Finally, we genotyped samples from 18 populations from Latin America using the AIMs panel and estimated variability in ancestry within and between these populations. This panel and its reference genotype information will be useful resources to explore population history of admixture in Latin America and to correct for the potential effects of population stratification in admixed samples in the region.Most individuals throughout the Americas are admixed descendants of Native American, European, and African ancestors. Complex historical factors have resulted in varying proportions of ancestral contributions between individuals within and among ethnic groups. We developed a panel of 446 ancestry informative markers (AIMs) optimized to estimate ancestral proportions in individuals and populations throughout Latin America. We used genome-wide data from 953 individuals from diverse African, European, and Native American populations to select AIMs optimized for each of the three main continental populations that form the basis of modern Latin American populations. We selected markers on the basis of locus-specific branch length to be informative, well distributed throughout the genome, capable of being genotyped on widely available commercial platforms, and applicable throughout the Americas by minimizing within-continent heterogeneity. We then validated the panel in samples from four admixed populations by comparing ancestry estimates based on the AIMs panel to estimates based on genome-wide association study (GWAS) data. The panel provided balanced discriminatory power among the three ancestral populations and accurate estimates of individual ancestry proportions , whose allele frequency varies between the ancestral groups. Once determined, these ancestral proportions can be correlated with normal phenotypes, can be associated with disease, can be used to control for confounding due to population stratification, or can inform on the history of admixture in a population. In this study, we identified a panel of AIMs relevant to Latin American populations, validated the panel by comparing estimates of ancestry using the panel to ancestry determined from genome-wide data, and tested the panel in a diverse set of populations from the Americas. The panel of AIMs produces ancestry estimates that are highly accurate and appropriately controlled for population stratification, and it was used to genotype 18 populations from throughout Latin America. We have made the panel of AIMs available to any researcher interested in estimating ancestral proportions for populations from the Americas. Most individuals from the Americas are admixed descendants of Native American, European, and African ancestors. Complex historical factors have resulted in varying proportions of ancestral contributions between individuals within and between ethnic groups In addition to providing estimates of individual's ancestral history, admixture proportions can be correlated to physiologic measurements such as spirometric measurements of lung function Several groups have described panels of AIMs designed to estimate individual ancestry and to control for the effects of population stratification in Latino populations In this paper, we describe a three-stage approach to developing a panel of 446 Ancestry Informative Markers (AIMs) optimized to characterize admixture throughout Latin America. In the first stage, we used genome-wide data from two African populations, three European populations, and six Native American populations to select AIMs that were informative, evenly distributed throughout the genome, and portable, having little within-continent heterogeneity. In the second stage, we validated the panel of AIMs in four admixed samples by comparing the ancestry estimates based on the AIMs panel with ancestry estimates based on genome-wide data. In the final stage, using these AIMs, we genotyped samples from 18 additional populations originating throughout the Americas to estimate ancestry differences within and between populations and to determine the onset of admixture for each group.nI statistic was 43.8, 44.0, and 44.0 for Africans, Europeans, and Native Americans, respectively. Because the mean locus specific branch length for European ancestry was lower than for African or Native American ancestry, there are 202 European AIMs with a median LSBL stF of 0.37 (25: 75 percentiles 0.35–0.41) and a median LSBL nI of 0.21 (25:75 percentiles 0.20–0.23). There are 115 African AIMs with a median LSBL stF of 0.63 (25: 75 percentiles 0.61–0.66) and a median LSBL nI of 0.37 (25:75 percentiles 0.36–0.40). The 129 Native American AIMs have a median LSBL stF of 0.56 (25: 75 percentiles 0.54–0.61) and a median LSBL nI of 0.33 (25:75 percentiles 0.32–0.36). The informativeness of the AIMs panel is summarized in A total of 446 AIMs were identified; the panel is presented in its entirety in 2) between ancestry estimates using AIMs and ancestry from GWAS data is high in most cases, especially for Native American and European ancestry in all three Mexican samples and European and African ancestry in the Puerto Rican sample. The correlation coefficient was lower for estimates of ancestry where there was less variance in the true ancestral proportion.2) between the genome-wide estimates and the estimates based on the panel of AIMs, as well as the mean differences, mean absolute differences and root mean square errors. As expected, reducing the number of AIMs in the panel results in decreasing correlation and increasing error of the ancestry estimates compared to the estimates produced with genome-wide data. Performance of the 194 AIMs panel, and to a lesser extent the 88 AIMs panel is comparable to performance of the 314 AIMs panel. The correlations between the estimates based on 22 AIMs and those based on genome-wide estimates are considerably worse, particularly for the estimates of African ancestry in Mexicans and Native American ancestry in Puerto Ricans, which are the ancestral components with the least amount of variance between subjects.We investigated the effect of the number of AIMs on the accuracy of the estimates of ancestry, using the parents of Puerto Rican subjects with asthma from the GALA study (n = 803) We evaluated the utility of the different panels of AIMs to control for the effects of population stratification in the Mexico City sample, which had previously been shown to have significant population stratification The panel of AIMs was carried forward to genotype a total of 373 individuals from 18 populations throughout the Americas using the Sequenom platform. Generally speaking, the platform performed well, though 75 SNPs were excluded due to lower call rates . The final analysis was based on 325 markers. Among the SNPs meeting quality control criteria, the average call rate was 91.7% . Two additional populations (Coyas and Mapuches) were genotyped but excluded from the analysis due to the low quality of the samples. Four additional individuals were excluded due to genotyping call rates of <90%.The two Afro-Colombian samples included in this study had a median African ancestry of 0.76 (0.64: 0.83) for Chocó and 0.54 (0.46: 0.69) for Mulaló. Finally, the Mestizo samples from Colombia, Venezuela and Northern and Southern Chile showed a relatively high dispersion in Native American and European admixture proportions. With the exception of some Venezuelans from Maracaibo, on the Caribbean coast, most of these individuals had small (<10%) African contributions.We also estimated the average number of generations since admixture for the Mestizo and African descendant samples . GeneralIn this study, we developed, validated, and tested a novel panel of AIMs designed to accurately estimate the ancestral components  of contemporary Latin American populations. We developed a new algorithm (provided in the web resources online) capable of taking genome-wide data from multiple populations within each continental group and identifying the most informative, well-balanced and portable markers to estimate ancestry proportions.−5: 6.2%) is not surprising. However, a small amount of European admixture would be expected to result in an underestimate of the information content of our AIMs. Although we did not include Native American populations from English-speaking North America for our analysis, our selection of markers excluded those with significant heterogeneity between Native American populations. Thus, we have no reason to believe the markers cannot be applied to North American populations, though the use of these markers for populations outside of Latin America should be pursued with caution.The ancestral samples used to identify the AIMs represented a wide variety of populations within each continental group. Specifically, we used six samples from Mesoamerica and the South American Andes as representatives of the ancestral Native American populations that make up modern Latin Americans. Our Native American samples had a median Native American ancestry of 97.7% (25: 75 range 93.2% to 100%) based on ancestry ascertainments using genomewide data. Given the history of European colonization in the Americas, a small amount of European genetic admixture . Historical records and genetic analyses indicate that most of the slaves imported into the Americas originated in West Africa By excluding markers with significant within-continent heterogeneity, the selected panel of AIMs should be broadly portable to populations from throughout the Americas. Moreover, the exclusion of markers exhibiting substantial within-continent heterogeneity serves to ensure that there is relatively little bias in the estimates of ancestral allele frequency. This is because any bias would have had to occur in all of the ancestral populations within a given continent, at a similar magnitude and in the same direction.On the other hand, by design, the AIMs panel would not be expected to differentiate within-continent population substructure. Indeed, we found that the eight Native American populations genotyped with the AIMs panel were indistinguishable in principal component space beyond the first principal component, which represented the degree of European admixture (data not shown). There are several reasons we chose to exclude markers that could have potentially been used to differentiate within-continent substructure. First, the principal reason for designing this panel was for identifying continental ancestry proportions in admixed samples, as continental admixture is the most important source of population structure in Latin Americans. Secondly, because we had a limited number of Native American ancestral groups available for study, we would have only been able to generate AIMs that distinguished Mesoamerican populations from Andean populations. Third, the use of heterogeneity filters was an important element of quality control, as it served to filter out alleles with extreme frequencies due to bias. Finally, because the genetic differences within continental groups are smaller than between continental groups, we would have required many more markers to accurately determine within-continent substructure.et al of diverse Mexican Mestizo populations (55.2%±15.4%) We validated the panel of AIMs by comparing ancestry estimates derived from the subset of AIMs to estimates derived from genome-wide data in four Latin American populations, three from Mexico and one from Puerto Rico. Overall, the ancestral estimates for both Puerto Ricans and all Mexican groups were consistent with previously published literature