Mus musculus) is a particularly well-suited model system to approach such questions, since it has a defined history of splits into subspecies and populations and since extensive genome information is available. We have used high-density single-nucleotide polymorphism (SNP) typing arrays to assess genomic patterns of positive selection and introgression of alleles in two natural populations of each of the subspecies M. m. domesticus and M. m. musculus. Applying different statistical procedures, we find a large number of regions subject to apparent selective sweeps, indicating frequent positive selection on rare alleles or novel mutations. Genes in the regions include well-studied imprinted loci (e.g. Plagl1/Zac1), homologues of human genes involved in adaptations  or in genetic diseases (e.g. Huntingtin and Parkin). Haplotype matching between the two subspecies reveals a large number of haplotypes that show patterns of introgression from specific populations of the respective other subspecies, with at least 10% of the genome being affected by partial or full introgression. Using neutral simulations for comparison, we find that the size and the fraction of introgressed haplotypes are not compatible with a pure migration or incomplete lineage sorting model. Hence, it appears that introgressed haplotypes can rise in frequency due to positive selection and thus can contribute to the adaptive genomic landscape of natural populations. Our data support the notion that natural genomes are subject to complex adaptive processes, including the introgression of haplotypes from other differentiated populations or species at a larger scale than previously assumed for animals. This implies that some of the admixture found in inbred strains of mice may also have a natural origin.General parameters of selection, such as the frequency and strength of positive selection in natural populations or the role of introgression, are still insufficiently understood. The house mouse ( Although there is abundant evidence for phenotypic adaptation in natural populations, it is still a challenge to understand the underlying genetic processes. House mice have colonized the world in several successive waves, the most recent ones in the wake of the spread of human agriculture and trans-oceanic shipping. They have adapted to many habitats and climates, and their populations provide a rich source of opportunities for studying the impact of adaptation and positive selection on the genome. By scanning the whole genome of four natural populations of mice, we detect abundant evidence for recent positive selection, including loci that are known to be involved in genetic diseases in humans. Unexpectedly, we also find a high proportion of gene exchange between populations that have long been separated. This finding supports the notion that hybridization and transfer of alleles can significantly contribute to new genetic material subject to positive selection. Genomic approaches allow an increasingly deeper insight into the forces that shape the evolution of genomes in populations. While it is evident that genome evolution depends on a balance of mutation, neutral evolution, negative selection and adaptation, the relative importance of each of these parameters is still only partly understood. Systematic genome scans for selective sweeps have shown that loci under recent positive selection can be readily detected in natural populations, but also that sweep signatures may be generated through drift effects associated with population bottlenecks or other demographic factors Another factor of increasing relevance for understanding the genetic composition of natural populations is allelic introgression from other subspecies or closely related species. While it is well known that hybridization between differentiated populations and species is relevant for the evolution of many plant species, the realization that similar processes are ongoing in animal populations has only come recently Mus musculus) to study the genetics of adaptations Mus in the past million years M. m. domesticus and M. m. musculus have separated about 300,000–500,000 years ago, which reflects in numbers of generations and relative molecular divergence the split of chimpanzees and humans. However, they are still considered subspecies, since they can be crossed and do not live in sympatry. On the other hand, hybrid males are often sterile We use natural populations of house mice  and Western Germany (Ger) and are derived from a colonization wave of Western Europe starting about 3,000 years ago M. m. musculus populations from the Czech Republic (Cze) and Kazakhstan (Kaz) is older but lies most likely still within the time frame of agricultural expansion of humans For the present study, we use two natural populations each from musculus . The twoM. m. domesticus and M. m. musculus since they are the main subspecies relevant for laboratory strains of house mice. However, we found in our analysis that calling SNPs in M. m. musculus appears less reliable and we have therefore refined the statistical approaches to retrieve reliable genotype calls. We use these data to apply a number of statistical procedures for detecting positive selection whereby each procedure has its merits and problems for such screens. Our data from a systematic, genome wide screen for introgressed regions provide a major new insight into the frequency of introgression of chromosomal regions between specific populations. To test whether the observed frequencies of introgressed regions within a population as well as their lengths can be explained by neutral processes alone, we compare our results to coalescent simulations testing different migration parameters and suggest that both, selective sweeps caused by rare alleles or new mutations, as well as adaptive introgression of haplotypes shape the composition of the genomes of mice.We use the Affymetrix Mouse Diversity Genotyping Array M. m. domesticus, although a number of SNPs polymorphic outside of this subspecies were added. Nevertheless, for samples with increasing genetic distance to M. m. domesticus, we saw an increase in apparently false heterozygote calls. We applied therefore the additional filtering steps described in the methods section. This yielded a list of approx. 