS)   based onReynold's genetic distance matrix between pairs of populations  formed tAlu element were carried out using the Arlequin 3.11 programme [CT, FSC and FST), a simultaneous contrast was carried out, taking into account the nine genetic markers included in the analysis, whereby for each component the critical global level (Pn) was calculated based on the critical levels (pi) obtained for each of the nine Alu elements (n). The statistic used is defined by the formula:In order to determine if the genetic variation is structured spatially, in the first comparison the MDS and NJ analyses were complemented with an analysis of molecular variance (AMOVA) from Excoffier et al. (1992) . The AMOrogramme . With thwhose distribution fits a chi-square distribution with 2n degrees of freedom.ri, which is given by the formulaIn order to study the geographic model of the genetic structure of the populations from the European Mediterranean area, in the second level of analysis the model of Harpending and Ward (1982)  was usedpi and P, respectively, are the frequency of the Alu insertion in the population i, and in all of the populations included in the analysis as a whole.where In turn, Harpending and Ward propose an island model of population structure in which there is a lineal relationship between heterozygosity and the distance to the centroid:hi and H correspond to the heterozygosity value in the population i and in all of the populations as a whole.where All of the authors responsible for producing this work have made an equal contribution, and they have agreed on the final manuscript.Table 1. This file contains the data on insertion frequencies and genetic diversity of the Galician population.Click here for fileTable 2. Results of the AMOVA analysis in three population groups based on a geographical criterion and according to nine Alu markersClick here for fileTable 3. Number of inhabitants in the districts of Galicia and the contribution made by each of them to the population sampleClick here for file


Glossina palpalis gambiensis from the Niayes area, controversies remain as to the best strategy implement, i.e. “eradication” versus “suppression.” To inform this debate, we used population genetics to measure genetic differentiation between G. palpalis gambiensis from the Niayes and those from the southern tsetse belt (Missira).The Government of Senegal has initiated the “Projet de lutte contre les glossines dans les Niayes” to remove the trypanosomosis problem from this area in a sustainable way. Due to past failures to sustainably eradicate CO1 DNA, and geometric morphometrics of the wings) were used on 153 individuals and revealed that the G. p. gambiensis populations of the Niayes were genetically isolated from the nearest proximate known population of Missira. The genetic differentiation measured between these two areas (θ = 0.12 using microsatellites) was equivalent to a between-taxa differentiation. We also demonstrated that within the Niayes, the population from Dakar – Hann was isolated from the others and had probably experienced a bottleneck.Three different markers  help decision on appropriate tsetse control strategies and (ii) find other possible discontinuities in tsetse distribution. Glossina palpalis gambiensis. To reach this objective, two different strategies may be used: suppression (decrease in tsetse densities) or eradication . For eradication, the approach has to be area-wide, i.e. the control effort targets an entire pest population within a circumscribed area, to avoid any possible reinvasion. Three different tools  were used, and all showed an absence of gene flow between G. p. gambiensis from the Niayes and from the nearest known population in the south east of the country (Missira). This genetic isolation of the target population leads to the recommendation that an eradication strategy for the Niayes populations is advisable. This kind of study may be extended to other areas on other tsetse species.Tsetse flies transmit trypanosomes to humans (sleeping sickness) and animals (nagana). Controlling these vectors is a very efficient way to contain these diseases. There are several strategies and methods that can be used for control, each being more or less efficient depending on several factors. The Government of Senegal wants to sustainably eliminate trypanosomosis from the Niayes region by controlling the tsetse vector,  Glossina palpalis gambiensis Vanderplank, which is a major vector of the debilitating diseases Human African Trypanosomosis (HAT) or sleeping sickness, and African Animal Trypanosomosis (AAT) or nagana , or if a sequential control strategy can be contemplated ., which will also depend on their respective history, including effective population sizes and possible bottlenecks.The level of isolation of the targeted tsetse populations