There was strong correlation between ancestral estimates obtained from the AIMs panel and those obtained from GWAS, providing strong support for the use of the AIMs panel to accurately estimate ancestry. For over 95 percent of the samples, the estimates of ancestry using AIMs were within 10% of the value obtained using GWAS data.2) represents the proportion of variance in the outcome variable , explained by the predictor (estimates of ancestry using AIMs), in cases where there is more limited variance in the outcome variable such as estimates of African ancestry in Mexicans and Native American ancestry in Puerto Ricans, we observe a lower R2. Nonetheless, measures of individual error in estimate, such as the root mean squared error, are comparable for all three ancestral estimates in both Puerto Ricans and Mexicans, suggesting that the panel performs consistently across all ancestral components, and in most cases, the estimate of ancestry using AIMs lies within 10% of the true measure of ancestry, as can be seen in The correlation was lower for the minor ancestral components (African ancestry in Mexican populations and Native American ancestry in Puerto Rican populations). This reflected the more limited between-subject variance in the minor ancestral component. Since the coefficient of determination , but it can be overestimated much more substantially. Conversely, the major ancestral component cannot be overestimated by more than the difference between 100% and its true value, but it can be significantly underestimated. This effect is most notable in We used the panel of AIMs to genotype 373 individuals from 18 Latin American populations. The samples were very diverse, and included individuals from several indigenous groups, African descendants and Mestizos from five different countries. Generally speaking there is strong concordance between ethnicity and admixture estimates. Specifically, seven out of eight indigenous samples showed a high degree of Native American ancestry. In particular, the four isolated groups from Venezuela  showed very little evidence of European or African admixture. The three indigenous groups from Colombia  had average Native American proportions higher than 80%, and a relatively small European contribution. That our AIMs panel could effectively estimate ancestry in lowland South American Native American populations (such as those in Venezuela) despite the fact that our AIMs were derived from Mesoamerican and Andean populations is reassuring and demonstrates that our strategy of excluding markers with significant heterogeneity ensures the generalizability of the markers. The indigenous Wichi from Argentina had considerably lower Native American ancestry and higher European ancestry  than the indigenous groups from Venezuela and Colombia. This is consistent with a recent study of Y-chromosomes that found widespread European paternal ancestry among Amerindian groups, including the Wichi, in Argentina In Bolivia, we found that the individuals from the Departments of Beni, Cochabamba and the Altiplano region of the La Paz Department had, on average, high Native American contributions. However, in the subtropical area of Yungas, many of the individuals recruited in the small community of Tocaña and one of the individuals recruited in the nearby town of Coroico had high African ancestry . The subtropical Yungas region is home to several scattered Afro-Bolivian communities. These Afro-Bolivians are the descendants of African slaves who were brought to work on the Potosi mines and coca plantations Two additional groups of African descent were included in this study, the Mulaló and Chocó from Colombia. African slaves were brought to Colombia early during the colonial period for gold mining, sugar cultivation, and cattle ranching. The proportion of African ancestry in these two Afro-Colombian groups was slightly lower than in the Afro-Bolivian community . Unlike the Afro-Bolivian sample from the Yungas region, in which most of the non-African contribution came primarily from indigenous groups, the two Afro-Colombian samples had similar European and Native American ancestral contributions . This hiet al, who examined thirteen Mestizo populations in Latin America and found extensive variation in Native American and European ancestry and relatively low levels of African ancestry Our samples from the four Mestizo populations from Chile, Colombia, and Venezuela showed a wide variability in the ancestral proportions, though the primary ancestral contributions were European and Native American. Only some of the subjects from Maracaibo, on the Caribbean coast of Venezuela, had greater than 10% African ancestry, as did some of the Puerto Rican subjects used to validate the AIMs. This is unsurprising, given that the rest of our Mestizo populations are from Mexico, Chile and the Northwest of Colombia, areas where the slave trade was not prominent. This is consistent with the findings of Wang One striking finding in this paper is the rich ancestral variation in the Americas, even within a single country. For example, among the six Colombian populations examined , median Native American ancestry varied between 0.13 in the Chocó and 0.86 in the Coyaima, African ancestry varied between 0.02 in the three Amerindian populations and 0.74 in the Chocó, and European ancestry varied between .09 in Coyaima and 0.52 in the Colombian mestizos. Likewise, even among the Bolivians in a single administrative department (state), there was a wide variation in African and Native American ancestry . These pIn summary, we have developed and validated a panel of 446 AIMs to estimate European, Native American and African admixture proportions. The markers were selected to have low heterogeneity within continents, in order to be portable throughout the Americas. This panel was specifically designed to provide accurate individual admixture estimates and to control for the effects of population stratification in association studies in admixed populations. The use of this panel will minimize the risk of false positives in candidate gene studies, or in research efforts designed to replicate signals identified in genome-wide association studies, even in studies with substantial population stratification.2 of 0.9 or higher) and have mean absolute error under 5%. Panels with 314, 194 and 88 AIMs all adequately controlled for the effects of population stratification in the Mexico City sample. The inflation factor (lambda) was reduced from 1.40 when using sex and age as covariates, to less than 1.04 when incorporating ancestry estimates based on genome-wide data and panels of 314, 194 and 88 AIMs, and reasonable control for population stratification could be achieved with even smaller panels.Our analysis of subsets of this panel has shown that to successfully control for population stratification in association studies, panels with 314, 194 and even 88 AIMs provide adequate estimates of the ancestral proportions with greatest variance that are strongly correlated with the genome-wide estimates  are likely to have been filtered out. Finally, when we applied our panel to new populations, it produced credible ancestry estimates, which compare favorably to ancestry ascertained from genomewide data not subject to ascertainment bias.This panel is intended to be an important resource for the community and we have provided both the source code for the algorithm to generate the AIMs, as well as allele frequency data and anonymized ancestral African, European, and shuffled Native American genotype information. We hope that investigators can use the selected panel of AIMs, which can be easily genotyped on readily available platforms, as a cost-effective tool to estimate continental ancestry in modern populations of the Americas.Informed consent was obtained for all subjects in all phases of this study, with input from local communities. These studies were approved by local institutional review boards and the relevant offices at each institution contributing samples  and East African  as well as Northern European  and Southern European  individuals One hundred and thirty-one Native American subjects, from Mesoamerica  A summary of the populations used for this study and genotyping platforms is given in 2 test for Hardy-Weinberg equilibrium at a significance threshold of 10−5.Four major quality control tests were performed on the data using the program plink stFnI2≥0.1) or within a predefined physical distance (≤500 kb pairs) of previously selected AIMs. This ensures maximum independent informativeness and that the AIMs were well distributed throughout the genome. In addition, in order for potential AIMs to be applicable to all subpopulations within a continental group, potential AIMs were also excluded if there was evidence of significant allele frequency heterogeneity between the samples representing each ancestral group . A script in the Python programming language that implements this algorithm and ancestral population allele frequency data are available for download.Markers representing the intersection of the genotyping platforms used to genotype the ancestral populations, which met quality control criteria  were used as a basis for selecting AIMs. In order to validate the panel of AIMs, estimates of ancestry using the panel were compared to estimates of ancestry using genome-wide data. Four admixed samples were used for validation. The first two datasets were parents of Puerto Rican and Mexican subjects with asthma genotyped on the Affymetrix 6.0 GeneChip as part of the Genetics of Asthma in Latino Americans (GALA) study 2>0.8. Genotypes from ancestral populations described above defined the ancestral clusters relevant to Latin Americans. We also estimated ancestry using the panel of AIMs identified with the protocol above. The performance of the AIMs panel was established by calculating the correlation coefficient (R2) and measures of discordance .We implemented a three-population model to estimate individual ancestry proportions from genome-wide data using the program ADMIXTURE Using the validated AIMs panel, we genotyped 18 populations collected from Bolivia, Colombia, Venezuela, Argentina, and Chile. A description of the origin of the samples is provided in Subjects were genotyped on a Sequenom platform with the 400 most informative AIMs identified in phase I. AIMs were included in the final analysis if they had a genotyping call rate greater than 95% and Hardy-Weinberg equilibrium in each population individually. We required that all samples had genotyping missing data rates of <10%. Sample population groups were excluded if they have average genotyping data rates of <10%File merging, strand flipping, allele frequency determination, linkage disequilibrium calculations, and identity by descent estimations were performed with the program plink http://bts.ucsf.edu/burchard/.Source code for the AIMs selection script is available at Figure S1QQ plots of genetic association studies of Diabetes in Mexicans, using nested sets of AIMs.(PDF)Click here for additional data file.Figure S2Origin of Mexican samples from MGDP-INMEGEN. Locations in purple correspond to Native American populations; those in red correspond to admixed MGDP-INMEGEN populations used for validation.(PDF)Click here for additional data file.Table S1AIMs used.(DOCX)Click here for additional data file.Text S1Supplemental methods, including detailed ethics statement and description of samples obtained for genotyping of populations throughout the Americas.(DOCX)Click here for additional data file.


