Sciurus vulgaris) in several European countries due to the introduction of the American grey squirrel (S. carolinensis) and the predicted arrival of the grey squirrel in France in the near future has lead to the development of a preventative conservation project in this country. In this study, we conducted an extensive survey of mitochondrial DNA variation in French red squirrels using a fragment of the mitochondrial D-loop and we compared the results with previously published data from other European populations. Our main aims were: (1) to determine whether genetically differentiated populations, which could represent prioritized units for conservation purposes, were present in France and (2) to determine whether the French population, which is currently largely undisturbed, could provide information on the postglacial recolonization history of the species. We found that French D-loop haplotypes show almost no tendency to cluster by geographic origin, be it region or country, suggesting that French red squirrels have not been isolated from other populations during an evolutionarily significant period and that they do not constitute an Evolutionary Significant Unit. The French red squirrels showed strong signals of population expansion, the opposite to what is observed in most other European populations, making them of particular interest to study the postglacial expansion history of the species.The decline of the red squirrel ( Sciurus vulgaris is a well-known animal found in many types of coniferous, deciduous and mixed forests of Eurasia S. carolinensisThe red squirrel S. vulgaris has been divided into numerous subspecies primarily based on coat color and morphological variation. The number of these subspecies varies according to the authors: more than 40 have been described by Corbet et al.S. vulgaris in its European range by analyzing a sample of 236 individuals from 15 European countries with mitochondrial and microsatellite nuclear markers. While microsatellite genotypes formed three clusters corresponding to three of the subspecies recognized by Sidorowicz Across its geographical range, S. vulgaris populations are threatened by the American grey squirrel S. Carolinensis will arrive from Italy in the coming decades et al.The red squirrel genetic diversity has also been studied at a finer geographic scale in small isolated populations in Belgium th century S. vulgaris are of recent continental Europe ancestry, probably the result of translocations of individuals from continental Europe Second, to our knowledge, there are as yet no fine scale genetic studies on red squirrel genetic diversity in regions where the populations have not been disturbed by interspecific competition, habitat loss or fragmentation. In France, the grey squirrel is currently not present and the total forest area has been increasing since the middle of the 19In this study, we examined mitochondrial DNA variation among French red squirrels and compared it with those of other European red squirrels with two objectives: to establish whether ESUs are present in France, and to determine whether French red squirrels could be used to improve our understanding of the postglacial expansion history of the species.Tissue samples from dead red squirrels originating from 11 French regions  were colAll conducted experiments complied with the current laws of France. In this study we obtained ministerial authorization to transport corpses of this protected species. This derogation was approved by the French “Comité National de Protection de la Nature” (CNPN).Sciurus vulgaris (GenBank Accession no. AJ238588). The fragment was amplified by PCR using the primers Lpro-SQL and SQR-SQR designed by Trizio et al.++, 1 µM of each primer and 1.25 units of Taq polymerase (Qiagen). Thermocycle conditions were 94°C for 30 s, 48°C for 30 s, and 72°C for 1 min, for a total of 35 cycles. Purified template DNA was sequenced on both strands with the PCR primers, using standard Sanger sequencing techniques. All sequences were proof read and aligned manually, generating a 516 bp alignment for 110 French red squirrels. Sequences were deposited into the GenBank database under accession numbers JX645360 to JX645469.DNA was extracted from 50 mg of tissue with a Macherey Nagel Nucleospin tissue kit, following the manufacturer’s protocol. Extracted DNA was resuspended in 100 ml elution buffer. Mitochondrial DNA variation was assayed by the amplification of a 516 bp fragment of the D-loop, matching nucleotide positions 15464–15979 of the published sequence of the complete mtDNA genome of ST, and to perform neutrality tests. To assess past demographic history, we calculated two standard neutrality tests, Tajima’s D SS. The significance of these two statistics tests was estimated by 10 000 coalescent simulations, using DNAsp5. The demographic history of French red squirrels was also inferred by a pairwise mismatch distribution analysis between individuals We used DNAsp5 to analyze sequence variations , to calculate genetic differentiation using Fθ = Ne µ (where Ne is the effective population size and µ the mutation rate) and g  i.e. the population has undergone an expansion was determined by using the likelihood surface produced by fluctuation. The analysis was repeated ten times and the mean and standard deviation of θ and g were calculated from the results of these ten runs.We also used a coalescent approach to validate the patterns of demographic history revealed by the mismatch distributions We tested whether Slatkin’s isolation-by-distance model S. vulgaris haplotypes. Nucleotide sequences of red squirrels from several other European countries were downloaded from GenBank and added to our data set, generating a 252 bp D-loop alignment. To determine which model of nucleotide substitution is the most appropriate for this dataset, we used MEGA version 5 S. lis, the closest relative of S. vulgarisWe used the maximum likelihood method to assess the phylogenetic relationships among Finally, in order to compare genetic variability and neutrality test values for our French samples with those obtained for other European samples in previous studies, we calculated these estimators as described above but using only the 252 bp alignment. We did not include populations whose individuals were sampled from a unique location in order to be able to compare values, except for the Calabria population because of its particular interest.et al.Sequences of a mtDNA D-loop fragment (516 bp) were successfully determined for 110 French red squirrels. This fragment encompasses the 252 bp and 395 D-loop fragments used in previous studies ST = 0.167; P<0.001), and between the majority of pairs of populations (not shown). A mantel test performed with the Arlequin software was not significant .The mismatch distribution of the D-loop sequences showed an unimodal distribution , was less than 0.01.FLUCTUATE was used to jointly estimate the parameters The model of nucleotide substitution that best fit the 252 bp alignment of 110 French squirrels and 32 European squirrels sequences was a Hasegawa-Kishino-Yano (HKY85) model D and Fu’s Fs neutrality tests are significantly negative for the French population  showed that samples from Calabria and Sweden had low levels of variation compared to samples from other countries . Tajima’pulation , as was et al.D and Fu’s Fs neutrality tests have significantly negative values. This indicates that the French D-loop haplotypes show an excess of singletons and an excess of haplotypes, features that are observed following a demographic expansion. Finally, the coalescent simulations performed with FLUCTUATE to estimate the demographic history of the French red squirrel also demonstrated that an expansion took place. Our analysis of French red squirrel therefore strongly supports the hypothesis by Grill et al.We found that French D-loop haplotypes do not show any tendency to cluster by geographic origin, similar to what was described for other countries or in Europe as a whole et al.et al.D and Fu’s SF tests, suggesting that it is of particular interest to understand the postglacial expansion history of the species.We observed a high haplotype and nucleotide diversity among French populations, in agreement with the results of previous studies (e.g. S. v. fuscoater subspecies, together with the majority of the mainland European populations Another objective of our study was to establish whether the French red squirrels were genetically distinct from other European populations. As described in previous genetic studies of red squirrels


The European and African contribution to the pre-existing Native American background has influenced the complex genetic pool of Colombia. Because colonisation was not homogeneous in this country, current populations are, therefore, expected to have different proportions of Native American, European and African ancestral contributions. The aim of this work was to examine 11 urban admixed populations and a Native American group, called Pastos, for 32 X chromosome indel markers to expand the current knowledge concerning the genetic background of Colombia. The results revealed a highly diverse genetic background comprising all admixed populations, harbouring important X chromosome contributions from all continental source populations. In addition, Colombia is genetically sub-structured, with different proportions of European and African influxes depending on the regions. The samples from the North Pacific and Caribbean coasts have a high African ancestry, showing the highest levels of diversity. The sample from the South Andean region showed the lowest diversity and significantly higher proportion of Native American ancestry than the other samples from the North Pacific and Caribbean coasts, Central-West and Central-East Andean regions, and the Orinoquian region. The results of admixture analysis using X-chromosomal markers suggest that the high proportion of African ancestry in the North Pacific coast was primarily male driven. These men have joined to females with higher Native American and European ancestry . This high proportion of male-mediated African contributions is atypical of colonial settings, suggesting that the admixture occurred during a period when African people were no longer enslaved. In the remaining regions, the African contribution was primarily female-mediated, whereas the European counterpart was primarily male driven and the Native American ancestry contribution was not gender biased. The study of the genetic diversity of human populations is important to reveal different aspects associated with the history of these individuals, which, in turn, is highly significant in many applied fields, namely clinical and forensic genetics. In the clinical field, genetic research studies are designed to identify associations between some alleles and/or genotypes and diseases in different areas America was the last continent to be colonised by people from Northeast Asia. After arriving in North America, there were at least three subsequent dispersions responsible for the colonisation of the continent, from North to South, reaching the most southern regions in South America In an attempt to identify important signs of American colonisation and characterise the history of present-day populations, different types of genetic markers were used, including autosomal, mtDNA, and Y- and X chromosome-specific polymorphisms e.g. .Similar to most populations in South America, Colombia has a complex history of colonisation, harbouring a gene pool comprising not only the contributions of different Native American groups but also great contributions from European and African people.The genetic study of the populations in Colombia has been the focus of previous studies, using different types of markers in different populations or regions of the country Indels are length variation polymorphisms produced by the insertion or deletion of one or more nucleotides. Indels are abundant, representing approximately 20% of all polymorphisms in the human genome The use of autosomal markers is typically the best strategy to disclose admixture patterns in populations, as these markers account for both paternal and maternal inheritance, unlike the results from linked uniparental markers. Although markers on the X chromosome represent the differential female versus male contribution, these markers can nevertheless be useful to reveal differences in populations that