will be an important parameter to guide the Government of Senegal to select the most optimal control strategy. Here we report population genetic analyses of microsatellite and mtDNA markers combined with morphometrics of  country  to assesIn the Niayes region, four tsetse populations were sampled using Vavoua traps: Dakar Hann which is a swamp forest harbouring an animal park within the city of Dakar, Diacsaw Peul, an area of riparian thicket where tsetse and cattle are in intense contact, Sebikotan and Pout, which are mango and citrus-tree plantations where tsetse and people are in close contact. The tsetse flies collected in the Niaye area and analyzed in the present study are the sole property of the Senegalese authorities. They were collected by the national veterinary services through official mission orders, in one wildlife park (Parc de Hann) and three private sites, with the oral consent of the owners. No written consent is mandatory for tsetse fly collection in Senegal. In the south-eastern part of the country, the area of Missira was sampled: it is the nearest known infested area from the Niayes, according to a detailed tsetse survey implemented as part of the baseline data collection of the Niayes tsetse Control project see . Areas bIn total 153 tsetse individuals were analysed originating from Diacsaw Peul , 8 males (M)), Dakar Hann , Sebikotan , Missira , and Pout .A total of 10 microsatellite markers were used (preceded by “X” for X-linked loci): X55.3 F-statistics F-statistics can be defined. FIS is a measure of the inbreeding of individuals (hence I) resulting from non random union of gametes within each sub-population (hence S). FST is a measure of the relatedness between individuals resulting from non-random distribution of individuals among sub-populations, relative to the total population; FST quantifies the differentiation between sub-populations in the total population (hence S and T). FIT is a measure of the inbreeding of individuals resulting both from non-random union of gametes within sub-populations and from population structure . These F-statistics are classically estimated by Weir and Cockerham 's unbiased estimators f (for FIS), θ (for FST) and F (for FIT) ierfstat version 0.03–2 F-statistics from any number of hierarchical levels Wright's F-statistics was tested by randomization (10000 permutations in each case). The significance of FIS was tested by randomizing alleles between individuals within sub-samples. The significance of FST was tested by randomizing individuals among sub-samples. These tests were performed with Fstat. The significance of FSR, i.e., the homozygosity due to subdivision into subsamples within the Niayes region, was tested by randomising individuals among subsamples of the Niayes. The significance of FRT, which measures the relative homozygosity due to the geographical separation between the Niayes region and Missira was tested by randomizing subsamples  between the two regions. These two last tests were performed with Hierfstat. For FIS the statistic used was directly the f (unbiased estimator of FIS). For other differentiation measures , the statistic used was the maximum likelihood ratio GThe significance of the FIS>0) may be due to the presence of null alleles. We used Micro-Checker 2.2.3 np at each locus according to Brookfield's second method Non random association of alleles within individuals  across loci with a FIS based analyses males were coded as missing data at these loci, otherwise these loci were coded as homozygous .Some loci are X-linked. For C-S&ED) In order to visualize the partition in genetic differentiation among all samples, a NJTree dendrogram was constructed with the software MEGA 3.1 r) can be estimated as p and q are frequencies of allele A at locus 1 and allele B at locus 2, respectively and D is the so called −FIS/(1+FIS) Effective population sizes were computed with three methods. Estim 1.2 led e.g.  Burrow'sFor intra locus based methods , loci showing evidence of stuttering and null alleles were removed.EH) with the heterozygosity that would be expected for a sample taken in a population at mutation/drift equilibrium with the same size and allele number (EQH). As allele number decreases faster than heterozygosity, a bottleneck is signed by EH > EQH in subsequent generations ottleneck v1.2 software Signatures of bottleneck events were investigated by comparing the expected heterozygosity for a sample (eN-pb). From the τ12eN-pb and 2τ2eN-pb generations before sampling with τ1 = 0.1 and τ2 = 2.5.The method of bottleneck detection described by Cornuet and Luikart sH , assuming no recombination were calculated using equations from STF was calculated according to equation 3 in STH was calculated according to equation 2–4, and K*ST according to equations 7–11 in STH is a haplotype frequency based