North Africa is considered a distinct geographic and ethnic entity within Africa. Although modern humans originated in this Continent, studies of mitochondrial DNA (mtDNA) and Y-chromosome genealogical markers provide evidence that the North African gene pool has been shaped by the back-migration of several Eurasian lineages in Paleolithic and Neolithic times. More recent influences from sub-Saharan Africa and Mediterranean Europe are also evident. The presence of East-West and North-South haplogroup frequency gradients strongly reinforces the genetic complexity of this region. However, this genetic scenario is beset with a notable gap, which is the lack of consistent information for Algeria, the largest country in the Maghreb. To fill this gap, we analyzed a sample of 240 unrelated subjects from a northwest Algeria cosmopolitan population using mtDNA sequences and Y-chromosome biallelic polymorphisms, focusing on the fine dissection of haplogroups E and R, which are the most prevalent in North Africa and Europe respectively. The Eurasian component in Algeria reached 80% for mtDNA and 90% for Y-chromosome. However, within them, the North African genetic component for mtDNA  is significantly smaller than the paternal . The unexpected presence of the European-derived Y-chromosome lineages R-M412, R-S116, R-U152 and R-M529 in Algeria and the rest of the Maghreb could be the counterparts of the mtDNA H1, H3 and V subgroups, pointing to direct maritime contacts between the European and North African sides of the western Mediterranean. Female influx of sub-Saharan Africans into Algeria (20%) is also significantly greater than the male (10%). In spite of these sexual asymmetries, the Algerian uniparental profiles faithfully correlate between each other and with the geography. On geographic, archaeological and historical grounds, Northwest Africa is considered a distinct spatial-temporal entity Population genetic studies, mainly those using the non-recombining Y-chromosome and mitochondrial DNA (mtDNA) uniparental polymorphisms, have provided insight into not only the structure and relationships among North African and neighboring populations but also the most probable origin and date of past immigrations and expansions of several informative lineages in the area. From the beginning, a prominent mtDNA Euroasiatic genetic component was observed in the Northern areas occupied by Morocco These results suggest that the Arabization phenomenon was mainly an acculturation process of the indigenous Berber population. However, the significantly higher presence of the prominently Arab Y-chromosome J-M267 haplogroup in cosmopolitan compared to rural samples pointed to a substantial male-biased Arab influence in North Africa and the Levant The impressive genetic information gathered from North Africa is beset with a notable gap, the lack of consistent information for the Algerian populations. Algeria is the largest country of the Maghreb and, in fact, the largest country of the whole continent. Although at mtDNA sequencing level the first North African sample studied was from an Algerian Berber-speaking Mozabite population Blood samples were anonymously obtained from a total of 240 unrelated healthy adult residents in northwest Algeria (Oran area) after informed written consent. This study was approved by the research ethics committee of the University of La Laguna. DNA was extracted using the standard salting-out method Mitochondrial DNA (mtDNA) sequence analysis was performed on the regulatory hypervariable segment I region (HVS-1). Haplogroup diagnostic mutations were analyzed using PCR-RFLPs and/or SNaPshot multiplex reactions Samples belonging to haplogroup H were assorted into H subgroups (H1–H15 subhaplogroups) using two SNaPshot multiplex reactions as described by Quintáns et al. RFLP analyses and subhaplogroup H nomenclature were as in Loogväli et al. http://www.isogg.org/tree/). However, for markers subdividing E-M78 and R-M343 the nomenclature proposed by Cruciani et al. For updated subdivision of haplogroup E we amplified and analyzed SNPs V12, V13, V22, V32 and V65 as specified by Cruciani et al. In addition to our samples, a total of 18050 mtDNA HVS-1 sequences from the Mediterranean basin (12675), Middle East (4106) and Caucasus (1269) and a total of 19343 Y-chromosome samples from the same areas  were included in the statistical analyses. To overcome microdifferentiation effects, samples were collapsed into larger areas or whole countries. In order to keep the highest SNP phylogenetic resolution for the Y-chromosome analysis, sub-haplogroup subdivisions in total samples were, sometimes, extrapolated from partial samples of the same area in which these sub-haplogroups were typed Haplogroup diversities (h) were calculated according to Nei ST distances between populations were calculated from mtDNA and Y-chromosome haplogroup frequencies, and their significance assessed by a nonparametric permutation test as implemented in the Arlequin program Pairwise FPhylogenetic relationships among mtDNA HVS-1 sequences of subhaplogroup M1 were established using the reduced median network algorithm Pairwise comparisons between our Algerian sample and two ones published previously Of all North African populations, Eurasian lineages are the most frequent in Algeria (80%) while sub-Saharan Africa origin accounts for the remaining 20%. At least two Eurasian lineages, M1 and U6, had Paleolithic implantation and subsequent expansions in North Africa, reaching the Sahel and Sudan belts. It seems that the main focus of distribution of U6 was in the Northwest and M1 in the Northeast areas of the Continent The geographic origins and gradients of some of these haplogroups are graphically reflected in the PCA plot . The firST distances between populations were calculated , European (0.036±0.003) and Middle East (0.021±0.003) populations. Within North Africa, the Algerian lowest genetic distance is observed for Tunisia (FST = 0.016) and the greatest for Egypt (0.026). Italy (FST = 0.032) and the Balkans (FST = 0.032) are the closest areas within the European peninsulas while France is the most distant European region (FST = 0.042). Finally, for the Middle East, the Levant (FST = 0.014) seems to be the most similar and the Arabian Peninsula (FST = 0.033) the most different. In fact, removing the pairwise comparison between Algeria and Arabia, the mean FST value for the Middle East drops considerably (FST = 0.018+0.001) rising the mean distance of Algeria from Europe significantly compared to that from the Middle East (p<0.01).Pairwise based FResults for the sub-typing of haplogroups E-M78 and R-M343 in the Iberian Peninsula and Northwest African countries including Algeria are presented in For the R-M343 subdivision, the Iberian Peninsula reflects a genuine European profile Supplementary ST distances between populations were calculated , with Europeans (0.197+0.019) and with West Asians (0.159+0.011) also reflects its geographic position. Within North Africa, its lowest distance is to W. Sahara-Mauritania (FST = 0.023) and the greatest to Libya (FST = 0.108). Italy (FST = 0.155) is the closest of the European peninsulas and Iberia the most distant (FST = 0.247), while for West Asia, the Levant (FST = 0.128) is the most similar area to Algeria and the Caucasus the most different (FST = 0.194).Pairwise based FBased on genome-wide genetic analysis, up to five differentiated genetic components  were recently detected in the ancestry of North African populations Strong microdifferentiation has been detected for both uniparental markers in Tunisia Y-chromosome haplogroup J-M267 frequency is also the highest in Algeria. The presence of this clear Middle Eastern haplogroup in other areas has been attributed to prehistoric spreads The unexpected presence of the European male lineages R-M412, R-S116, R-U152 and R-M529 in the Mahgreb could be the male counterpart of the maternal gene flow signaled by the mtDNA haplogroups H1, H3 and HV0. In fact, there are several haplogroups with clear geographical origins from European or North African sides of the Mediterranean, but also present on the opposite side. This could be used to estimate the respective levels of gene flow between areas, assuming that their present day frequencies in the source countries were the same when they spread to the other Mediterranean shore. Thus, mean frequency values for the native North African male clusters E-M81 and E-V65 in the Maghreb , are 40.03±11.66 and 3.40±0.60 respectively. The mean values for the same markers in western-central Mediterranean Europe  are 1.86±1.28 and 0.26±0.8 respectively. Taken together, these values would suggest around 5% male Maghreb input in Mediterranean Europe. In turn, E-V13, R-M412, R-S116, and R-U152 could be used to infer the male European input in the Maghreb, giving a value around 8%. Applying the same reasoning, mtDNA U6 and M1 frequencies on the European side would indicate the maternal gene flow from the Maghreb, the estimated value being around 10%. However, when we tried to calculate the European maternal input into the Maghreb using the H1, H3 and HV0 haplogroups, we realized that their respective mean frequencies in Mediterranean Europe  are within the same range as those found in the Maghreb . This