470,000 high quality SNPs, 264,000 of which were polymorphic in M. m. domesticus and 167,000 were polymorphic in M. m. musculus. This difference reflects the ascertainment bias towards M. m. domesticus, since the M. m. musculus populations are not less polymorphic in unbiased analyses The Affymetrix Mouse Diversity Genotyping Array (MDGA) was designed on the basis of SNP differences between several laboratory mouse strains, both traditional and wild-derived inbred strains M. m. domesticus populations, in line with the somewhat biased representation of SNPs.The overall distance analysis shows that the populations and the individuals within the populations are mostly well separated, i.e. represent unrelated animals as expected based on the sampling scheme applied . The dis2 against physical distance for up to 100 kb. The strongest decay is seen in the M. m. musculus population from Kaz. This is in line with the observation that the Kaz population has a higher genetic diversity M. m. domesticus populations is similar to LD estimates for a wild Arizona M. m. domesticus population Assessing linkage disequilibrium allows estimating average sizes of haplotypes that could become subject to selection in a population. Linkage disequilibrium was therefore calculated for all four populations. M. m. musculus population from the Czech Republic shows a slower decay than the Kaz population. We ascribe this in part to the presence of large introgressed regions from the other subspecies in this population (see below) and have therefore produced a second version of the LD plot excluding all regions showing signs of introgression for all populations . Simulation results for the different parameters and lists of candidate regions are provided in We used the XP-CLR statistic that evaluates allele frequency differentiation between populations M. m. domesticus, 20 kb for M. m. musculus - see methods) to have a value above 3.5. These candidate regions are listed in The XP-CLR statistic was used to visualize the distribution of potential sweep regions across the chromosomes . All regM. m. musculus populations have on average larger introgressed regions, with the largest of approximately 20MB in the Cze population. When all regions are considered, a total of 25% of the genome is subject to mutual introgression between the sub-species and 9.6% when the Cze population is disregarded. The regions are distributed across all chromosomes . We applied Hapmix with the same settings as used for analysis of the empirical data to the simulated data sets. We can estimate significance of this observation by assuming a migration rate of 4Nm = 1. At this migration rate simulations match the proportion of the genome subject to introgression in the Czech population while for all other populations a greater fraction of the genome would be expected to have introgressed leaving p values more conservative. Nevertheless the observed frequency of introgressed haplotypes is significantly higher in all populations compared to neutral simulations .The introgressed regions could also reflect incomplete lineage sorting since the split of the sub-species, as it has been proposed in the analysis of several specific loci GO analysis of genes covered by introgressed regions  showed sTo visualize selective sweeps and introgressed regions in the population and chromosome context, we generated UCSC browser custom tracks based on phased data from the individuals in each population. M. m. domesticus/M. m. musculus boundary does appear to play a larger role than previously expected and that the frequency and size distribution of the introgressed fragments can not be explained by a neutral introgression model. This suggests a general interplay between selection and introgression shaping the genomes, which is difficult to disentangle. Still, we discuss these factors in turn in the following, since we have used different approaches to detect them.Our analysis provides an insight into the complex interplay between selection, introgression and drift in natural populations of the house mouse. We cannot even claim that we have identified all complexities, since the deme structure of mice with local inbreeding is difficult to capture in standard statistical models. However, our sampling scheme for the local populations was designed to get a representative sample across a whole area, which is more likely to represent a more long-term stable population sample A number of statistical methods have been developed to infer selection from SNP data in population surveys. The best datasets for applying such tests were so far available for human populations The XP-CLR statistic appears currently best suited to deal with this complexity . Moreover, the possible influence of adaptive introgression was not tested by these authors.There is an ongoing discussion on whether inbred laboratory mouse strains are ultimately derived from a mixture of subspecies crossed in during some phase of the inbreeding process The GO analysis did provide some enrichment terms of gene classes for introgressed regions, but closer analysis showed that this is due to the fact that a few regions encode gene clusters, such as olfactory genes or bitter taste receptor genes. This leads to a relative over-representation of terms like “sensory perception” or “G-protein coupled receptor activity” for the whole statistic, although this is only due to these clusters. Overall, we see no general pattern of types of genes subject to introgression across the genome. Still, the fact that the gene clusters mentioned are involved in introgression is of particular interest, since they code for genes that are very likely subject to population specific adaptations, as has also been noted previously Our data suggest that the genome of natural populations is not only shaped by drift and selection, but also by introgression from sister-species. As discussed above, there is increasing evidence that this occurs also in other animal species, including even humans, where haplotypes from Neandertals and Denisovans have introgressed in some populations All animal work followed the legal requirements, was registered under number V312-72241.123-34 (97-8/07) and approved by the ethics commission of the Ministerium für Landwirtschaft, Umwelt und ländliche Räume, Kiel (Germany) on 27. 