present higher maternal differentiation.The aim of the present study was to examine a set of 32 X-indel markers to improve the current knowledge concerning the genetic background of Colombian populations. The typing of these markers in different populations from Colombia revealed differences among the admixture processes in the studied populations, based on genetic distances, ancestry proportions and linkage disequilibrium patterns.http://www.uma.net/e/policy/b3.htm).Written informed consent was obtained from all participants for cooperation in this study under strictly confidential conditions. The present study was approved through the Ethics Committee at the University of Antioquia , in accordance with the ethical principles of the 2000 Helsinki Declaration of the World Medical Association  in 12 different Colombian population groups .Genomic DNA was extracted using a standard salting-out procedure All samples were typed for a panel of 32 X-indels using a previously described protocol The PCR products were prepared for subsequent analysis, adding 1 µL of amplified product to 12 µL of Hi-Di™ Formamide (Applied Biosystems (AB), Foster City, CA) and 0.15 µL of internal size standard GeneScan™ 500 LIZ® (AB). Capillary electrophoresis and detection were performed on a 3130 Series Genetic Analyser using a G5 filter set and POP-4™ (AB) polymer. The resulting electropherograms were analysed, and the genotypes were assigned using GeneMapper v3.2 (AB).FST) and the Analysis of Molecular Variance (AMOVA), as well as the corresponding non-differentiation p-values, were assessed using ARLEQUIN software v3.5.1.3 FST matrix was represented using STATISTICA v7.0 software .The gene diversity values, observed and expected heterozygosities, Linkage Disequilibrium and Hardy-Weinberg equilibrium tests were all calculated using ARLEQUIN software v3.5.1.3 For comparison purposes and inferences on admixture, genotypic data from African and European reference samples were obtained from a previous study including the same 32 X-indels Use population Information to test for migrants” option was used with the Linkage model; the allele frequencies were correlated and updated using only individuals with POPFLAG = 1, in this case the African, European The apportionment of genetic ancestral contributions was estimated using the STRUCTURE v2.3.3 software The 32 X-indel profiles obtained for the 869 Colombian samples are listed in The allele frequencies were calculated in both male and female samples, and no significant differences were observed. Thus, all chromosomes from males and females were pooled in a single sample for each population and used for population comparisons.p-values are listed in A comparison was made between all samples from the 12 populations in Colombia and two published reference samples from Europe and Africa Significant differences were detected between the samples from the Native American group of Pastos and all admixed populations, except Nariño and Casanare. The genetic distance between Pastos and Casanare is nevertheless high, indicating that the observed result can be due to the small sample size of Casanare.Significant differences were also observed in all pairwise comparisons of the 12 different samples from the Colombian populations and the reference samples from Europe and Africa.Comparisons between pairs of Colombian admixed population samples showed significant differentiation values between the Chocó, Cartagena and Nariño populations in most pairwise comparisons with the remaining samples. In eleven pairwise comparisons, significant differences were found between Chocó and nine other Colombian samples. Cartagena and Nariño showed a significant differentiation in six out of eleven comparisons see . Chocó iMoreover, the position of Norte de Santander in the plot was displaced in a direction that did not correspond to any of the three African, Native American and European source populations. This sample was expected to group with the samples from the Central-East Andean region, which was not the case. Nevertheless, the observed values for the genetic distance were not high enough to exclude the hypothesis that no significant differences exist between Norte de Santander and the other three populations from the Central-East Andean region . In summBecause differences were not observed between the samples from Boyacá-Cundinamarca, Huila and Santander, all located in the Central-East Andean region and the samples from Arauca, Meta and Casanare in the Orinoquian region, these samples were grouped. Subsequently, all samples were classified according to the geographic region using the same criteria as in previous studies FCT of 0.0142 . A low percentage of variation was observed among populations within groups . This result demonstrates the consistency of the groups considered.After grouping the samples according to geographic region, AMOVA revealed a significant proportion of variation between groups, with a fixation index p-values were all above 0.0044 for a total of 384 tests, for 32 markers tested in 12 populations). The Hardy-Weinberg equilibrium was also tested after pooling the female samples from the Central-East Andean and Orinoquian regions, and no statistically significant deviations were detected from the expected genotypic distributions in any of the tested regions in Colombia (data not shown).The generated data were used to test Hardy-Weinberg equilibrium in the female samples. For a significance level of 0.00156, obtained using Bonferroni’s correction for multiple tests (32 per population), no significant deviations for the genotypic distributions were observed within any population  was tested in male samples from each Colombian region. p-values of LD were previously observed in Europeans and Africans p-values in three other samples. The samples from the Central-East Andean region showed clear signs of association between all allele pairs inside a large block of 896,139 bp, including MID357, MID356, MID3703, and MID3774. This large linkage block was not observed in the African and European samples studied by Pereira et al. A significant association could be expected for MID356 and MID357, considering that these loci are just 5.2 Kb (0.0198 cM) apart at the X chromosome see  and thatThe close proximity of the loci MID3690, MID3719, and MID2089 (spanning approximately 174 Kb) suggests that these three markers might form a LD block For the remaining loci, no significant LD values were observed in most pairwise tests (data not shown). An exception was observed in the samples obtained from Nariño and Pastos , where sThe large LD block observed in the Central-East Andean region can be attributed to recent admixture.For the admixture analysis, we used previously published reference population data for Africans and Europeans The reference and study populations were simultaneously analysed to determine the ethnic contributions to all Colombian samples. The results obtained using the 32 X-indel polymorphisms showed similar African, European and Native American contributions to most regions , except Most South American populations were subjected to an intensive admixture, primarily comprising individuals from Europe and, to a lesser extent, Africa during the last 500 years.Due to the inheritance properties of the X chromosome, mothers have a higher contribution to the offspring than the fathers. Therefore, in a population with the same number of males and females, two times more female than male ancestral contribution to the overall gene pool is expected after sex-biased admixture events To infer the gene flow patterns in the studied populations, we compared the interethnic admixture estimates obtained for the X-indels with those previously published for a group of 52 autosomal SNPs In general, the results showed a lower contribution from European individuals to the X-chromosomal than to the autosomal gene pool, consistent with previous results on mtDNA and Y chromosome lineage markers, showing sex-biased mating between European men and Native and African women in many admixed populations throughout South America e.g. . MoreoveA more detailed regional analysis revealed that in the North Pacific coast, the apportionment of African ancestry was lower for X-indels than for autosomal SNPs, while for the European and Native American contributions, the inverse was observed. These findings and those from previous data for lineage markers In the remaining regions, X-indels showed higher African and lower European contributions compared to autosomal SNPs; similar estimates were observed for the Native Americans. This pattern is consistent with a gene flow primarily involving males with higher European and lower African ancestry than the females, with no differences between Native American ancestry proportions in men and women.Comparing the results from different source markers can be important to disclose population events that act differentially in males and females. Therefore, 12 different Colombian samples were analysed using a set of 32 X-chromosomal indels, and the results were compared with previously reported data for 52 autosomal SNPs in Colombian populations Notably, the African ancestry values estimated in the present study were higher than those previously reported FST was obtained for the X-indels than for the 52 autosomal SNPs. The admixture analyses corroborate the results of the analysis of genetic distances, showing a higher African contribution for the autosomal than for the X-chromosomal markers. These results support a higher African ancestry of the males contributing to this population in contrast with a higher Native American ancestry of the females. A high African contribution was also detected in the population from the Caribbean region but, due to the lack of autosomal data, we could not confirm the same trend of a higher X-chromosomal than autosomal African ancestry. However, for the Caribbean region, Vergara et al. The Chocó population (in the North Pacific coast) was least distant to the African reference population, and a slightly higher Data from lineage markers previously showed that Nariño, in the Southwest Andean region, has an almost complete Native American mtDNA gene pool and a predominance of Y-chromosomes from Europe No significant differences in the interethnic admixture were observed among the remaining populations. Although a higher European ancestry was confirmed using autosomal markers, all populations presented identical European and Native American X-chromosomal contributions, which were slightly higher than the African contribution.Clear signs of associations between the loci in a large block of 896,139 bp  were observed in the sample from the Central-East Andean region, emphasising the importance of population admixture history in creating LD. Thus, in future studies, it would be interesting to increase the number of samples and populations analysed to assess the potential of this linkage block for dating admixture events in American populations.Figure S1Shepard diagram for the two-dimensional MDS plot represented in . This Scatterplot shows the reproduced distances plotted on the vertical (y) axis versus the original values plotted on the horizontal (x) axis. The small deviation of the reproduced distances to the step-line indicates a good fit.(PDF)Click here for additional data file.Table S1List of 32 X-indel genotypes from the samples included in the present study.(XLS)Click here for additional data file.Table S2Genetic distances (FST) between the Colombian populations , Africa and Europe, and the corresponding non-differentiation p-values . Significant p-values are indicated in red, with a significance level of 0.0008 (after applying Bonferroni’s correction for multiple tests).(DOCX)Click here for additional data file.Table S3Allele frequencies of 32 X-Indel markers in samples from a Native American group (Pastos) and from six Colombian regions: South-West Andean Region (Nariño); Central-West Andean Region (Antioquia); Central-East Andean Region ; Orinoquian Region ; North Colombian Pacific Coast (Chocó); Caribbean Region (Cartagena). In the table it is represented the frequency of the shorter allele, called allele 1 .(DOCX)Click here for additional data file.Table S4List of significant p-values of LD for polymorphic loci separated by more than 1 Kb. The results are sorted according to the distances between the two loci in the pair.(DOCX)Click here for additional data file.