would imply a 100% European contribution to the maternal pool of the Maghreb. The fact that the three markers show similar frequencies on both sides rules out stochastic processes as a possible explanation, but further analyses, based on complete mtDNA sequences, are mandatory to investigate alternative scenarios.ST distance between Algeria and other Maghreb countries for Y-chromosome (0.061) is nearly three times higher than for mtDNA (0.023), 5 times higher when based on distances between Algeria and Europe and nearly 8 times higher when involving Middle East populations. Gender specific demographic features were used to explain these differences Genetic and geographic distances faithfully correlate for both uniparental markers , indicatRecently, it has been reported that the sub-Saharan African gene flow to Tunisia shows a strong sex bias, involving a significantly larger female contribution (p<0.0001) The inclusion of Algeria offers a more complete view of the North African genetic landscape from maternal and paternal perspectives, showing not only spatial gradients for some lineages but also sexual asymmetry in the relative affinities between populations. Perhaps, the strongest sexual discrepancy refers to the time of the main Middle East and European spreads into North Africa, whereas from the Y-chromosome perspective they seem to have occurred since the Neolithic onwards Table S1HVS-1 sequences, SNPs and RFLPs analysis results for the North-west Algerian population (N = 240).(XLS)Click here for additional data file.Table S2mtDNA Haplogroup frequencies (%) in the studied populations.(XLS)Click here for additional data file.Table S3References of the populations studied in (XLS)Click here for additional data file.Table S4Distribution of H subhaplogroup frequencies (%) in the studied populations.(XLS)Click here for additional data file.Table S5References of the populations studied in (XLS)Click here for additional data file.Table S6Y-chromosome haplogroup frequencies (%) in the studied populations.(XLS)Click here for additional data file.Table S7References of the populations studied in (XLS)Click here for additional data file.Table S8ST for mtDNA  and Y-chromosome  haplogroups.Pairwise linearized F(XLS)Click here for additional data file.Table S9MtDNA and Y-chromosome geographic haplogroup assignation.(XLS)Click here for additional data file.


Styela clava Herdman, 1882 is consideredto be native to Japan, Korea, northern China and the Russian Federation in theNW Pacific, but it has spread globally over the last 80 years and is nowestablished as an introduced species on the east and west coasts of NorthAmerica, Europe, Australia and New Zealand. In eastern Canada it reachessufficient density to be a serious pest to aquaculture concerns. We sequenced afragment of the cytochrome oxidase subunit I mitochondrial gene (COI) from atotal of 554 individuals to examine the genetic relationships of 20 S.clava populations sampled throughout the introduced and nativeranges, in order to investigate invasive population characteristics. The datapresented here show a moderate level of genetic diversity throughout thenorthern hemisphere. The southern hemisphere  displaysa greater amount of haplotype and nucleotide diversity in comparison. Thisspecies, like many other invasive species, shows a range of genetic diversitiesamong introduced populations independent of the age of incursion. The successfulestablishment of this species appears to be associated with multiple incursionsin many locations, while other locations appear to have experienced rapidexpansion from a potentially small population with reduced genetic diversity.These contrasting patterns create difficulties when attempting to manage andmitigate a species that continues to spread among ports and marinas around theworld.The solitary ascidian  An important aspect of biodiversity conservation and sustainability of marineresources is the mitigation of non-indigenous species (NIS). To-date, there havebeen no reported extinctions of native marine species caused by exotic invadersMicrocosmus squamiger. The firstintroduction of this species was recorded in1983 and it appears that extensivesharing of haplotypes has since occurred among populations Pyura praeputialis, first recorded in 1985, andBotryllus schlosseri, first recorded in the 1830's,displayed genetic differentiation among native and introduced populations andbetween harbours and bays for this same gene Undariapinnatifida, first recorded outside of Asia in 1971, exhibit highgenetic differentiation but low diversity, while introduced populations exhibitrelatively high diversity Rapanavenosa, first recorded in 1940s, displayed high genetic diversitywithin its native range but was monomorphic throughout invaded Europe Balanus glandula. This species, recorded outside of NorthAmerica since the 1960's, exhibits marked genetic structure within its nativeand invaded ranges, which enabled the identification of two different incursionpathways, one to Argentina and another to Japan Carcinus maenus, a ubiquitous marine invader since itstransport outside of Europe in the early 19th century, has beencomprehensively studied The combined availability of high-throughput molecular techniques and analyses of theresulting data based on explicit evolutionary models has caused a recent surge inthe number of studies seeking to use genetic patterns to assess invasion pathwaysand the evolution of invasiveness Styela clava Herdman, 1882 is one of manynon-indigenous marine tunicates that now dominate fouling communities of ports andmarinas around the world S. clava is native to the NW Pacificcoastal regions of Japan, Korea, northern China and the Russian Federation. Thefirst recorded introduction beyond this range was in California, probably in the1920s (Lambert & Lambert 1998). In the Atlantic, this species was first observedin Britain, in Plymouth Sound and the adjacent Lynher River Estuary, in 1953 S. clava are now recorded throughout the northernhemisphere, including Atlantic Europe plus a recent record from the Mediterraneanbasin The solitary ascidian Styela clava used microsatellite markers andmtDNA to assess the role of human-mediated transport on regional expansion of thisintroduced species in England Styela clava. Both studies suggested that extensive admixturewas occurring through human-mediated transport within the regional locations. Inaddition, the New Zealand study showed that recreational vessels and commercial portvessels were both introducing new populations into New Zealand waters andrecreational vessels were also responsible for post-border expansion of S.clava independent of the port populations S. clava using microsatellite markers also showed no evidenceof reduced genetic diversity due to founder events throughout Europe, althoughevidence of population expansion and/or sub-structure was observed in manypopulations Two recent studies of Styela clava, we assessedgenetic diversity and its distribution among populations in an extensive globaldataset of mitochondrial cytochrome oxidase I gene (COI) sequence. The long, welldocumented, chronology of global introductions makes this an ideal species to assessfounder effects and changes in diversity with respect to time of introduction, aswell as connectivity among populations. Based on the three recent molecular studiesof this species, our expectation was that the mitochondrial diversity would not bereduced in newer incursions and that admixture among populations would be high formany populations due to their derivation from multiple sources. However, geneticdifferentiation among geographic regions was also expected, based on thedifferentiation of genotypes between the two native populations In this study of the solitary ascidian Styela clava from three different labswere aligned. Protocols for sample collection and DNA extraction can be found inGoldstien et al. A sub-set of partial fragments of the mitochondrial DNA oxidase subunit Imitochondrial gene (COI) for apriori population groupings was also tested using Analysis ofMolecular Variance (AMOVA) ST and Nei's average number of pairwise differences in Arlequin v. 3.0 . Finally, to determine therelationship between ages since the introduction was reported and the geneticdiversity for the populations; diversity measures Theta(S) and haplotypicrichness, a Pearson's correlation test was done using XLstat . A Non-linear regression was also performed in XLstat using the leastsquares method and default functions.All sequences were checked in the respective laboratories and were broughttogether for alignment. Alignments and haplotypes were identified and confirmedmanually in Bioedit v. 5.0.6 S. clava individuals, derived from 20 populationsaround the world , while haplotype diversity was moderate, ranging between 1 and 13haplotypes per population. New Zealand and Japan were the most genetically diversepopulations and contributed most of the diversity to the total data set, as shownwith π, Theta(S), and haplotypic richness . The Lyt network  shows th network .Two populations from the east (Otsuchi Bay) and west (Tsukumo Bay) coast of Honshu,mainland Japan, represent the native populations in this study, albeit a small areaof the entire range. Both of these populations exhibit high genetic diversity , and