genetic differentiation statistic that does not take into account the number of differences separating different haplotypes. STH = 1−(S/HTH), where SH is the weighted average of subpopulation genetic diversities and HT is the estimated haplotype diversity of the total population. STK* = 1−(S/KTK), where SK* is a weighted average of the log corrected average number of sequence difference in the populations being compared, and TK is the average number of difference between sequences. A permutation test, in which haplotypes or sequences were randomly assigned to the different localities 10000 times, was used to test the significance of STH and STK*) COI from G. p. gambiensis  were generated using the algorithm of A portion of the 5′ end of the mitochondrial gene COI was amplified, purified and sequenced using the primers CI-J-2195 In total, the number of analysed wings was 20, 21, 18, 34 and 18 for Diacsaw Peul, Dakar Hann, Sebikotan, Missira and Pout respectively. These analysed individuals were all also analysed by the molecular markers.MD), which were calculated between populations. The statistical significance of Mahalanobis distances was estimated by 1,000-runs permutation tests Wings were dry-mounted between two microscope slides and scanned with a scanner. From this picture, 10 landmarks defined by vein intersections were recorded as previously described C-S&ED and Kst*, between C-S&ED and MD, and between Kst* and MD.To evaluate the correlation between distance matrices we undertook three Mantel tests sH = 0.5) was lower than the one found in Missira (sH = 0.7) .The per-locus number of alleles was 3.4, 3.7, 3.9 and 4 for Diacsaw Peul, Dakar Hann, Sebikotan and Pout respectively , but it was 8.7 for Missira. The Wilcoxon test confirmed that mean genetic diversity in the Niayes . FIS were only due to null alleles. However, both loci seemed to suffer from stuttering. Without these four loci, the six remaining loci did not result in rejection of the hypothesis of panmixia .There was a global and significant homozygote excess over all loci .Among the 36 possible tests, four pairs of loci displayed a significant linkage at the 5% level of significance. This proportion (4/36) is not different from the proportion expected under the null hypothesis . The mean θ value between Missira and all the Niayes populations was high and significant, θ = 0.12 . Within the Niayes, the mean θ value between Dakar Hann and the three others was also high and significant θ = 0.12 , which is of the same order of magnitude as the one between Missira and the Niayes. This is illustrated in FSR = 0.069, P-value = 0.001) and an absence of significant contribution of region . This latter point is due to the fact that Dakar-Hann is as different from the other Niayes subsamples, than Niayes are from the remote Missira subsample.The overall mean A constant seemed to be that Diacsaw Peul, Dakar-Hann and Sebikotan displayed very small effective population sizes (3 to 28), and Pout and Missira appeared much bigger . However, some of the computations led to infinite population sizes and sometimes the different methods did not converge to a reliable estimator see , which sP-values of 0.001, 0.001 and 0.116 for IAM, TPM and SMM mutation models respectively; and Pout with P-values 0.012, 0.348 and 0.935 for IAM, TPM and SMM  ranges the bottleneck between 3.2 and 80 generations ago. With the 95% confidence interval upper limits (i.e. 23) the ranges increases to [5–115]. This bottleneck provides a good explanation for the genetic isolation noticed for this site during the population differentiation analysis.Only two subsamples provided some evidence for a bottleneck: Dakar-Hann with  SMM see . These vcytochrome oxidase 1 (COI) are shown in The statistics of genetic differentiation between the Niayes population samples from Sebikotan, Pout, Dakar Hann and Diasca Peuhl and the south-eastern Senegal site Missira based on 738 nucleotides of mitochondrial An analysis of the shape of the wings was conducted by using Procustes analysis to compare wing shape among the five different populations. All Mahalanobis distances between populations were significant except between Pout and Sebikotan. This is illustrated by reclassification scores which were over 70% indicating differentiation between populations see . MissiraThe correlation between the genetic distances and morphometrric distances were all strong and significant see . The strG. p. gambiensis population of the Niayes and those of the main tsetse belt in the south-eastern part of Senegal. Using three different kinds of markers, i.e. microsatellite DNA, mitochondrial DNA and geometric morphometrics, the data led to the conclusion that the G. p. gambiensis population from the Niayes can be considered isolated with very little risk of re-invasion should the population