The post-genomics era has ushered in a renewed interest in genomic diversity studies particularly with regard to definition of polymorphisms and their functional significance. An understanding of the functionality of these large sequences and their variability between populations and individuals appears to be the next logical step to provide meaning to the voluminous human genome sequences. Population-based description of polymorphisms, especially in functionally active gene complexes, such as the major histocompatibility complex (MHC), has gained immense importance in this era of ‘functional genomics’. Because of the large degree of polymorphism and extensive linkage of its various loci, gene complex within the human leukocyte antigen (HLA) region, which is the human equivalent of MHC, serves as a good mini genome model for anthropological investigations of genetic relatedness and migrations of different world populations. Three outstanding features of the human MHC, namely high polymorphism, tight linkage among the loci, and the non-random association of its various alleles make the system of particular interest from the perspective of population genetics.th century. This was the last major movement, which produced perceptible changes in the composition and culture of the indigenous population. The Muslims did not accept the Hindu religion and this led to large-scale conversions. The Europeans, Portuguese, Dutch, French, and British started their intrusions from the 15th century ad onwards, and the Indo-European community grew from these infusions. The Mongolians entered the eastern part of India from Southeast Asia. The Indian subcontinent therefore became the melting pot of the various racial groups who largely halted here and intermingled with the local population.The Indian subcontinent is a land of enormous cultural, geographical, and linguistic diversity, and harbors appreciably greater genetic diversity than any other comparable global region after Africa. The society comprises various tribal groups, ranked caste and other population groups that are largely endogamous. Why and how this genetic diversity arose in India is not clear? The “Out of Africa” migration model of modern humans suggests that a small population, most probably from East Africa expanded throughout Africa around 100 kya. This was followed by a second expansion between 60 and 40 kya into Asia and from there to other continents. and endeAs a result of evolutionary history, endogamy, and consanguinity, populations of the Indian subcontinent demonstrate high genetic differentiation and extensive sub-structuring. A recentHLA-based studies carried out in the Indian population by various groups. have higMost populations in the Indian subcontinent follows the Caucasoid pattern of HLA allelic distribution with variable admixture of genes from the Mongoloid and Aryan races;The observed extreme diversity in the HLA class I and class II region genes in these populations is due primarily to the existence of several ‘novel alleles’ and ‘unique haplotypes’ that could have arisen as a consequence of the founder effect, racial admixture, and geophysical or socioeconomic barriers contributing to the genetic drift;There is compelling evidence to suggest that selection pressure could have directed the generation of such novel alleles and unique haplotypes.The high level of variance in the repertoire of peptide presenting MHC molecules implies altered antigen presentation, immune responses, and HLA genetic profiles in relation to disease susceptibility and resistance.The extreme degree of polymorphism may pose limitations for the development of potentially global vaccines and may require development of novel vaccination protocols for effective therapeutic use in extremely diverse populations like those in the subcontinent.The existence of large variety of alleles and haplotypes, both unique and representative of other ethnic groups in the Indian subcontinent poses additional challenges in the transplantation context, particularly with regard to the hematopoietic stem cell transplantation.In view of the importance of HLA in defining genetic architecture of the human population, understanding the demographic history and population kinship along with unique constitution of MHC in ethnically well-defined populations has important implications on its immunobiology. This requires the use of advanced molecular technologies determining HLA diversity at high-resolution level, use of appropriate statistical methods for measuring the selection pressure , maximum likelihood analysis, Ewans–Watterson homozygosity, strict selection of sampling algorithm to avoid sampling errors for better representation of a population, and critical evaluation of the data in relation to other populations. Owing to its inherent genetic diversity and ‘unique’ MHC repertoire, data generated in the Indian subcontinent on diverse population groups could become an invaluable genomic resource for assessing the implications of new polymorphisms in the generation of immune response, defining susceptibility/resistance factors in diseases, survival in varying environmental conditions, design of universal drugs or vaccines and finally to generate an insight into the extent and comparative effect of different evolutionary forces like founder effect, isolation, divergence, genetic admixture, and mutations, as well as environmental selection on the generation and maintenance of polymorphism.


The archeology and history of the ancient Mediterranean have shown that this sea has been a permeable obstacle to human migration. Multiple cultural exchanges around the Mediterranean have taken place with presumably population admixtures. A gravitational territory of those migrations has been the Iberian Peninsula. Here we present a comprehensive analysis of the maternal gene pool, by means of control region sequencing and PCR-RFLP typing, of autochthonous Andalusians originating from the coastal provinces of Huelva and Granada, located respectively in the west and the east of the region.The mtDNA haplogroup composition of these two southern Spanish populations has revealed a wide spectrum of haplogroups from different geographical origins. The registered frequencies of Eurasian markers, together with the high incidence and diversification of African maternal lineages  among Huelva Andalusians when compared to its eastwards relatives of Granada and other Iberian populations, constitute relevant findings unknown up-to-date on the characteristics of mtDNA within Andalusia that testifies a female population substructure. Therefore, Andalusia must not be considered a single, unique population.The maternal legacy among Andalusians reflects distinctive local histories, pointing out the role of the westernmost territory of Peninsular Spain as a noticeable recipient of multiple and diverse human migrations. The obtained results underline the necessity of further research on genetic relationships in both sides of the western Mediterranean, using carefully collected samples from autochthonous individuals. Many studies have focused on recent North African gene flow towards Iberia, yet scientific attention should be now directed to thoroughly study the introduction of European genes in northwest Africa across the sea, in order to determine its magnitude, timescale and methods, and to compare them to those terrestrial movements from eastern Africa and southwestern Asia. The archeology and history of the ancient Mediterranean have shown that this sea has always been a permeable obstacle to human migration. Throughout prehistory onwards, people have traveled and moved across the Mediterranean and migrated in many directions. Scenarios of early human movements in the Mediterranean both over land and by sea are found in the obsidian trade of Melos island with Greece mainland , and evidences of seafaring and island occupations as Cyprus, Corsica, Sardinia and Majorca are dated ~8,500 BC ,2. ConseThe mitochondrial (mtDNA) genome is being one of the most widely used molecular tools in the knowledge of Iberian genetic diversity, commonly by the joint analysis of control region and some coding region variants. Even so, in the last years, the level of phylogenetic resolution in the study of mtDNA in Iberian populations is reaching the maximum by means of complete genome sequencing, paying special attention to particular maternal lineages with different ancestral roots in diverse continental areas ,10,13-17Within Peninsular Spain, the most thoroughly examined region in terms of human mtDNA genetic variation has been the Cantabrian cornice –in particular Galicia and the Basque area -21. GaliGiven the abundance of surveys dealing with the extent of genetic diversity both in Iberia and its islands (e.g. the Canary Islands), the absence of an exhaustive and monographic analysis addressed to know mtDNA profiles in locally defined Andalusian coastal populations is striking. The attractive prehistory and history of Andalusia, the widest and most populated region of Spain, makes its present-day human population a prominent objective to detect scenarios of population substructure and to examine the expected impact of African and other Mediterranean populations on the Iberian gene pool. Consistently with the geographical proximity between southern Spain and Africa, previous published studies have obtained results that show evidences of African-linked mtDNA lineages among Andalusians as well as high levels of diversity, either analyzing regional general samples -24 or foIn this paper, we provide novel data on mtDNA haplogroup (Hg) composition in autochthonous individuals from Huelva and Granada provinces, located respectively in the west and in the east of Andalusia region  whereas the latter is strongly linked to the Nazari kingdom, the Islam’s last possession in Iberia. Tartessos was an essential axis in Iberian protohistory, the foyer through which the Iberian Peninsula entered fully into Mediterranean history , other nine from Mediterranean Europe and Middle East, and from continental Europe and North Africa. The genetic position of Huelva sample (#5) at the lower right quadrant of the bidimensional space, and its proximity to Canary Islands (#8) might be well interpreted by the relevant weight of haplogroup U6 in its mtDNA pool. Noteworthy, genetic affinity is found between Castilians from Zamora  and northern Portuguese . This finding would be pointing out ancient admixtures before the emergence of the Portugal kingdom in the 12th century.Cluster C2 is also significantly determined by haplogroup H , L2 , L1 , U6 , and M (M1)  and it coherently encompasses 7 populations from North Africa. Finally, cluster C5 groups southwestern Asia samples, determined by haplogroups R0, M, HV*, N and K, whereas C6 is mostly shaped by populations from the Middle East and other neighboring populations as Syria (#32), Iraq (#34), Yemen (#41) and Egypt (#53); the haplogroups included in the category “Others” (lineages not frequently found in western Europe) are defining this last cluster. We also run an additional HCA plot (not shown) excluding populations from clusters C4, C5 and C6. The population topologies keep the same pattern that the original HCA and no improvement of the visual interpretation of data was reached.C3 is significantly characterized by high frequencies of the typically African mtDNA clades: L3  as well as their shape and position. In the U6 lineage network  movements. The population size in the south of the Iberian Peninsula –information based on archaeological sites – was already relevant since Paleolithic onwards , northern Moroccan province Mauretania Tingitania was grouped together with peninsular provinces to form the Diocesis Hispaniarum from which Tangier region (Morocco) and