sigThe statistical parsimony network  reflectsCT, 0.25) than for amonggeographic regions . The variation within groups(ΦSC) is also reduced for the BAPS groups indicating that these groupings contain less variationthan occurs within geographic regions .BAPS groupings  indicateStyela clava and to test the link between age ofincursion and genetic diversity of this widely introduced ascidian. The COI generevealed a moderate level of haplotype diversity with low to moderate nucleotide diversity (0.000–0.005), useful in identifyinggenetic similarities among populations. This level of haplotype diversity is similarto that observed for the star sea squirt, Botryllus schlosseri, forwhich 16 haplotypes were identified from 181 individuals throughout Europe Microcosmus squamiger, nowpresent in northern hemisphere locations CystodytesdellechiajeiMicrocosmus squamiger, however, exhibited low nucleotidediversity (0.002 – 0.008) compared to the other ascidians, excludingS. clava, while for Cystodytes dellechiajeinucleotide diversity was also high (0.006 – 0.08) The aim of this study was to assess genetic diversity and its distribution amongpopulations of S. clavasampled here exhibit significant genetic diversity and genetic differentiation fromeach other, and have high genetic diversity compared to most populations of theintroduced range, with only New Zealand populations exhibiting similar geneticdiversity , did show higher COI diversity than other sites on this coast  did not include this locality).Most of the haplotypes shared among northern hemisphere populations are also presentin the Otsuchi Bay population, while haplotypes shared between Tsukumo Bay andintroduced populations were also present in the Otsuchi Bay population . The higS. clava and the Otsuchi Bay sample. For instance,native populations of the amphipod Caprella mutica in Japan weregenetically diverse and all exhibited unique haplotypes that were not observed inany other locations in the data set Undaria pinnatifida, particularly in Europe but also in NewZealand and America, were genetically similar to aquaculture populations in Japanand Korea but less similar to natural Japanese populations Molecular data for other marine invasive species of NE Asian origin have displayedweaker links with Japanese populations than were observed between introducedpopulations of S. clava populations are geneticallydistinct from the northern hemisphere introduced populations and from the nativepopulations sampled, a pattern also observed for U. pinnatifidaS. clava exhibit a high number of unique haplotypesthat are likely to be present in a native region not sampled. The significantgenetic differentiation between the two New Zealand populations, Hauraki Gulf andLyttelton, suggests that these ports received founders from different sources, mostlikely from vessels arriving from different locations. The same result was shown formicrosatellite markers in New Zealand populations The southern hemisphere It has been suggested that the build-up of genetic diversity from multiple sources iscreating more successful invaders and that the founder effect may be overstated forNIS One of the difficulties for biosecurity management of marine invasions is our abilityto identify and date incursions. This species, like many others, shows a range ofgenetic diversities within populations and differentiation among them, independentof the age of incursion. Multiple incursions appear to be associated with thesuccessful establishment of this species in many locations, while other locationshave potentially experienced rapid expansion from a small founding population withreduced genetic diversity. The potential for multiple incursions blurs the linebetween founder events and the time required for genes to spread into establishedpopulations. These mixed patterns create difficulties when attempting to manage andmitigate a species that continues to spread.Table S1ST  and Nei'spairwise distance within populations  and corrected distance amongpopulations  of S.clava for mtDNA geneCOI.Pairwise comparisons for Φ(DOC)Click here for additional data file.


Leopardus pardalis) in the United States currently exhibit low levels of genetic diversity. One hypothesis for this observation is that habitat fragmentation, resulting from human induced changes in the landscape during the 20th century, created island populations with highly reduced gene flow and increased genetic drift and inbreeding. In an effort to investigate this, we used a portion of the mitochondrial control region and 11 autosomal microsatellite loci to examine historical levels of genetic diversity and infer temporal changes in ocelot populations between 1853 and 2005. Levels of genetic diversity were higher in historical ocelot populations than in extant populations from Texas. The earliest documented loss of mitochondrial haplotype diversity occurred at Laguna Atascosa National Wildlife Refuge. The second extant population inhabiting private lands in Willacy County retained higher levels of genetic diversity through the 1990s, but subsequently lost diversity over the next decade. A similar pattern was observed for autosomal microsatellite loci. This supports the argument that low levels of genetic diversity in Texas are related to human induced population reductions and fragmentation, both of which threaten the remaining ocelots in the United States. At this time, the best means of mitigating the continued erosion of genetic variation are translocation of individuals either from larger populations in Mexico to Texas, or between the Texas populations.Ocelots ( Leopardus pardalis) is a small (<15 kilogram), solitary, Neotropical felid th century, and by the 1960s the distribution became restricted to southern Texas The ocelot (e<14), high genetic drift , and reduced genetic variation  The two known ocelot populations in the United States are characterized by small effective population size  and extant ocelot populations (1984–2005) occupying the Tamaulipan Biotic Province (TBP) of southern Texas and northeastern Mexico. A portion of the mitochondrial DNA (mtDNA) control region and 11 nuclear microsatellite loci were used to address the following questions: (1) What were historical levels of genetic variation in ocelot populations in the United States? (2) Have levels of genetic variation changed in these populations over the last 100 years?Museum samples (n = 15) used in this study were generously provided by the National Museum of Natural History (NMNH) of the Smithsonian Institution. Museum accession numbers and information available on the specimens sampled are provided in the  = 40); (2) private ranches in northern Willacy County, Texas ; and (3) Los Ebanos Ranch, Mexico . Ten to 20 mg of crushed bone or diced skin was placed in 600 µl of Cell Lysis Solution, 3 µl of Proteinase K (20 mg/ml) was added, and the tubes were inverted 25X. Samples were incubated at 55°C for 3 days with the addition of 1.5 µl of Proteinase K every 24 hrs. Tubes were periodically inverted. After incubation, 3 µl of RNase Solution (4 mg/ml) was added, tubes were inverted 25X, and incubated for 15 min at 37°C. Samples were cooled to 4°C for 10 min and 200 µl Protein Precipitation Solution was added. Tubes were then vortexed for 20 sec and centrifuged at 16,000 g for 5 min. The supernatant was pipetted into 600 µl of 100% isopropanol with 1 µl of glycogen. Tubes were inverted 50X and centrifuged at 16,000 g for 3 min to pellet the DNA. The isopropanol was decanted and tubes were inverted on clean absorbent filter paper. The DNA pellet was washed with 600 µl of 70% ethanol, inverted 3X, and centrifuged at 16,000 g for 1 min. Ethanol was decanted, tubes were inverted on clean absorbent filter paper, and air dried for 20 min. The DNA pellet was hydrated in 40 µl of DNA Hydration Solution at 65°C for 1 h and stored at 4°C.All DNA extractions and PCR preparations of museum samples were conducted in a lab dedicated to genetic analyses of museum samples and physically separated from areas where work with modern DNA was performed. There were no contemporary samples of any species present in the museum lab. The equipment used for the museum samples, including pipetmen, centrifuge, refrigerator, freezer, tube racks, and heat blocks was either new, or decontaminated and subsequently used solely for museum work. Prior to each extraction or PCR preparation, all surfaces, bench tops, and equipment were cleaned with 20% bleach. Irradiation from a UV hood lamp was then used to destroy any remaining potential DNA contaminants on tubes, tips, racks, and pipetmen. Personnel working with ancient DNA wore clothes and shoes not previously worn in a laboratory where modern DNA was handled. Aerosol tips were used for all extractions and PCR preparations, which were performed in a UV sterilized hood. Negative controls were included with every extraction and monitored to ensure that no trace contamination was present.We did not repeat the sequencing or genotyping of museum samples; however, the following evidence suggests that we had data of sufficient quality for analysis: (1) We did not observe any contamination in negative controls. (2) The two new haplotypes observed in museum samples were never previously detected in contemporary samples and were sequenced from