eventually be eradicated. These data corroborate the results of the entomological baseline data collection and observations in the field indicating the absence of G. p. gambiensis in the 120 km long area between Missira and the Niayes . On this basis, eradication of G. p. gambiensis from the Niayes can therefore be recommended as an appropriate control strategy. The data from this study, which was part of a comprehensive baseline data collection effort, confirmed that eradication can be recommended as an appropriate control strategy, and as such the study greatly assisted in the decision making on which strategy to select. Although the isolation of the target population is not an absolute prerequisite for AW-IPM, tackling a continuous pest populations is actually more complex, requiring more resources and a long-term, regional commitment . It is also noteworthy that some of the microsatellite loci used on the individuals in this study amplified poorly, a behaviour not recorded in earlier studies of G. p. gambiensisG. p. gambiensis since such cryptic species have been suspected and shown to exist in closely related taxa Looking at G. p. gambiensis from the Niayes and those which should be released if they do not originate from the same area (e.g. from Burkina Faso). Although previous studies with G. p. gambiensis originating from Mali and Burkina Faso revealed no mating barriers between these populations , nor did another study between G. p. gambiensis and G. p. palpalisG. p. gambiensis  and an annual precipitation below 400–500 mm might have induced the development of some level of xerotolerance of these relatively small populations In case the Government of Senegal decides to integrate the sterile insect technique (SIT) as part of the AW-IPM (area-wide integrated pest management) approach, it would be advisable that prior to the release of sterile males, experiments be conducted to assess mating compatibility between the G. p. gambiensis haplotypes have probably been lost from the Niayes region, leaving just two haplotypes remaining today. The low diversity was also observed at microsatellite loci with lower genetic diversity than in Missira. The three different markers used in this study generally showed good agreement in the differentiations observed, as can be seen by the high and significant correlations coefficients computed between them.The second question that has relevance for a future AW-IPM programme was to know whether within the Niayes region, the four populations showed any genetic differentiation, or if they constituted a single, panmictic (i.e random mating) population. Low haplotype diversity of the populations of this area is in agreement with a previous Single Strand Conformation Polymorphism-based study on mitochondrial DNA haplotypes in the Niayes region of Senegal FST estimates and the small sample sizes, the effect of which is probably increased by historical events such as the probable bottleneck that affected the Dakar-Hann population. This may explain why only microsatellite data suggest that the population from Dakar Hann is so much differentiated from all the others, including the geographically closest ones. From an operational point of view however, the population of Dakar Hann is probably also isolated from the three others, since the bottleneck signature was still visible, suggesting very limited exchanges (if any) with the other populations. Field data also corroborate this since the population of Dakar Hann is located within an animal park of this huge city which can be seen as an isolated refuge for this tsetse population. This further suggests that an elimination operation may probably be implemented on this population.The only apparent discrepancy between microsatellites on one hand and COI based and morphometrics based distances on the other hand probably comes from the variance expected for paired The results also suggest that the samples from Diacsaw Peul, Sebikotane and Pout, although showing some genetic differentiation, are not completely isolated from each other. This is also consistent with ecological data since it appears that the maximum distance between the forest patches of this area is less than 2km (data not shown.).G. p. gambiensis and Trypanosomosis in the Niayes region of Senegal. In the near future, it should be encouraged to carry out such studies prior to the selection of target areas or the choice of control strategies, and these should be part of the overall collection of baseline data Click here for additional data file.Table S1Effective population size estimates according to the different methods described.(0.02 MB XLS)Click here for additional data file.Table S2Probabilities of detecting bottleneck in each population according to microsatellite mutation model.(0.02 MB XLS)Click here for additional data file.