Andalusia were the first territories to be Christianized at the beginning of the 4th century. Therefore, it seems unlikely that U6a lineages located in the network core were the result of the posterior Islam expansion in Iberia, despite the multiple invasions occurred from the Maghreb to the Peninsula during this period. Interestingly, other new genetic data would support that hypothesis. The outstanding presence of European specific haplogroups in the maternal gene pool of contemporary northwestern African human populations may account for the occurrence of migrations from Europe, being the Iberian Peninsula an important source of that gene flow [Thereafter, during the colonial expansion on early 1ene flow ,30,32. TVia de la Plata (“silver way”) which acquired a remarkable military and trade importance not only during Roman Empire, yet before and after it, since it connected important cities and mining deposits. This road might have permitted population movements from the south- and north-western Spain during long periods. These maternal lineages shared among western Spanish populations may correspond to women supporting military contingents or trading. Since Portuguese populations are absent from the L1b core, and all the Portuguese sequences belonging to this lineage are derived, Spanish and Portuguese genetic histories may have been rather different for this mitochondrial sub-haplogroup.In the sub-Saharan L macrohaplogroup network Figure B, lineagLineage L2a, the second most represented in the network  shows nth-19th centuries) as the source of L lineages in Portugal, the main destination of black slaves in Europe. In this way, some authors have discarded a likewise input move for U6 and L haplogroups in the Peninsula, arguing that both mtDNA clusters show different geographic distributions and frequencies in autochthonous populations from Portugal and Spain. Some sub-Saharan genes coming from Ibero-America would have been introduced in Iberia as well. The presence and the relatively high frequencies of the African immunoglobulin allotype GM*1,17 23′ 5* in western Spanish populations has been partially interpreted in the same frame [Some authors  pointed me frame ,55.Alboran route, currents and winds go from Europe to Africa most of the year, and the high Rif and Sierra Nevada Mountains permit a permanent sight of land during the journey. So, it seems likely that the island was known among Paleolithic hunter-gatherers, due to its richness in sea mammals, big fishes and an extensive intertidal zone, probably used by humans, for its rich fauna food [the Taforalt harpoon, [Migrations between Maghreb and Iberian Peninsula had to take place across the sea since Paleolithic and the transport of women implied the use of bigger watercrafts than those crewed only by men. Sea navigation might have been commonly practiced as a fishing technique, without losing sight of the land for safety. The shortest distance between Europe and Africa is located at the Gibraltar Strait, and such distance markedly increases as one move towards east, reaching an approximately steady separation of around 150 km. Winds and ocean currents are very variable and dangerous around the Strait, and an alternative though not excluding calmer route might have been those that connects Cape Three Forks (Morocco) with the Andalusian coast, between Malaga and Almeria, in which the small Alboran Island  is halfway, and could be used as a stopover. Within this una food . Furtherharpoon, ) or evidHomo sapiens does not imply that peopling of the northwestern side of that continent took place from the inside exclusively.The obtained results underline the necessity of further research on genetic relationships in both sides of the western Mediterranean, using carefully collected samples from autochthonous individuals. Such studies should include different markers analyzed in a high genetic resolution, since migrations have been numerous in the region through a long period of the human history, presumably starting in Paleolithic times. Many studies have focused on recent North African gene flow towards Iberia, yet scientific attention should be now directed to thoroughly study the introduction of European genes in northwest Africa across the sea, in order to determine its magnitude, timescale and methods, and to compare them to those terrestrial movements from eastern Africa and southwestern Asia. The African origin of modern We have analyzed 279 mtDNA sequences from healthy, unrelated individuals of both sexes with geographic origins in Huelva (n = 158) and Granada provinces (n = 121). Further details on geography, history and demography of the studied populations can be found in ,9.For sampling processes, which must be considered as a critical activity in this kind of works, demographic stability and historical criteria were taken into account in the selection of the localities; touristic coastal areas, as well as the capital cities of the provinces were discarded. Each participant was informed about the aims of this research and full informed consent was obtained from all donors. Maternal ancestry of donors was recorded for a minimum of three generations born in the same province. The analyzed subjects were selected from a bigger sample collected during 2004–2008 by members of this research team, helped by local health workers. Andalusian DNA stock samples are available in our Laboratory of Molecular Anthropology . The present research project and protocols have been officially approved by the Bioethics Committee of the Complutense University of Madrid.Genomic DNA was isolated from blood samples (≈5-7 ml) by means of a standard proteinase-K digestion followed by phenol-chloroform extraction and ethanol precipitation.For each sample, mtDNA hypervariable region I (HVS-I) and part of HVS-II (≈820 bp) were amplified using primer pairs F15973 and R296 . SequenThere were two individuals from Granada that could not be classified in any concrete lineage, neither by control sequences nor by RFLPs. Therefore, they were categorized as NR* since we could only confirm the polymorphism 10873.mtDNA control region sequences obtained were aligned by using BioEdit v.7 software  and onlyD) [FS) [r) [FST indexes between the two Andalusian populations were also calculated , and their significance was evaluated with a nonparametric permutation test. Previous tests were performed using Arlequin 3.5 software [2 test was performed and the corrected typified residuals were analyzed to assess differences in haplogroup profiles (IBM SPSS Statistics 19).Within-population genetic diversity parameters of the studied populations were calculated. To check deviations from selective neutrality, and thus to assess evidence for presumable population expansion, we employed the Tajima’s (D)  and Fu  [FS)  statistiS) [r)  and by tS) [r) . Moleculsoftware . In addihttp://www.fluxus-engineering.com). The most parsimonious tree was constructed by hand, and confirmed by the Network MP-calculation option. We followed the weighting scheme proposed by [To construct maternal genealogies, a median-joining network  was infeposed by  for all posed by ,45. Moreposed by .Système Portable pour l’Analyse des Données, [Likewise, dataset of 53 selected populations drawn from the literature was used for performing an Analysis of Molecular Variance (AMOVA) . This teDonnées, ). This mSpatial Analyst Extension). This contour map was constructed using Inverse Distance Weighted method (IDW), which assumes that the influence of the variable being mapped (haplogroup U6 frequency) decreases with distance from the sampled locations. We used a power of two for the analysis.Finally, the geographical distribution of lineage U6 in the Mediterranean space was represented by a surface interpolation map built with ArcGIS 10.1 . Coding region polymorphisms are reported in the hierarchical order as tested. GenBank accession numbers for control region sequences are included.Click here for fileIB: Iberia, EUR: Europe, NME: Near/Middle East, SWA: Southwest Asia, NAF: North Africa.A set of selected human populations used for mtDNA comparative analyses. The geographic regions established are Click here for fileMedian-joining network showing the phylogenetic relationship of the 197 Andalusian mtDNA haplotypes. Haplogroups are shown in blue. The mutated positions reported in the branches refer to the reference sequence rCRS . All polymorphisms are control region mutations, except those in red (coding region haplogroup diagnostic positions). Transversions are specified with the base change after the mutation. Suffix “i” indicates an insertion, “d” a deletion and “Y” heteroplasmy (C/T). Retromutations are indicated with an exclamation mark. Size of circles is proportional to the haplotype frequency. Huelva and Granada samples are marked in red and green, respectively.Click here for file


Campylopterus curvipennis, a lekking hummingbird with an extraordinary vocal variability across Mesoamerica.Mesoamerica is one of the most threatened biodiversity hotspots in the world, yet we are far from understanding the geologic history and the processes driving population divergence and speciation for most endemic taxa. In species with highly differentiated populations selective and/or neutral factors can induce rapid changes to traits involved in mate choice, promoting reproductive isolation between allopatric populations that can eventually lead to speciation. We present the results of genetic differentiation, and explore drift and selection effects in promoting acoustic and morphological divergence among populations of C. c. curvipennis, C. c. excellens, and C. c. pampa disjunctly distributed in the Sierra Madre Oriental, the Tuxtlas region and the Yucatan Peninsula, respectively. Sequence mtDNA and microsatellite data were congruent with two diversification events: an old vicariance event at the Isthmus of Tehuantepec (c. 1.4 Ma), and a more recent Pleistocene split, isolating populations in the Tuxtlas region. Hummingbirds of the excellens group were larger, and those of the pampa group had shorter bills, and lineages that have been isolated the longest shared fewer syllables and differed in spectral and temporal traits of a shared syllable. Coalescent simulations showed that fixation of song types has occurred faster than expected under neutrality but the null hypothesis that morphological divergence resulted from drift was not rejected.Analyses of two mitochondrial genes and ten microsatellite loci genotyped for 160 individuals revealed the presence of three lineages with no contemporary gene flow: Our phylogeographic analyses uncovered the presence of three Mesoamerican wedge-tailed sabrewing lineages, which diverged at different time scales. These results highlight the importance of the Isthmus of Tehuantepec and more recent Pleistocene climatic events in driving isolation and population divergence. Coalescent analyses of the evolution of phenotypic traits suggest that selection is driving song evolution in wedge-tailed sabrewings but drift could not be rejected as a possibility for morphological divergence. Mesoamerica is considered one of the largest and most important biodiversity hotspots in the world, based mainly on the global proportion of vertebrate endemism and the loss of the original primary vegetation cover . NonetheCampylopterus curvipennis), a sexually monochromatic, size dimorphic hummingbird species complex commonly found in montane cloud forests and humid tropical forests [Campylopterus curvipennis curvipennis (SMO), C. c. excellens (TUX) and C. c. pampa (YUC) [c. 30 out of 320 extant hummingbird species known as lek breeders [The wedge-tailed sabrewing  . Wedge-tbreeders , and becbreeders . In addibreeders . Their cbreeders ,19.Species with highly differentiated