both strands. (3) We detected microsatellite alleles in museum samples that were not previously observed in contemporary samples. (4) We did not observe any genotypes with more than 2 alleles, which would have suggested cross-contamination. (5) For low quality DNA samples the most common errors are PCR failure and allele drop out, both of which would reduce diversity observed in museum samples; therefore, our study provides a conservative estimate of historical ocelot variation.Felis silvestris catus) mitochondrial genome FCU20753. This is located in the central conserved region between repetitive sequences I and II Mitochondrial sequence data for museum specimens were generated during this study . SequencTaq buffer , 0.25 units of HotMaster Taq, 0.25 µM of forward primer, 0.25 µM of reverse primer, 0.1 µg/1 µl bovine serum albumin , and 10–20 ng DNA template. Reaction conditions included an initial denaturing step of 94°C for 1 min followed by 50 cycles of 94°C for 15 sec, 54°C for 30 sec, 72°C for 60 sec, and a final extension step of 72°C for 2 min. PCR products were sequenced using an ABI BigDye v. 1.1 Terminator Kit  and an ABI 3100 automated sequencer by the Laboratory of Plant Genomics and Technology or an ABI3730 in the Molecular Cytogenetics and Genomics Laboratory, Texas A&M University. Sequences were obtained in both directions, then aligned and edited using SEQUENCHER v. 3.0 .The precautions taken to ensure that all sequences obtained from museum samples were not contaminants are described in the section above. We performed PCR amplification in 10 µl volumes containing 0.2 mM of each dNTP, 1X PCR HotMaster Taq buffer, 0.25 units of HotMaster Taq, 0.24 µM forward primer, 0.24 µM reverse primer, 0.1 µg/µl bovine serum albumin, and 5–20 ng DNA template. Conditions for PCR amplification included an initial denaturing step of 94°C for 1 min, 50 cycles of 94°C for 30 sec, 53°C for 30 sec, 72°C for 60 sec, and a final extension step of 72°C for 2 min. PCR products were visualized on a 1.2% agarose gel stained with ethidium bromide (0.5 µg/ml) and then exposed to ultraviolet light. Samples with visible PCR product were genotyped with an ABI 3100 automated sequencer in the Laboratory of Plant Genomics and Technology and sized using GENOTYPER v. 2.0 (Applied Biosystems).Eleven microsatellite loci  previously isolated and mapped in the domestic cat were used Sequence alignments were performed in CLUSTAL-X st estimates in ARLEQUIN. Estimates of Fst were tested for significance against the null distribution of Fst values obtained from 1,000 permutations of haplotypes under the null model of panmixia in ARLEQUIN.Two methods were used to compare mtDNA variation in ocelot populations. An exact test for population differentiation based on haplotype frequencies was implemented in ARLEQUIN. Population structure was tested using pairwise Fe), observed heterozygosity (Ho), number of alleles (AN), and number of private alleles (AP1) were estimated using GENALEX v. 6.0 R1) was estimated in FSTAT v. 2.3.9 and using the rarefaction approach (AR2) in HP-RARE v. 1.1 P2) were also estimated using the rarefaction approach in HP-RARE to take into account different sample sizes. Pairwise Fst between populations was estimated using AMOVA analysis and tested for significance with 9,999 permutations in GENALEX.Measures of genetic variability, including expected heterozygosity  sampled between 1991 and 1998  were comparable to those from the extant Los Ebanos population. In Willacy, haplotype diversity decreased from H = 0.536 (SD = 0.123) in 1984–1990, to H = 0.233 (SD = 0.126) in 1994–1998, and to H = 0 in 2005 (SD = 0.00029) in 1984–1990 and decreased to π = 0.00056 (SD = 0.00023) in 1994–1998 and to π = 0 in 2005 . Nucleotide diversity of the Los Ebanos population was also significantly higher than the other populations sampled (P<0.05). The historical samples from Texas had higher nucleotide diversity than the contemporary Willacy and Cameron populations.Estimates of haplotype diversity in Los Ebanos, historical Texas samples, and Willacy 1984–1990 were not significantly different. However, haplotype diversity of the Willacy population in 1994–1998 was significantly lower than in Los Ebanos (st = 0.159 (P = 0.028) (st = −0.025 (P = 0.357). Haplotype frequencies of the historical Texas samples were most similar to the Willacy population and most divergent from the Cameron population.The greatest level of genetic differentiation was between the Cameron 1996–1998 and Los Ebanos 1991–1998 populations with F= 0.028) . The lowEleven microsatellite loci were genotyped in the historical specimens and compared to contemporary samples. The microsatellite data set was archived in DRYAD. However, not all loci were successfully genotyped in every museum sample; therefore, data from only a mean of 5 samples per locus were obtained. Genotypes for the same 11 loci were also obtained for a subset of ocelots from contemporary populations . After BN = 2.46, SE = 0.37) and highest in Los Ebanos  were comparable to Los Ebanos. Mean number of alleles was not significantly different between Cameron and Willacy; however, both were significantly (P<0.05) different from Los Ebanos and the historical Texas samples. The number of alleles was not significantly different between Los Ebanos and historical Texas samples. Allelic richness estimated directly from the population samples and using the rarefaction method in Texas was highest in the historical population and lowest in Cameron . In the Texas historical sample, He was also significantly higher than in Willacy (P>0.05). With only a mean of 5 samples per locus, the sample size for the historical population was not high enough to allow rigorous tests for genetic divergence. However, pair-wise Fst was estimated between each population to examine the consistency of the microsatellite data set with the mtDNA data. The most divergent populations were Cameron and historical Texas, with Fst = 0.367 (P<0.001), and the lowest divergence was between Willacy and Los Ebanos with Fst = 0.146 (P<0.001)  .n = 10 and Willacy (1) 1984–1991, n = 8, (2) 1996–1998, n = 10, and (3) 2001–2005, n = 10. There was a reduction in both allele number and He in each of the populations  1986–1989, n = 10, (2) 1996–1998, n = 10, and (3) 2001–2005, ulations . Camerone<14 in Cameron and Ne<4 in Willacy) Comparisons of contemporary ocelot populations to historical specimens provided strong evidence for a loss of genetic diversity in Texas over the last 100 years. The historical Texas population examined had haplotype and nucleotide diversity comparable to a contemporary population in northern Mexico, and substantially higher than what remains in Texas. Two of the haplotypes observed in museum specimens were no longer detected among extant ocelots. By the 1980s, the federally protected Cameron population at Laguna Atascosa National Wildlife Refuge lost all variability at the mtDNA control region sequenced in this study. The population on private lands in Willacy County still retained 2 haplotypes through mid-1990s; however, by 2005 it was also fixed for the haplotype present in Cameron. These observations are consistent with the small effective population size previously estimated in Texas circa 1995  Puma concolor coryi) Lutra lutra) Tympanuchus cupido pinnatus) Tympanuchus cupido cupido) Mustela nigripes) Canis lupus) Alopex lagopus) Human development and habitat modifications were extensive during the 20Bufo calamita) DaphniaMelitaea cinxia) Rabidosa punctulata and Rabidosa rabida) Pan troglodytes) Panthera tigris) Phoca vitulina) Puma concolor) The rapid loss of genetic diversity observed in these species may influence their future persistence. Inbreeding depression in small populations has been found to negatively affect many traits including larval growth rates in the natterjack toad , or reciprocally between the two remnant Texas populations, is a practical method for restoring a portion of the historical genetic diversity. This is the most feasible immediate strategy for combating isolation, inbreeding, and genetic drift because landscape restoration of thornshrub corridors required to increase natural connectivity is predicted to take a long time (i.e. >20 years) Northeastern Mexico is the most appropriate source of individuals for translocations to Texas based on the higher genetic diversity, ecological similarity between these localities, and their close phylogenetic relationship Reciprocal translocations between the Willacy and Cameron populations should be implemented immediately because this would mimic natural connectivity, reduce inbreeding and drift, and restore a substantial amount of historical variation, while being financially, logistically, and politically more feasible than translocations from foreign countries. The endangered ocelot is the last Neotropical felid that still has viable breeding populations in the United States. Management strategies should incorporate restoration of genetic diversity and population connectivity to ensure persistence and expansion of these remnant populations in order to achieve recovery of the ocelot in its northernmost range country.Table S1Information on the ocelot museum specimens sampled during this study.(XLSX)Click here for additional data file.