Reticulitermes taxonomy in recent years, there is still much discussion about phylogenetic relationships. This study investigated the evolution from intra- to interspecific phylogeny in the island subspecies Reticulitermes lucifugus corsicus and threw new light on this phenomenon. An integrative approach based on microsatellites and mitochondrial and nuclear DNA sequences was used to analyze samples taken from a wide area around the Tyrrhenian sea and showed how the subspecies evolved from its origins to its most recent form on continental coasts.Although much research has been carried out into European According to mitochondrial phylogeny and molecular clock calculations, island and continental taxa diverged significantly by vicariance in the Pleistocene glacial period. However, more recently, numerous migrations, certainly human-mediated, affected the structure of the populations. This study provided evidence of direct hybridization and multiple introgressions which occurred in several hybrid areas. Analysis using STRUCTURE based on microsatellite data identified a population in Provence (France) which differed considerably (Fst = 0.477) from populations on the island of Corsica and in Tuscany in the Italian peninsula. This new population, principally distributed in urban areas, is highly heterogeneous especially within the ITS2 regions where homogenization by concerted evolution does not appear to have been completed.This study provides an unusual picture of genetic interaction between termite populations in the Tyrrhenian area and suggests that more attention should be paid to the role of introgression and human impact on the recent evolution of European termites. Reticulitermes lucifugus species was first called Termes lucifugum and for a long time was the only recognized species in Europe. Since then, extensive studies of the Reticulitermes genus in this region have shown the existence of six sibling species and a Tyrrhenian subspecies [The entomologist P. Rossi  describebspecies . The prebspecies -10.Reticulitermes lucifugus corsicus, a subspecies of a European subterranean termite [et al. found the species in two urban areas [ termite , is a span areas  and Marian areas ) and in R. lucifugus corsicus has still not been clearly defined and is known from subsidiary descriptions in general publications [R. lucifugus corsicus has been classified as an endemic subspecies of the islands of Corsica and Sardinia. Biometric data clearly puts the Corsican termites closer to continental R. lucifugus and only quantitative differences can be noted from chemical data – defensive secretions of the soldiers [R. lucifugus [ications ,11,12. Asoldiers  and cutisoldiers . Only thucifugus ,4,8, as R. lucifugus corsicus diverged from the Italian R. lucifugus. The geological history of the area is very complex [et al. [et al. [Reticulitermes genus. This area contains populations of termites that could possibly shed light on the past and recent evolution of the species complex, from the first speciation events to possible hybridization. More than two centuries after Rossi's discovery, Tuscany could once more be a region that may provide useful information for the study of European termites.These characteristic differences probably developed after isolation on the islands and confirm that the taxon is endemic. However, it is difficult to date accurately at what stage  complex -17. The  complex , later s [et al.  and Luch [et al.  is that  [et al. ,20 in thReticulitermes lucifugus corsicus termite evolved, from its origins to its recent appearance in continental coastal areas. An integrative approach was used to clarify the phylogenetic relationship with the Italian R. lucifugus termite and to detect any hybrid forms, using various genetic markers [This phylogeographic study in the Tyrrhenian area reveals how the island  markers , such aslucifugus (L) and corsicus (C) taxa matching sequences already in Genbank [The COII sequences of 60 samples  found in many different localities, clade C had lower genetic variability than clade L , which had a 'Corsican' nuclear haplotype. This lack of concordance between nuclear and cytoplasmic genotypes could be an example of the asymmetrical introgression already described by Avise et al [The nuclear sequences (ITS2), which generally evolve more slowly than the organelle genome ,23, werese et al .R. lucifugus and Marseille-C: R. l. corsicus) were found in Marseille  as well as a colony with a mix of both haplotypes for the same individual , and Follonica  in Tuscany may both originate from an admixture between subspecies. The second run (K = 3) showed a new population nested in the R. lucifugus population in Provence, France. The Bastia colony (Corsica) formed a separate cluster for K = 4 and particularly for K = 5. The Marseille-C colony was partly assigned to this Bastia group . Finally, by over-estimating K, the program attributed colonies to populations. This could indicate genetic variability within the R. lucifugus peninsular group. However, this illustrated the homogeneity in the populations in the island samples , colonies that had apparently been introduced (Bastia) and colonies in Provence .Five microsatellite loci were analyzed. Heterozygosis was often low (Mean Ho = 0.16) with a mean of 3.83 alleles per locus  also appeared between the two groups. Axes 2 (12.42%) and 3 (10.49%) separated R. lucifugus colonies into two populations: one from Italy and one from Provence. Close to these clusters, the Bastia colony formed a separate population