populations may represent the early stages of the speciation process. In these populations, drift, ecological selection or both can induce changes in traits involved in mate choice, promoting reproductive isolation between allopatric populations that can eventually lead to speciation -23. The C. curvipennis. The use of both bi-parentally inherited molecular markers with different mutation rates is important because it allows us to make inferences at different temporal scales and provides more reliable insight into the historical processes involved in the evolution of taxa than studies based on the analyses of a single marker do. In addition to genetic data  infer the processes behind its evolutionary history, specifically assessing the role of the Isthmus of Tehuantepec as a barrier, and (iii) evaluate the role of drift and selection in driving phenotypic (morphology and song) divergence in wedge-tailed sabrewings. Patterns resulting from intraspecific geographical variation in phenotypic traits and genetic markers should provide insights into the factors driving population differentiation and ultimately speciation in Mesoamerica.To better understand the patterns of diversification and endemism in Mesoamerica, we use mitochondrial (mtDNA) and nuclear (microsatellites) DNA data to infer the processes behind the evolutionary history of ocesses; ), we useFrom 160 wedge-tailed sabrewings we obtained mtDNA sequences that contained 81 polymorphic sites (38 in the ATPase and 43 in the control region) of the 1,407 bp of the genes analyzed. No insertions or deletions were present; therefore, variants were identified based solely on nucleotide substitutions. Sixty-three haplotypes were identified for the 22 wedge-tailed sabrewing populations. Haplotype and nucleotide diversity are summarized in Table curvipennis, SMO) and eastern  groups on either side of the Isthmus of Tehuantepec , corresponding to disjunct western  and 0.52 (0.13-1.21) Mya, respectively. These results suggest that the split between the Sierra Madre Oriental and Yucatan Peninsula clades may have occurred during the mid-Pleistocene. With respect to SMO, for the TUX clade the constant clock TMRCA estimated divergence times of 614,000 (0.12-1.55 Mya) and 202,000 (0.05-0.47 Mya) years ago for 2 and 5% substitution/My, respectively. This implies that the split between the TUX and SMO clades may have occurred more recently in the late Pleistocene.The haplotype network showed a strong phylogeographic structure among three groups , but not among populations or areas within the SMO  Figure . Of the curvipennis, SMO; excellens, TUX; and pampa, YUC) with the highest percentage of the variation being explained by differences among groups (90.41%) and the lowest by differences among populations within groups  , but not among sampling localities within the fragmented areas of the SMO . Pairwise ST Fand ST Rwere quantitatively similar but we only report ST Rvalues because the stepwise mutation model implemented in this estimate is more appropriate for microsatellites [Additional file ST Rvalues among sampling localities ranged from 0.001 for Xil/Ord to 0.42 for Risc/Nov. As with the pairwise ST Fvalues for the mitochondrial genes, estimates of ST Rwere significant for almost all comparisons between localities from TUX versus the rest of the localities and comparisons between localities from YUC versus the rest after Bonferroni corrections. Values from comparisons between sampling localities within SMO were not statistically significant. Comparisons between samples from Bec and Gar (from the YUC region) versus the rest were not significant in most cases after Bonferroni corrections probably due to sample size, affecting statistical power.Significant genetic subdivision was detected among sampling localities  was at K = 2 ; in the analysis where the TUX samples were excluded two clusters were detected for the SMO samples with no evidence of genetic clustering at this level. These results suggest a hierarchical clustering pattern where YUC samples are highly divergent and samples from TUX are sub-structured in a cluster that contains samples from SMO.Results of the STRUCTURE analysis corroborated population substructure. Using all genotypes together the log likelihood was highest for 2 Figure . One clu2 Figure . HoweverAmong-group comparisons of gene flow (M) from the MIGRATE analysis ranged from 0.49 (TUX to YUC) to 1.98 (TUX to SMO), however, all comparisons were less than or not significantly greater than 1.0 , 0.004-4.21) and close to 1 in the opposite direction TUX-SMO , suggesting that divergence took place in the presence of gene flow from the SMO to the Tuxtlas region. In contrast, low migration rates following the split were estimated for populations east and west of the Isthmus of Tehuantepec , indicating that the population split occurred in the absence of gene flow, i.e. this geographic barrier was not permeable to migrants. Assuming a mutation rate for microsatellites of 2.96 × 10-3, estimates of effective population size indicate that the ancestral population was significantly larger  than the populations after divergence. The TUX group had the lowest population size  followed by YUC  and SMO .Our simulations to test whether genetic groups might have originated in the face of gene flow were consistent across replicates, and produced confident posterior probability peaks for the parameters estimated. When testing for migration following the split between the SMO and TUX groups, migration rates were higher than 1 in the SMO-TUX direction , and these differences were significant for each morphological variable  was further examined for possible allometric effects using the residuals of this relationship, and the differences between lineages were maintained . For females the MANOVA also showed significant lineage differences , and the results of the univariate ANOVAs were all significant  are significantly larger than those of the curvipennis (SMO) and pampa (YUC) groups, and that pampa individuals have shorter bills.The MANOVA showed significant lineage differences in male morphology , but not in the second and third . PC1 was mainly explained by minimum and peak frequency of note 1, duration of note 3, and frequency range, peak frequency and duration of note 4.A total of 344 syllable types were detected across populations. Songs were very versatile and no successive syllable repetitions were detected except for vocalizations recorded in the YUC region from the n Figure . Based on Figure . In addin Figure . On averSTF) and morphological distances for males , even when controlled for geographic distance . However, the relationships between pairwise ST Rand morphology were not significant. In the case of females none of the relationships were significant. Regarding acoustic distances, Mantel test showed a strong positive correlation between genetic and song sharing distance , and also for the genetic and common syllable distance . These relationships were maintained when geographic distance was accounted for . These analyses indicate that more genetically divergent lineages shared fewer syllable types, and differed acoustically in the common syllable, independently of distance. Lastly, we did not find a significant relationship between morphological and acoustic distances versus habitat-related (climate and topography) distances for either males or females.Mantel tests showed a strong positive correlation between genetic  that nuclear genes would be fixed in these populations under neutrality, suggesting that drift cannot be rejected as a possibility for morphological divergence [Additional file Coalescent simulations  of morphs value for reconstructed trees was equal to 8 when considering four populations for vocal characters. The upper 95% CL for time since population divergence was 0.48e Ngenerations assuming a dichotomous branching model of divergence and 0.68e Ngenerations assuming a simultaneous model. When branch lengths were reduced four times, an s value of 3 occurred in none of simulated trees, suggesting that there is a very low probability (P < 0.0001) that nuclear genes would be fixed in different populations under neutrality [Additional file The coalescent simulations of the song and mtDNA sequence data from four populations with large sample sizes of sequences in which song characters are fixed  were used to differentiate the roles of selection and genetic drift in song evolution. The observed pampa group in the Yucatan Peninsula, the curvipennis group along the Sierra Madre Oriental, and the excellens group in the Tuxtlas region. The patterns of genetic differentiation inferred here for C. curvipennis are consistent with the results for other codistributed avian species in montane cloud forests of the Sierra Madre Oriental and the Tuxtlas region such as Chlorospingus ophthalmicus [Buarremon finches [pampa group when K = 2 and excellens when K = 3, indicating a lesser degree of divergence between the TUX and SMO genotypes in comparison to those of YUC. Consistent with the lack of haplotype sharing and high pairwise ST Rvalues, the results of gene flow measures based on microsatellites indicate that the SMO, TUX and YUC populations exchange no migrants. Both mtDNA and biparentally inherited microsatellite loci are spatially structured between allopatric populations of wedge-tailed sabrewings suggesting that gene flow is restricted and that C. c. curvipennis, C. c. excellens, and C. c. pampa constitute genetically unique populations.Phylogeographic analyses on mtDNA sequence data and microsatellites were highly congruent identifying three wedge-tailed sabrewing lineages: the halmicus  and Buar finches . Even th finches . Similarpampa and the curvipennis-excellens groups is consistent with the hypothesis that the Isthmus of Tehuantepec was a major vicariant event and has been a significant habitat barrier to dispersal, as observed in previous mtDNA phylogeographical studies with montane bird species [c. 6 Ma; [It is possible that allopatric fragmentation has affected the genetic structure of wedge-tailed sabrewings. Although the existence of several mutational steps in a haplotype network is indeed indicative of allopatric fragmentation, it could also result from non-sampled haplotypes from intermediate populations. The latter is not the case here, as we sampled populations covering the entire geographical range of the species complex [Additional file  species ,9-11; hoc. 6 Ma; ), periodc. 