Procambarus clarkii) was introduced to China in the early 20th century. It has been spread to almost all forms of fresh water bodies including lakes, rivers and even paddyfields in most provinces of China. To clarify issues such as the initial entry point(s), dispersal pattern, genetic diversity and genetic structure of Procambarus clarkii in China, the genetic structure and diversity of P. clarkii populations at 37 sampling sites  were analyzed using both mitochondrial gene sequences (COI and 16S rRNA) and 12 nuclear microsatellites. Multiple tests including phylogenetic analyses, Bayesian assignment and analysis of isolation by distance showed that (i) the population from Japan and those collected from China, particularly from NanJing (BGt and XG) and its some neighboring sites , have similar genetic composition, (ii) relatively high genetic diversity was detected in Chinese populations, (iii) the P. clarkii populations in China did not experience significant population expansions. Taken together, Nanjing, Jiangsu province is the presumed initial entry point, and human-mediated dispersal and adaptive variation are likely responsible for the observed genetic pattern of P. clarkii in China.The red swamp crayfish ( Procambarus clarkii), native to south central USA and northeastern Mexico, is one of the most notorious invasive species in the world P. clarkii is relatively weak Successful biological invasions require that non-indigenous species pass through a series of filtering stages including transport, release, population establishment, and in many cases, dispersal. A successful invader is characterized by a number of biological and ecological features for its dispersal and establishment in a new habitat P. clarkii was introduced to Nanjing, China from Japan in 1929 P. clarkii aquaculture has been developed rapidly, and this species has become one of the most important aquatic products in China P. clarkii. In general, the red swamp crayfish threatens local biodiversity in freshwater ecosystems, and their burrows result in dam damages and a huge loss of irrigation water, causing significant economic loss P. clarkii is considered as an important pest of wet-seeded rice (Oryza sativa) fields P. clarkii in China. A few articles have touched upon these topics to date Information on the population structure changes would help to understand the biological invasions of this alien species, and in some cases, would be useful for the establishment of possible methodologies for prevention and control of its invasions P. clarkii populations in China have relatively high genetic diversity, which would likely facilitate its invasion success to some extent, (ii) whether Nanjing was the initial point of entry of P. clarkii in China, (iii) whether the introduction of P. clarkii in China was derived from a single or multiple event(s). Here we used both mitochondrial gene sequences (COI and 16S rRNA) and 12 nuclear microsatellites to clarify dispersal pattern, genetic diversity and genetic structure of Procambarus clarkii in China.In this study, we aim to clarify the issues including: (i) whether the N = 1776). The mean observed heterozygosity (Ho), the mean expected heterozygosity (He), and the polymorphism information content (PIC) were 0.6723, 0.7913 and 0.7551, respectively. The Lo (Louisiana) and Sa (Saitama) population exhibited the highest genetic diversity among the 37 populations, while the ZX population showed the lowest  = 6.4–11.8, and expected heterozygosity (He) = 0.7002–0.8214. Deviations from HWE were observed at multiple loci in multiple populations. Most of the deviated cases showed significant heterozygote deficiency.The number of alleles ranged from three to 27 across all 12 microsatellite loci in 37 populations (e lowest . All theHd) was 0.403. The variance and the standard deviation of haplotype diversity was 0.00072 and 0.027, respectively. Under the Maximum Composite Likelihood model, the overall mean pairwise genetic distance of the six haplotypes was 0.011. Most individuals (231/313) had Hap_C2, which was mainly distributed among the P. clarkii populations collected in China and Japan. The haplotypes Hap_C3, Hap_C4, Hap_C5 and Hap_C6 were only detected in the population collected in the USA  were found. There were some base substitutions but no insertion or deletion was found. Twelve parsimony informative sites and three singleton variable sites were detected . The hap the USA .Hd) was 0.390, while the variance of haplotype diversity was 0.00077 and its standard deviation was 0.028. Under the Maximum Composite Likelihood model, the overall mean pairwise genetic distance of the three haplotypes was 0.003. Seventy-five percent of the examined individuals (212/284) had Hap_S2, which was mainly distributed among the populations collected in China and Japan. The Hap_S3 was only detected in the population collected from the USA, whereas the Hap_S1 was shared among in the populations collected in China, Japan and the USA , the nucleotide diversity was 0.00083. Only three haplotypes (GenBank accession numbers: JX120109- JX120111) were found. Two parsimony informative sites and one base deletion were detected . The hap the USA .Fst) in 37 populations of P. clarkii ranged from 0.023 between the populations XYw and XYc to 0.157 between the populations DY and YJ, NX (details not presented).Based on the microsatellite genotype data, the Nei's genetic distance (0.145) between the populations XYw and XYc was the lowest, while the Nei's genetic distance (0.999) between the populations ZX and Sa was the highest. The gene flow parameter among populations ranged from 1.284 between the populations DY and PYL to 10.595 between the populations XYw and XYc. The gene flow between the populations XYw and XYc occurred very frequently and its parameter value was far greater than those among other populations (range from 1.284 to 6.968). The coefficient of genetic differentiation (P. clarkii collected in Japan and the USA (Lo and Sa), and some Chinese populations. The other big clade included all the remaining P. clarkii populations collected in China  of microsatellite genotype data revealed that 91.26% of genetic variation could be explained by the variation within populations, whereas the remaining (8.74%) came from variation among populations . HoweverP = 0.710; mitochondrial COI sequences, r = 0.036, P = 0.688; mitochondrial 16S rRNA sequences, r = 0.043, P = 0.631). Thus, the crayfish in China did not show isolation by distance.Regressions of genetic distances against geographical distances were not significant , derived from mitochondrial COI sequences analysis and 0.860 (P = 0.567>0.05) from mitochondrial 16S rRNA sequences analysis, indicating that the P. clarkii did not significantly experience the population expansion.The detection of population expansion was performed using P. clarkiiP. clarkii dispersal. However, most of the P. clarkii populations in China are separated by geographic barriers , which could be major physical barriers to natural dispersal. However, we detected a high level of gene flow among populations separated by geographic barriers. For example, the gene flow between populations NBp and HF was 5.489  was only detected in the P. clarkii population in the USA  were the presumable initial entry points. And then, they started dispersing in the vicinity of Jiangsu province and along the Changjiang river basin. Nevertheless, of course more genetic data from source area need to be collected for investigating the initial entry point for P. clarkii invasion.It has been reported that red swamp crayfish were introduced to Nanjing, China from Japan in 1929  the USA , S2A. Si the USA , S2B. Se the USA . Third,  the USA . Fourth, the USA . TherefoP. clarkii may have been introduced only once from Japan to Nanjing in 1929 P. clarkii was likely introduced from multiple events. So, we speculated that unobserved multiple introduction events or cryptic invasions might exist.It is believed that Phalaris arundinacea, in North America were higher than those in the place of origin in Europe P. clarkii populations in China generally showed high genetic diversity. However, it was found that even if Baguazhou township, Nanjing, showed the highest allelic and gene diversity among the populations investigated in China by analysis of microsatellites, mitochondrial COI and 16S rRNA sequences. Thus, some unobserved multiple introduction events and cryptic invasions could not be excluded, although some documents reported that P. clarkii may have been introduced only once from Japan. As the Bayesian assignment showed, surprisingly, that populations NB, XG, BGt and CJr, and two more populations from Japan and the USA, were clustered together with extremely high membership coefficients. However, the other populations from China were grouped into another six genetic