12. 2007.Mus caroli, Mus famulus, Mus spretus, Mus cypriacus, and Mus macedonicus were processed. SNP typing microarrays (Affymetrix Mouse Diversity Genotyping Array) were processed according to the recommended procedures of the supplier.Mice were collected in Germany (in the Cologne-Bonn area), France , Czech Republic (around Studenec) and Kazakhstan (around Almaty) taking care to obtain a representative local sample as described in M. m. musculus samples), all SNPs called heterozygous were re-checked. Only those, which had an average intensity for both alleles higher than a cutoff, were retained. The cutoff was the first quantile (0.1) of homozygous calls in the reference (M. m. domesticus) samples, or when those were unavailable, prior homozygote cluster centers, distributed by Affymetrix, were used to determine the cutoffs. As this procedure still does not remove all false heterozygous calls, we conservatively excluded also from each population SNPs not concordant with Hardy-Weinberg equilibrium (one sided Fisher test with p<0.01). Protocol S1 provides additional details.First, we excluded from the analysis all SNPs flagged as unreliable in the array annotation file provided by Affymetrix. Because we found BRLMM-P calling (Affymetrix) to be susceptible to false heterozygote calls when a probe set is not compatible with a sample  were binned into groups followed by calculation of median r2 for each bin.We used the command line option of haploview st.Among the large number of available statistics to search for natural selection in genomic data, we chose a number of complementary statistics to suit our experimental setup. We focused our search on comparisons between sister populations from the same subspecies  to minimize effects of ascertainment bias and demography that could arise in cross-subspecies comparisons. We found that Rsb http://genepath.med.harvard.edu/reich). The following parameters were used: window size 0.02 cm, grid size 5 kb for M. m. domesticus populations and 20 kb for M. m. musculus, maximum # of SNPs within a window 50, correlation level from which the SNPs contribution to XP-CLR result was down weighted 0.95. Genetic positions based on XP-CLR values were calculated using scripts made available by st we used a sliding window approach  to detect candidate windows outside simulation defined cutoffs. Different cutoff levels are provided in Mus spretus, Mus cypriacus and Mus macedonicus to determine the ancestral state of SNPs. In cases were data for these three species were either not available or inconclusive, the ancestral state was inferred from either Mus famulus or Mus caroli, if possible. Heterozygous genotypes in the outgroup species were disregarded. Fst was calculated using the formula Fst = 1-Hs/Ht where Hs is the mean heterozygosity of the two populations and Ht is twice the product of the heterozygosities within populations. We also applied the correction for sample size developed by For Rsb, ΔDAF and FHapmix We applied Hapmix with standard settings but adjusted the recombination parameters to values observed in mouse as described in Time since admixture was set to 100 generations and the miscopying parameter to 0.0005, which we found to allow for optimal use of the resolution of the MWGDA to also detect smaller introgressed haplotypes with reasonable power by intense visual verification. The minimum per SNP certainty threshold to call a SNP introgressed was 0.9 (default setting).To estimate a false discovery rate (FDR) for introgressed regions we applied Hapmix with the same settings we used for empirical data to simulations without introgression (see next paragraph for demographic parameters). Because migration is set to zero in these simulations, the remaining regions that are detected as being introgressed reflect incomplete lineage sorting. Assuming a genome size of 2.7 Gb in mice FDR = 2.7*n/o, where n is the number of regions detected in 1 Gb of simulated data without migration and o is the number of observed introgressed regions in the empirical data.To determine cutoffs for sweep statistics under a neutral scenario we employed coalescent simulations with ms x in subpopulation i, and x = 0) and fixation (x = 1) in a subpopulation. We start our algorithm with an empty set of ascertained SNPs. We then choose a random SNP from the simulation data, add it to the ascertained set, and calculate the new s. If the new s is smaller than the s of the set without this SNP, the SNP is kept in the ascertained set, otherwise it is rejected. We then chose a random SNP from the ascertained set, remove it, and check whether this lowers s or not. If it does, the SNP is removed from the ascertained set, otherwise it is kept. We then choose again a random SNP from the simulation data and repeat the above process until no further improvement in s has been achieved over a substantial number of cycles, which we defined to be 10 times the number of segregating sites in the simulation sample.To resemble the ascertainment scheme of the chip design in our neutral simulations we extend an approach first introduced by Data visualization was done with the UCSC genome browser http://datadryad.org/)The SNP data are deposited under doi:10.5061/dryad.s3h97 at the DRYAD data repository Click here for additional data file.Protocol S1Statistics on the effects of filtering on SNP calling.(DOCX)Click here for additional data file.Table S1Results of sweep statistics and candidate gene lists.(XLS)Click here for additional data file.Table S2Gene Ontology (GO) over-representation analysis for sweep gene lists.(XLSX)Click here for additional data file.Table S3Introgressed regions and Gene Ontology (GO) over-representation analysis for introgressed gene lists.(XLSX)Click here for additional data file.