6 Ma; . The phyc. 6 Ma;  across tRegarding the most recent divergence between the SMO and TUX populations, the oscillations in climate during the Pleistocene caused a considerable expansion in the range of highland habitats and their descent to lower elevations ,36. ThesMorphological data for both males and females indicated that individuals from TUX were significantly larger than those from SMO and YUC, and individuals from SMO and TUX had longer bills than those from YUC. The lack of correspondence between climate and topographic conditions and morphological traits suggests that weak habitat selection pressures have been shaping morphological divergence. This inference should be made with caution, considering that habitat structure was characterized using only ground-based interpolated climate and topography data. Information about vegetation structure obtained using satellite and airborne remotely sensed images would offer a more powerful way for testing adaptive and nonadaptive hypotheses concerning the evolutionary processes that operate across the environmentally heterogeneous space occupied by wedge-tailed sabrewings. However, these data require substantial processing for applications in spatial analyses and are sensitive to cloud contamination ; these dC. c. curvipennis), TUX (C. c. excellens), and YUC (C. c. pampa) populations. Besides, independently of geographic distance, a significant positive relationship between genetic and morphological distances was found highlighting the idea that drift might facilitate phenotypic divergence when acting in concert with selection [When conducted a coalescent-based test using mtDNA sequence data to evaluate whether the observed phenotypic differentiation can be attributed to drift or some form of selection, we could not reject the possibility of drift in driving morphological differentiation of the SMO  through natural selection. Differences in culmen length are congruent with the differences in habitat characteristics of the Yucatan Peninsula  compared to the higher altitude and wetter habitats of the Sierra Madre Oriental and the Tuxtlas region, suggesting that populations adapted to different ecological conditions, including perhaps the presence of flowers with longer corollas. Differences in environmental variables provide the potential for ecological differentiation where different selection pressures might act to shape the relationship between pollinator and plant species . Howeverpampa lineage from the Yucatan Peninsula whose individuals live in deciduous tropical forest at a lower altitude and much drier conditions than populations from the other lineages, which inhabit cloud or tropical forests at higher altitudes that have wetter conditions. However, we found no significant correlations between acoustic and habitat-related distances (climate and topographic variables), which suggest that these conditions play a minor role in shaping song divergence.The divergence of mating-related acoustic signals has been described as being shaped by habitat-dependent selection in many bird species -47, becaIn contrast, we found a strong acoustic divergence pattern in syllable sharing between SMO populations and also between geographic areas, the latter corresponding to the genetic differentiation among the three lineages of wedge-tailed sabrewings. Each lineage had an exclusive assemblage of syllable types, and song sharing was lower between than within lineages. Also, the acoustic traits of a common syllable were more divergent between than within lineages. The coalescent analysis to evaluate whether vocal differentiation can be attributed to drift or selection, suggested that the fixation of song types assumed to be encoded by nuclear genes, has occurred faster than expected by genetic drift, providing evidence that selection is driving song evolution in wedge-tailed sabrewings. In lek-breeding systems where male birds are exposed to strong sexual selection pressures, the use of certain song types over others could increase their adaptation to social conditions and/or reproductive success. Although song could potentially play a role in increasing genetic polymorphisms and generating reproductive isolation and speciation through sexual selection ,48,49, fThe positive relationship between genetic and acoustic distances suggests that independently of geographic distance, the most genetically distant lineages shared fewer vocal elements and differed in the acoustic traits of the common syllable. This relationship is expected under a drift model of song evolution, where acoustic divergence is higher between populations that have been genetically isolated for the longest time . As in sThe information generated by our study allows us to make the following conservation recommendation. The genetic differentiation among the three genetic groups revealed from the mtDNA analyses suggests that these groups have been genetically isolated for a long period of time. This genetic isolation and the lack of contemporary gene flow among these groups were also supported by the microsatellite data. In addition, the ancestral effective population size was larger than the size of either genetic group after splitting as revealed by IMa analysis. Because SMO, YUC and TUX are distinct genetic groups with no genetic connection among them, conservation plans should consider the sites within each of them as independent, and future management plans should focus on conserving the genetic diversity of the three genetic groups.excellens group, the relatively small effective population size (one-third of that from SMO), and because the accelerated deforestation rates in the Tuxtlas region [Conservation of the Tuxtlas region is particularly important due to the restricted distribution of the s region  is a thrs region . This res region . Considec. 1.4 million years ago without gene flow during the process of divergence, as well as more recent climate events during the Pleistocene in driving isolation and population divergence of wedge-tailed sabrewings with gene flow from the SMO to the Tuxtlas populations. Coalescent analyses of the evolution of phenotypic traits suggest that the fixation of song types has occurred faster than would be expected by genetic drift, suggesting that the action of selection is driving song evolution in wedge-tailed sabrewings. However, the role of drift in driving morphological divergence could not be rejected. Finally, considering the accelerated deforestation rates in the Neotropics the conservation of the montane and tropical forest in this region is needed, and special attention must be paid to small isolated areas with genetic uniqueness such as the Tuxtlas region.The genetic and phylogeographic analyses of this study, based on mtDNA and microsatellites, uncover the presence of three lineages of Mesoamerican wedge-tailed sabrewings that exhibit no contemporary gene flow. These correspond to the disjunct distribution of populations at the Sierra Madre Oriental, the Tuxtlas region, and the Yucatan Peninsula. Our results highlight the importance of the Isthmus of Tehuantepec in generating population divergence Campylopterus curvipennis breeding seasons, covering the entire geographical range of the species complex: (i) 15 populations of C. curvipennis curvipennis (SMO), (ii) three populations of C. curvipennis excellens (TUX), and (iii) four populations of C. curvipennis pampa (YUC) (Table i) three populations from southern Tamaulipas and northern San Luis Potosí (nSMO herein); (ii) three populations from southern San Luis Potosí and Hidalgo (cSMO herein); and (iii) nine populations from Puebla and central Veracruz (sSMO herein)  where the temperature is lower. The excellens TUX populations are located in wetter semideciduous tropical forests at a lower elevation , and those of the pampa YUC populations are in drier deciduous tropical forests at a much lower elevation (c. 60-250 m above sea level) and higher temperatures  (see Acknowledgments for tissue loans).For genetic analysis, based on the disjunct geographical distribution of the species complex ,32, we sThree body measurements were obtained from mist-netted hummingbirds using a dial caliper to an accuracy of 0.1 mm and a wing ruler: exposed culmen (from the base of the bill to the tip of the upper mandible), wing chord , and tail length . All measurements were taken by CG. Sex determination was carried out in the laboratory by means of a polymerase chain reaction (PCR) using primers 2550F  and MSZ1Acoustic recordings were made for 64 males  at 11 of the 22 sampled populations . Amplification reactions  contained 0.72× buffer, 3.5 mM MgCl2, 0.14 mM of each dNTP, 0.2 μg/μl of BSA, 0.3 μM of each primer, 0.05 U Taq (Promega), and 2-5 μl of genomic DNA. PCR reactions were performed in a 2720 thermal cycler (Applied Biosystems) or in an Eppendorf Mastercycler thermocycler with the following temperature profile: initial denaturation at 94°C for 5 min; 35 cycles consisting of denaturation at 94°C for 1.5 min, annealing at 53-60°C for 1 min and an extension of 1.5 min at 72°C; and a final extension of 72°C for 7 min. PCR products were visualized on a 1% agarose gel stained with ethidium bromide, purified with the QIAquick PCR purification kit  and sequenced using the Big Dye cycle terminator kit. Sequences were visualized in a 310 automated sequencer (Applied Biosystems) and by Macrogen Inc. ATPase genes were sequenced in both directions and the control region only in the reverse direction using primer ARCO1R except when ambiguities were present in the sequences. Sequences were edited using SEQUENCHER 4.8 demo (Gene Codes) and then manually aligned with SE-AL 2.0a11 [HQ380686-HQ380755.We extracted genomic DNA from one of the feather samples using chelex , and thL 2.0a11 . All uniCampylopterus curvipennis  to root the tree. Alignment for the phylogenetic analyses is included as an additional file [Additional file We reconstructed intraspecific phylogenetic relationships among haplotypes using Bayesian inference in MRBAYES 3.12 . Data web obtained from fossil records and biogeographical events, demonstrating support for the 2% rule across taxonomic orders. Similar substitution rates have been described for other protein-coding mitochondrial DNA markers such as ATPase and ND2 [In the absence of appropriate internal calibration points for many groups of birds, the 2% divergence-per-My clock calibration has been widely used. However, the degree of heterogeneity of molecular evolution rates across lineages and genetic loci could confound the accuracy of divergence time estimates, making the use of the 2% rule controversial -71. In a and ND2 ,73-75. N and ND2 , Milá et and ND2 , and Bar and ND2 . Uncerta and ND2 ,78, so w and ND2 . TMRCA eA statistical parsimony haplotype network was obtained using the program TCS 1.2.1  using thHd) and nucleotide diversity (π). To estimate population genetic structure we conducted an analysis of molecular variance (AMOVA). We used the model of molecular evolution of Tamura and Nei with a gamma shape parameter = 1.2757 for the combined data set, to estimate genetic distances. A total of 16,000 permutations were estimated to determine the AMOVA's significance. Two AMOVAs were run, grouping sampling sites based on the fragmented geographical range of wedge-tailed sabrewings  within populations and loci in GENEPOP. Bonferroni corrections were applied to correct for multiple simultaneous comparisons [Expected and observed heterozygosity and mean number of alleles per locus  were calculated in GENEPOP 3.4 . Allelicparisons .ST R[ST F[ST Rpairwise values in PAUP 4.0 [To investigate population genetic structure we calculated global and pairwise RST  and pairRST [FST  in RSTCARST [FST  and ARLEPAUP 4.0 .K) through the multilocus genotypic data. We ran STRUCTURE without population information under the admixture model with correlated allele frequencies. Ten independent chains were run for each K, from K = 1 to K = 10. The length of the burn-in was 500,000 and the number of Markov chain Monte Carlo (MCMC) replications after the burn-in was 1,000,000. To determine an accurate number of clusters we calculated the statistic ΔK based on the rate of change in the log probability of data between successive K values following Evanno et al. [To examine geographic patterns of population genetic substructure, we performed Bayesian genetic clustering using STRUCTURE 2.2.3 , which io et al. .ST Fcalculations. We ran 10 short  and three long chains , after discarding the first 20,000 genealogies as a burn-in.Contemporary gene flow (M) among populations, and groups of populations, defined by mtDNA and microsatellite analyses, was estimated with a maximum likelihood coalescent approach using microsatellite data and MIGRATE v. 3 . The fir1, m2m), and effective population sizes (q) from the ancestral population and after the split occurred between the two groups in both analyses. Estimates of migration rates were converted to the effective number of immigrants per generation by using estimates of theta. For estimates of effective population sizes of each genetic group we assumed a mutation rate per generation of 2.96 × 10-3 [Both mtDNA and microsatellite genetic analyses indicated three genetically diverged groups. We investigated whether these groups of populations had diverged in the face of gene flow by using IMa . We perf6 × 10-3 .C. curvipennis complex, we performed a multivariate analysis of variance (MANOVA) with the three measurements taken as dependent variables, and lineages (see below) as fixed factors. One-way ANOVAs followed the MANOVA to determine lineage differences for each variable. To meet parametric analysis assumptions, variables were log transformed, but untransformed data are reported in figures. These analyses were performed for each sex separately due to size dimorphism using SUPERANOVA .To examine differences in morphological variation between groups of the http://www.birds.cornell.edu/raven with the parameters fully described in González and Ornelas [We generated spectrograms of recordings using RAVENPRO V. 1.4  Ornelas . We used Ornelas  for deta Ornelas ,19, we tc. 1 km2 [We used climate and topographic variables across all 22 sampling localities to test for habitat selection acting on morphology and acoustic traits. We used these variables as a broad level approximation of habitat and ecological variation potentially related to availability of floral nectar resources . We c. 1 km2 , in combc. 