clusters. If Nanjing was the only initial entry point and only one introduction event occurred in the 1920s, all or almost all genetic variation should be detected in the area surrounding Nanjing. Here, there might be two explanations for the results of assignment: (i) multiple introductions which might generate divergent invasive populations The high diversity of invasive species population could be caused by multiple introductions from one or more places P. clarkii populations in China had very high diversity as shown in the present study. Other studies on P. clarkii in China have also showed similar results Some exotic invasions succeed despite founder effects, and consequent low genetic diversity was often due to the invasive populations experiencing bottlenecks and genetic drift P. clarkii populations , number of effective alleles (Ne), the observed heterozygosity (OH) and the expected heterozygosity (He) were calculated using the program POPGENE version 1.31 P. clakii populations, the Neighbour-Joining and UPGMA dendrograms for populations based on the Nei’s genetic distance matrix were constructed using the software MEGA 5.05 Based on microsatellite data, the number of alleles (ad hoc method for estimating the number of genetic clusters Structure version 2.3.1 Correlation between genetic distance and geographic distance was assessed using IBDWS version 3.21 Fs and Li’s tests) was also considered by DnaSP 5.10. Analysis of molecular variance (AMOVA) was conducted in ARLEQUIN v3.5.1.2 to calculate the variance components and significance levels of variation within a population and among populations. Relationships among mtDNA COI and 16S rRNA haplotypes were examined respectively using a statistical parsimony haplotype network generated at the 95% connection limit with TCS version 1.21 Moreover, based on mitochondrial COI and 16S rRNA sequences, the variation sites, parsimony informative sites, number of haplotype, and nucleotide diversity were determined by using the soft DnaSP 5.10 Figure S1One sequence of haplotypes and distribution of all haplotypes obtained from mtDNA COI and 16S rRNA sequences.(PDF)Click here for additional data file.Figure S2Statistical parsimony networks of mtDNA COI (A) and 16S rRNA (B) sequences for P. clarkii samples.(PDF)Click here for additional data file.Figure S3UPGMA dendrogram of 37 P. clarkii populations based on Nei’s (1972) genetic distance (D).(PDF)Click here for additional data file.Table S1List of the populations of P. clarkii studied indicating the location, their country of origin, geographical position of sampling sites, genetic diversity at 12 microsatellite loci, and haplotype diversity at mtDNA COI and 16S rRNA sequences.(PDF)Click here for additional data file.


Necrophila japonica (Coleoptera: Silphidae), which exhibits flight muscle dimorphisms using phylogeographic approaches. N. japonica lives in both stable and unstable habitats, and the flight muscle dimorphisms may have been maintained through the use of these diverse habitats. We studied the distribution pattern of the proportion of individuals lacking flight muscles in relation to the genetic differentiation among geographic populations using an 842-base pair sequence of the COI-II gene. Both flight-capable and flightless individuals occurred over the distribution area, and the flight muscle condition showed no significant phylogeographic pattern. Several populations comprised flight-capable individuals only, whereas few comprised flightless ones only. Demographic expansion was suggested for major clades of COI-II haplotypes, and the genetic differentiation showed an isolation-by-distance pattern among the populations in Japan. The proportion of flightless individuals was higher in a population with a higher annual mean temperature and with higher genetic diversity among individuals. These results indicate that geographic expansion occurred recently while flight muscle dimorphisms have been maintained, that flight-capable individuals have colonized cooler  habitats, and that flightlessness has increased in long-persisting populations as suggested by high genetic diversity.Some insect species exhibit polymorphisms in flight muscles or wings, which provide opportunities for studying the factors that drive dispersal polymorphisms and the evolution of flightlessness in insects. We investigated the macroscale evolutionary pattern of flightlessness in the widespread Japanese beetle  Insects acquired flight capabilities approximately 400 million years ago . The abiSpecies of several insect orders exhibit variations in flight ability due to polymorphisms in flight muscles or wings. These may be caused by genetic variations (polymorphisms) and/or by phenotypic plasticity . In partNecrophila japonica, which inhabits the Japanese islands, exhibits flight muscle dimorphisms in both sexes , and individuals with flight muscles lay many eggs  . One speth sexes . Becauseth sexes , flightldominant . Indeed,dominant . We haveany eggs . Thus, hommunity . Flight ommunity , 2008b. ommunity .N. japonica by examining the relationships between the geographic pattern of flight muscle condition, genetic diversity within and among populations, and the phylogeographic pattern of this species in Japan. Since flight-capable individuals colonize peripheral or new habitats more frequently than flightless individuals  analysis. Note that, although we included the intrapopulation genetic diversity as an explanatory variable in the GLM analysis because it may be correlated with the proportion of flight-capable individuals, the causal relationship between these variables was not clearly determined. We calculated mean pairwise distances between individuals within a population as an index of genetic diversity. Climatic condition variables included annual mean temperature, annual rainfall, and maximum snow depth at each sampling site based on meteorological data from 1971 to 2000 . We perfST = 0.39; P < 0.001), although genetic variances were higher within populations than among populations . The Mantel test showed a significant isolation-by-geographic distance pattern  of haplotype and nucleotide diversities were 0.66 ± 0.26 and 0.0020 ± 0.0014, respectively . There wN. japonica were genetically differentiated among geographic regions. The largest clade was 3–1, whose haplotypes mainly occurred in northern and central Honshu. The haplotypes of clade 3–2 occurred in central and western Honshu, Shikoku, and Kyushu, while those of clade 3–3 were only found in central Honshu. The haplotypes of clade 3–4 mainly occurred in central Honshu, whereas clade 1–1, one of the one-step clades within clade 3–4, was found only in Hokkaido. The haplotypes from Hokkaido (clade 1–1) were closer to those from central Honshu than to those from northern Honshu (clade 3–1), which is adjacent to Hokkaido. All three-step clades exhibited nonsignificant SSD values, but clades 3–1, 3–2, and 3–4 had negative significant Fu's Fs, suggesting demographic expansion in these clades . Dimorphic populations consisting of beetles with and without flight muscles were found throughout the Japanese archipelago  of Silphinae in Japan  individuals occurred in most parts of the distribution area and in all clades of COI-II haplotypes. Thus, flight dimorphisms in N. japonica, which are likely maintained by inhabiting various habitats ranging from unstable river banks to relatively stable forests  individuals would more readily achieve colonization in peripheral or new habitats e.g., . Flight N. japonica might have inhabited refugia in south Hokkaido during the last glacial maximum, as suggested for a flightless silphine species, S. perforata  populations were flightless, and dimorphic populations occurred in the intermediate zone. Populations in different regions possessed distinct COI lineages, which may have diverged during the Pliocene and Pleistocene , another beetle species exhibiting flight muscle dimorphism in Japan, dimorphic populations occur widely, but monomorphic flight-capable populations tend to exist in the northern-most and southern-most regions (N. japonica.Flight-muscle dimorphisms in insects may not always reveal a phylogeographic pattern consistent with that of in Japan . Southeristocene . In P. l regions . AlthougN. japonica, it is necessary to conduct additional studies such as microscale field surveys and breeding experiments to clarify the genetics behind flight-muscle dimorphisms and phylogeographic analysis using multiple nuclear loci to corroborate the results from COI-II gene sequences. Given that loss of flight appears to have occurred in diverse insect lineages, such studies will provide new insight into the mechanism of flight loss and related ecological evolution in insects, one of the major processes of insect evolution (To further elucidate the mechanism of flight loss in volution .