which was much more similar to the population in Italy. Finally, the lucifugus Italian group included colonies originating from unexpected geographical regions such as Valle d'Alesani (Corsica), Vitrolles-A and Marseille-O (Provence). Based on these results, the genetic differentiation between the three populations was calculated using F-statistics , but, among R. lucifugus taxon, the Provencal population is also highly separated to the Italian population (Fst = 0.265).The results obtained using factorial correspondence analysis (FCA) also showed a similar distribution into 4 populations Figure . Axis 1 Reticulitermes form three clearly distinct clades with a high level of divergence. The genetic distances between the island clades  and the mainland clade (L) are similar  period (18 kyr to 12 kyr) [Reticulitermes populations, Luchetti et al. argued that the social and geographical structure of populations could significantly increase the mtDNA substitution rate [Reticulitermes taxa have older origins than previously thought.The large number of mutations that have accumulated between the three clades shows a long-term separation without any interbreeding. The different hypotheses suggesting that all present-day European  refugia ,8,12,18  [et al.  and Hewi [et al. . More re 12 kyr) . This ti 12 kyr) . Some en 12 kyr) -35 and f 12 kyr) ,37. Many 12 kyr) ,39. To jion rate ,40. But ion rate  which woR. l. corsicus is also a possibility, with ancestral origins from an isolated population in an Ice Age refugium. Thus, each taxon would have been separated from taxa from different refuges . This could explain why the three clades have almost the same number of substitutions (about 20 nucleotides) and consequently the same divergence time. As the Corsican mitotype is closer to the Calabran (19 nucleotides) than to the Tuscan (23 nucleotides), the populations in Corsica and Sardinian may have started to diverge before recolonization of Italy.It is, however, difficult to determine whether dispersion or vicariance play a greater role in cladogenic events ,42. The et al. [Reticulitermes, as the divergence times are similar [R. lucifugus could correspond to a 'phalanx' expansion mode [The expansion on the continent is confirmed by mitochondrial and microsatellite data. Colonization was slow and may have lasted several thousand years. At the mitotype level, this expansion through the Italian peninsula generated a South/North cline, already reported by Uva et al. . The exp similar . Moreoveion mode . The micion mode ). HoweveReticulitermes were introduced into unexpected zones by human transport [R. lucifugus in Bordeaux (France) , R. grassei in Devon (UK) ([R. santonensis in Hamburg (Germany) and Santiago (Chile) [R. urbis in France and Italy . Three colonies -Bastia, Viareggio and Marseille-C- appear to have been introduced. Of these, the colony in Bastia is of particular interest as it forms an entity, neither entirely Provencal   and the presence of microsatellite alleles characteristic of Tuscany along the East coast of Corsica  could also indicate relatively recent hybridizations. In general, these traces of hybridization tend to diminish and even disappear by backcross with local populations.Coptotermes gestroi (Rhinotermitidae) by Jenkins et al [In Southern France, the Provence region may be a specific new hybrid zone. Among the multiple copies of the ITS2 gene, some colonies Figure  have a mns et al . The conns et al , and in R. lucifugus and R. l. corsicus groups , by various taxonomic criteria  and also by this complex phyletic evolution . Its diReticulitermes adapt. Allowing horizontal transfers of the genetic material, better adapted forms could appear in some small populations [Reticulitermes as urban pests and study how they are adapting to cities – as for urban colonies in Provence. After thousands of years of speciation under paleogeographical constraints, human impact [Reticulitermes evolution.Nevertheless, evidence of introgression in hybrid zones could influence how ulations . The coln impact ,5,55,56-Reticulitermes samples from the Tyrrhenian region were analyzed. Some samples had been collected recently in Corsica (France) and Tuscany , others came from old collections that had been kept in a laboratory (Uva 1999) or from ad hoc donations. Of the 61 collection sites detailed in Table Various tory Uva 999 or frtory Uva 999 or frtory Uva 999 or fret al. [Total DNA was extracted from a single termite preserved in alcohol, using a modified version of the method described by Kocher et al. . PCR ampReticulitermes genus were used – mitochondrial sequences – cytochrome oxydase II gene (COII) [Two types of sequencing commonly used to study the phylogeny of the e (COII) ,6,58- ane (COII) ,58  were genotyped at six microsatellite loci previously isolated in the species flavipes  and R. stonensis  was then performed using genetix 4.05.2 software to classify the populations in different clusters . genepopTL did most of the sampling. TL, NC, DL and SD did the DNA sequencing and microsatellite genotyping. TL and NC analyzed the data and wrote the paper. AGB conceived and coordinated the study. All the authors read and approved the final manuscript.Analyzed microsatellite sequences characteristics and variability. N = number of individuals, Na = number of polymorph alleles and Ho = mean heterozygosis rate observed for each locus in the R. lucifugus and R. l. corsicus colonies.Click here for fileList of primers and PCR profiles used for amplification and sequencing.Click here for file