1 km2  for eachversus genetic distances (ST Ffor sequence data and ST Rfor microsatellites), controlling for geographic distance with partial Mantel tests. To test for the habitat-selection model, we compared matrices of morphology and acoustic distances (acoustic measurements) with habitat-related (climate and topography) distances.We followed Ruegg et al.  to examiIn order to test whether the degree of male phenotypic differentiation  was caused by drift or divergent selection, we performed an indirect coalescent-based simulation method developed by Masta and Maddison . BecauseIn order to implement this method with our data, we performed two different analyses: in the first analysis for morphological characters we considered three representative populations  corresps statistic of Slatkin and Maddison [s values indicate greater levels of incomplete lineage sorting, suggesting a more recent population divergence, and smaller s values indicate lower levels of incomplete lineage sorting suggesting older population divergence. Then, we compared the observed s value against s values obtained from simulated trees to estimate time since population divergence. This comparison was performed with computer simulations generating 10,000 gene trees within a population tree by gene coalescence. We set e N= 500 in each population  and vocal (s = 3) characters. Estimates of generations since population divergence were divided by four considering that nuclear genes have four times the population size of mitochondrial genes. Estimates of s, branch lengths and coalescent simulations were performed in MESQUITE v. 2.73 [We first calculated the Maddison , a measuaddison, ), and th v. 2.73 .CG and JFO conceived of and designed the study. CG carried out the fieldwork, generated the molecular data, and conducted all genetic and phenotypic analyses with input from CGR and JFO. CG and JFO wrote the manuscript with contributions from all authors, who read and approved the final manuscript.Campylopterus curvipennisPairwise comparisons between populations of . Pairwise ST Fvalues  of mtDNA and pairwise ST Rvalues  of microsatellites between populations. Values statistically significant at P < 0.001 are indicated in bold.Click here for fileSTRNeighbour-joining tree of sampled locations based on pairwise . Analysis obtained from microsatellite data showing YUC group clustering together in a basal position, and sampling localities of SMO group clustering in a geographically unresolved clade. Sampled locations with only one individual were excluded from the analysis.Click here for fileResults of the coalescent-based simulation on phenotypic traits. Estimates of s of Slatkin and Maddison from 10,000 coalescent simulated gene trees to test the probability of complete sorting of vocal characters (A and B) and morphological characters (C and D) in wedge-tailed sabrewing populations. Regarding vocal characters, there is a low probability of fixation in nuclear genes under neutrality regardless of the assumption of dichotomous branching (A) or the simultaneous model of divergence (B). In contrast, the probabilities that nuclear genes would be fixed under neutrality are high for morphological characters, regardless of the assumption of dichotomous branching (C) or the simultaneous model of divergence (D). This suggests that divergent selection has caused the pattern of vocal variation among populations, but the null hypothesis that morphological divergence resulted from drift was not rejected. Arrows indicate the expected value of s in a completely sorted tree.Click here for fileCampylopterus curvipennis species complexGeographic distribution of . Distribution of Campylopterus curvipennis species complex based on museum (MZFC) and bibliographic records [C. c. curvipennis, yellow = C. c. excellens, red = C. c. pampa. records ,59, showClick here for fileAlignment of ATPase 6-8 and control region mtDNA sequences. Alignment of the concatenated ATPase 6-8 (1-875 bp) and control region (876-1407 bp) mtDNA sequences for 160 individuals of Campylopterus curvipennis and three outgroups used in the phylogenetic analyses.Click here for file


To carry out a deeper forensic and anthropological evaluation of the short tandem repeat (STR) D9S1120 in five Mestizo populations and eight Amerindian groups from Mexico.ST-values were compared with those previously obtained with nine STRs of the Combined DNA Index System (CODIS-STRs).We amplified the STR D9S1120 based on primers and conditions described by Phillips et al, followed by capillary electrophoresis in the genetic analyzer ABI Prism 310. Genotypes were analyzed with the GeneMapper ID software. In each population we estimated statistical parameters of forensic importance and Hardy-Weinberg equilibrium. Heterozygosity and FST = 2.81%; P < 0.0001), larger than between Mestizos . D9S1120 showed less genetic diversity but better population differentiation estimates than CODIS-STRs between Amerindian groups and between Amerindians and Mestizos, but not between Mestizo groups.Amerindian and Mestizo populations showed high frequencies of the allele 9 and 16, respectively. Population structure analysis (AMOVA) showed a significant differentiation between Amerindian groups (FThis study evaluated the ability of D9S1120 to be used for human identification purposes and demonstrated its anthropological potential to differentiate Mestizos and Amerindian populations. Sample analysis of the Human Genome Diversity Project-Centre d’Etude du Polymorphisme Humain (HGDP-CEPH) panel with 377 microsatellites or short tandem repeats (STR) conducted in five Native Amerindian populations  found a high frequency of a small allele (275 basepairs) at the tetra-nucleotide locus D9S1120, which was absent in 47 other worldwide populations . FinallyThe forensic potential of D9S1120 for detecting Native American ancestry was evaluated in a third study, which typed three native and two admixed populations from Colombia and three non-American populations ,14. The DNA was extracted from fresh blood samples by salting-out method . Its quaST distances and exact test P values including previously published population data (Het) and population differentiation (FST) based on D9S1120 polymorphism with those previously estimated with nine CODIS-STRs in Mexican Mestizos from Chihuahua, Jalisco, Yucatan, and Veracruz , but the allele 10 was exclusively observed in the Purépecha group. The increased allele number by population was related to the presence of alleles 13 and 19, which were observed in Mestizos from Chihuahua (north), Jalisco (west), and the Maya group (southeast). The smallest allele number was observed in the Tojolobal  and Lacandon  native groups. The modal alleles in Amerindian groups were 9 (38.2%) and 16 (30.5%), but in the Tojolabal and Maya groups the allele 16 was prevalent over the allele 9   and 3. IP value (P = 0.0364), however this was not significant after Bonferroni correction. In order to record representative D9S1120 genotypes for the main Mexican population groups, we obtained a ratio of genotype frequencies (RGF) between Mestizos and Amerindians and vice-versa  and were clearly separated in the MDS plot (P < 0.01), but were similar to most of Mexican Mestizos, except those from Yucatan (P = 0.0032) (data not shown).We estimated genetic distances and Fst  D9S1120  and repr D9S1120 . MexicanMDS plot . No diffMDS plot . Among nMDS plot , which eMDS plot . SimilarP > 0.01; data not shown) and to the majority of Mexican Amerindian populations, except Lacandones. Individually, the Colombian Awa-Kuaikier population was different from Tarahumaras, Purépechas, Tzotziles, and Lacandones, whereas the Pijao group was different only from Lacandones and Tojolabales. Lacandones was the most distinct and markedly differentiated Mexican Amerindian group . However, Amerindians showed larger and significant inter-population differentiation  than Mestizos . When we clustered Mestizos vs Amerindians, the differentiation both between the groups  and within the groups was significant , indicating that the population clustering was not robust. Although differentiation between Amerindians from Mexico and those from Colombia , differentiation within the groups was . Finally, when population structure was assessed between Amerindians (Mexicans plus Colombians) and populations from Europe, Asia, and Africa .AMOVA based on D9S1120 showed a significant inter-population differentiation among all the Mexican populations (FST) based on D9S1120 were compared with those previously obtained using 9 CODIS-STRs from the corresponding Mexican populations . D9S1120 showed lower genetic diversity than most of the nine CODIS-STRs in most of the populations (The genetic diversity represented by heterozygosity (Het) and the population differentiation coefficient . In Mestizos, this assumption was evaluated in view of the previously described increasing northwest to southeast gradient of Amerindian ancestry and the opposite gradient of European ancestry. This pattern has been consistently obtained with different genetic systems, such as CODIS-STRs, Y-STRs, and genome-wide single nucleotide polymorphisms analysis ,14,22. W9RA was prevalent in the majority of Mexican Amerindian groups, except in the Mayas and Tojolobales, where the modal allele was 16 . There are contrasting explanations for such allele distribution. In the Mayas, the large number of alleles and the observed heterozygosity suggests admixture, which is in agreement with previous reports on elevated gene flow  and on cThe forensic parameters estimated in this study confirm that D9S1120 can be a useful tool for human identification and molecular anthropology. On average, genetic diversity values (Ho) were larger in Mestizos than Amerindian groups  and 3. IAn interesting finding was that Mestizos and Amerindian groups from Mexico could not be differentiated from their counterparts from Colombia , which i


The leatherback turtle (Dermochelys coriacea) in the Dominican Republic appeared to be isolated from Awala-Yalimapo, Cayenne, Trinidad and St. Croix but connected with other Caribbean populations. Two distinct nesting populations of hawksbill turtles (Eremochelys imbricata) were detected in the Dominican Republic and exhibited interesting patterns of connectivity with other nesting sites and juvenile and adult male foraging aggregations. The green sea turtle (Chelonia mydas) has almost been extirpated from the Dominican Republic and limited inference could be made from our samples. Finally, results were compared with Lagrangian drifting buoys and published Lagrangian virtual particles that travelled through the Dominican Republic and Caribbean waters. Conservation implications of sink-source effects or genetic isolation derived from these complex inter-connections are discussed for each species and population.Nesting by three species of marine turtles persists in the Dominican Republic, despite historic threats and long-term population decline. We conducted a genetic survey of marine turtles in the Dominican Republic in order to link them with other rookeries around the Caribbean. We sequenced a 740 Many marine vertebrates have complex life cycles requiring the use of different habitats that are often spread over wide spatio-temporal scales and usually result in a network of connections among different populations and between populations and distant feeding grounds Chelonia mydas), loggerhead (Caretta caretta) and leatherback (Dermochelys coriacea) turtles during the 1980s Eretmochelys imbricata) shell was exported to Japan between 1970 and 1986 The Dominican Republic has, for many years, presented a gap of knowledge in marine turtle biology and conservation. Although it has been suggested as an important nesting area for several marine turtle species The consequences of the accumulation of these threats to the Dominican Republic nesting populations were not properly addressed until a recent study mixed stock point of view nesting population point of view Genetic markers have been widely used to establish links between populations and feeding grounds and to infer relative exposure to threats Other approaches have involved the study of ocean currents, and have demonstrated that they may play a crucial role in defining hatchling and juvenile marine turtle dispersion Here we combine genetic data from the Dominican Republic turtle nesting population with published genetic data from other nesting populations and feeding grounds, as well as tracks of passive drifter buoys and satellite tracking of adult turtles to: 1) determine if Dominican Republic turtle nesting populations are isolated from others in the region; 2) determine if mtDNA sequences reflect population size reductions; and 3) determine which feeding grounds are used by Dominican Republic marine turtles.During the 2007 and 2008 nesting seasons (from March to October), samples from three nesting marine turtle species were collected within an extensive monitoring project to assess nesting marine turtle abundance and distribution in the Dominican Republic bp fragment of the mtDNA control region using the primers LCM15382 (5′-GCTTAACCCTAAAGCATTGG-3′) and H950 (5′GTCTCGGATTTAGGGGTTT-3′) bp (leatherback sea turtle), 491bp (green sea turtle) or 384bp/480bp (hawksbill sea turtle). Our 25 µL polymerase chain reaction (PCR) included the following: genomic DNA, 1× PCR Buffer, 2 mM MgCl2, 0.12 mM dNTP, 0.2, µm of each primer and 0.04 U/µL of Taq polymerase. After an initial 5 min denaturing step (94°C), our PCR protocol consisted of 35 cycles of the following temperature regime: 1 min at 94°C (denaturing), 1 min at 52°C  and 90 s at 72°C (extension). In addition, we included a final extension step of 10 min at 72°C. Following PCR, we removed single-stranded DNA by digesting 5 µL of PCR product with 2 µL of a combined Exonuclease I and Shrimp Alkaline Phosphatase solution (ExoSAP-IT®). The reaction mixture was incubated for 15 min at 37°C, followed by other 15 min incubation at 80°C to inactivate the two enzymes. We sequenced both forward and reverse strands using the BigDyeTM Primer Cycle Sequencing Kit (Applied Biosystems) run on an automated DNA sequencer (ABI PRISM 3100). For each sequencing reaction, we used 2 µL of our PCR product in a 10 µL reaction mix under the following conditions: 1 m denaturing step at 96a followed by 25 cycles consisting of an initial denaturing of 10 s at 96°C, 5 s at 50°C  and 4 m at 60°C (extension). Products were purified by ethanol precipitation before enter the sequencer.DNA was extracted using the QIAamp extraction kit (QIAGEN®) following the manufacturer’s instructions. We amplified a ∼740bp) haplotypes previously described for the leatherback turtle bp) haplotypes described in those manuscripts that used the same or similar primers in the leatherback turtle http://accstr.ufl.edu/) that includes all published haplotypes. Posterior statistical analyses were carried out with short, long sequences or both depending on the data found in the published literature.Sequences were aligned by eye using the program BioEdit 5.0.9 Differences in haplotype frequencies of samples from the same species at different locations within Dominican Republic were assessed using a Chi-square test. Values were compared to the distributions observed by randomizing individuals among populations using Monte-Carlo resampling h) and nucleotide diversity (π) st) using Arlequin 3.0 In order to assess the genetic diversity compared to the other Atlantic populations we calculated haplotype diversity  (bp) haplotype frequencies, as this analysis requires detailed information of haplotype frequencies of nesting populations and feeding areas and the dataset regarding putative feeding grounds available in the literature is most extensive for this fragment  . Additiofragment . Nestingfragment  or long fragment . As we cfragment  and S3.http://www.aoml.noaa.gov/). An initial query within the locations database was undertaken in order to select all locations plotted near (<50 km) either the Jaragua National Park (DRJ/DR) or Saona Island (DRS) nesting populations. Then, all buoys with at least one location obtained near one of these Dominican Republic nesting areas were selected as an evidence of passive arrival or departure from the area.In order to simulate hatchling turtle dispersal, data describing the tracks of satellite-tracked surface drifter buoys were obtained from the Global Drifter Program of the National Oceanic and Atmospheric Administration . These Lagrangian buoys are periodically released throughout the year at varying locations and are tracked by satellite , providing several positional fixes per day  and exhibited the haplotype CM-A5  which isbp fragment or the 711bp fragment (h (SD) = 0.176 (0.074), π (SD) = 0.0014 (0.0012)] with the exceptions of some populations outside the Caribbean like Awala-Yalimapo , Cayenne , French West Indies , Saint Croix  and Trinidad . No recent expansion was suggested for Dominican Republic leatherback population, independently of the length of the marker used . As previous studies with the 496bp fragment indicated bp fragment yielded similar results  and hence were treated as two independent units for all posterior analysis. Genetic variability of the two Dominican Republic populations was similar or higher than other nesting populations . No recent expansion was suggested for either populations irrespective of the length of the marker used . Analysis of genetic structuring of Dominican Republic nesting beaches in relation to other populations in the Caribbean showed deep levels of differentiation with the exceptions of the DRS population that showed no significant differences to the proximate nesting aggregation of Mona Island in Puerto Rico (PRV), the populations of Belize (BLZ), Barbados Windward (BWI) and U.S. Virgin Islands (USV)  . The simare test . The lacp 83.9%;  and the  79.61%;  fragment 79.61%; .rookery centred ‘many-to-many’ mixed stock analysis for Dominican Republic juveniles suggested that turtles originating from the two nesting areas are likely to have been distributed in foraging grounds across the Caribbean, although the 95% confidence intervals were very high   between Conservation planning for mobile species such as marine turtles, depends on robust spatio-temporal information about the RMU of interest ca. 260 green turtles nesting per year in the 1980s Green turtles in the Dominican Republic bp sequences did not change the number of haplotypes in the Dominican Republic in contrast to previous studies, where some 496bp haplotypes were subdivided into multiple 711bp haplotypes bp sequences to other nesting and feeding areas is needed in order to test if longer sequences improve resolution of population structuring as they have for the loggerhead turtle The leatherback turtle is thought to be the least philopatric of the marine turtles The connection between the Dominican Republic leatherback population with several larger populations within the Atlantic would favour putative recovery through immigration Previous studies in the Caribbean have shown that the hawksbill turtle exhibits a high degree of philopatry resulting in fine scale population structure bp)/EiA23 (720bp) haplotype, the second by the high frequency of the F (380bp)/EiA11 (720bp) haplotype and the last by the high frequency of the A (380bp)/EiA1 (720bp) haplotype . The nesting aggregation in Mona Island, Puerto Rico aplotype . Based oThe structuring of the Caribbean stocks has strong implications for the conservation of the hawksbill nesting populations of the Dominican Republic. The DRJ population is completely isolated, suggesting it is unlikely to receive females by immigration. The small population size Regardless of female philopatry, hawksbill turtles appear to be highly mobile during the juvenile developmental phase. Blumenthal et al. rookery centred mixed stock analysis, did not support this hypothesis, as it predicted an homogeneous distribution of juveniles originating in both Dominican Republic nesting populations. Such mixed stock analysis usually yields wide confidence intervals in marine turtles due to the existence of common haplotypes and results may be taken with caution. Furthermore, the population sizes of the two Dominican Republic nesting populations mixed stock centred analysis. For instance, the juvenile feeding ground located near DRJ present study), but it also receives turtles from Cuba. This means that any extreme mortality of this juvenile aggregation would have an impact on these populations.Considering all these results and the putative dispersal mechanisms of the species The contribution of both Dominican Republic nesting populations to the Puerto Rico male aggregation is very clear. Satellite tracks of adult males and females in Puerto Rico showed that adults move from Puerto Rico and arrive to the vicinity of DRJ and DRS and remained there, possibly to breed Historical measures of the genetic variability would be desirable in order to detect a recent loss of the genetic variability in the Dominican Republic nesting populations caused by the recent population decline One of the milestones in the conservation of endangered species is the detection and quantification of threats affecting declining populations. However, the contextualisation of these potential conservation sinks in a wider regional area is necessary when complex life histories and complex populations structures are present, such as in sea turtles Table S1Haplotype frequencies of Caribbean Hawksbill marine turtle nesting populations. Using the short (380 bp) fragment and including nesting population size (nests/year) used as a baseline for the Mixed Stock Analysis. Puerto Rico data from PRB (DOCX)Click here for additional data file.Table S2Haplotype frequencies of Caribbean Hawksbill marine turtle feeding grounds. Using the short (380 bp) fragment.(DOCX)Click here for additional data file.Table S3Haplotype frequencies of Caribbean Hawksbill marine turtle nesting populations. Using the long (720 bp) fragment and including nesting population size (nests/year) used as a baseline for the Mixed Stock Analysis. PS: Present Study.(DOCX)Click here for additional data file.