Cricetus cricetus) was found in the Małopolska Upland in Poland. A total of 142 common hamsters were captured between 2005 and 2009. All hamsters were genotyped at 17 microsatellite loci and partial sequences of the mitochondrial (mtDNA) control region were obtained. No mixed populations with mtDNA haplotypes of both lineages were found. The distance between marginal populations was about 20 km; no hamsters were found in the area between. A principal components analysis (PCA) was performed on microsatellite data and the greatest change in PC1 scores was found between marginal samples. To define the habitat components responsible for the phylogeographic break, we compared the habitat composition of sites occupied by hamsters with those from which hamsters were absent. We found that hamsters avoided forested areas and sandy soils. The area of the potential barrier was characterized by a high proportion of woodland and unfavorable soils in comparison with neighboring areas inhabited by hamsters. They cannot settle in this area due to their high winter mortality in shallow burrows and high predation in the fields adjacent to forests.In anthropogenically disturbed habitats, natural barriers still exist and have to be recognized, as they are important for conservation measures. Areas of phylogeographic breaks within a species are often stabilized in inhospitable regions which act as natural barriers. An area of contact between phylogeographic lineages of the common hamster ( The current distribution of a species and its geographic range limits are the outcome of a balance between several ecological and evolutionary processes  were also obtained from all animals. The PCR profiles for microsatellite and ctr amplification, the method of microsatellite analysis, and sequencing reactions for ctr were performed as described previously . All hamsters were genotyped at 17 microsatellite loci developed for the common hamster: Ccrμ3, 4, 10, 11, 12, 13, 15, 17, 19 and 20  with 500,000 iterations and a 50,000 burn-in periods. The admixture model and correlated allele frequencies were used in simulations. The admixture model represents populations which have recently or currently had enough gene flow for individuals to have ancestors in more than one population. The correlated allele frequencies model assumes that the populations originated from a common ancestor population in the past and the differences between them are the result of drift that has occurred since that time. Populations that have diverged not long ago have more similar allele frequencies. Although presently, the gene flow between common hamster populations might be lower considering the population crash, this problem is quite recent  for origin population assignment  for each individual from each sampled population  and heterozygosities (Ho and He). The tests for departures from the Hardy–Weinberg equilibrium for the populations were also computed in Arlequin. The Fis value based on Weir and Cockerham  was calcPopulation differentiation based on Fst values between pairs of populations was calculated in Arlequin. Statistical significance was estimated with 10,000 permutations. The model of isolation by distance (IBD) was tested in Isolde implemented in Genepop (Raymond and Rousset http://www.geocities.com/miroslavdressler/surgemain.htm). Barriers were identified as zones of maximum slope following the contour lines of the PC1 scores. PCA analysis was also used to visualize the population differentiation in the area of contact and to get additional support information for the number of genetic populations established by Structure.Principal components analysis (PCA) was performed on microsatellite data in GenAlEx 6.0  in Rundom Pro 3.14  were polymorphic with Po2 and Po5 haplotypes. However, the nucleotide diversity value for the total P3 lineage in the Małopolska Upland is very low  between the genetic (Fst/1-Fst) and the geographic distance was found.Pairwise Fst values between populations ranged from 0.14 to 0.35, and all the estimates were statistically significant. The Fst values between pairs of populations of different phylogroups was over 0.2, save cluster 2E1 + 3E1 and 2P3 (the maximum slope following the contour lines), suggesting that there is a barrier to gene flow in this area. This high gradient region of genetic change closely corresponds to the area where no hamsters were found in two consecutive years.The first and second PCA axes explained 38.61% and 32.51%, respectively, of the total variability at microsatellite loci. A synthetic genetic map for the first axis of PCA scores was superimposed on a geographic map of the study area in Fig. The differences in habitat composition between the occupied and the nonoccupied squares are given in Table An area that forms a barrier between phylogeographic lineages of the common hamster was found in the Małopolska Upland. Although the time of contact in this area is not known, we would like to stress that it is unlikely to be connected with the recent population crash. On the distribution map of the common hamster in Poland created by Surdacki  and baseThe lineages could come into contact here not earlier than the end of the Last Glaciation, as hamsters of the E1 lineage appeared in the Małopolska Upland quite late following the demographic expansion of this group in southeastern Poland, which was dated roughly to the end of the Last Glaciation and the beginning of the Neolithic , although not always assigned with a high probability to one particular population, were always assigned to the expected phylogroup. On the other hand, there is an obvious similarity in microsatellite diversity between the phylogeographic lineages. The difference in microsatellite diversity between the phylogroups found by AMOVA, although significant, was very small, about 3.5% (Banaszek et al. Tscherskia triton (Song et al. Cricetus cricetus on the basis of relatedness analysis (Banaszek and Ziomek P3 and E1 populations in comparison with the values for other population pairs may indicate this opportunity of gene flow.The gene flow between hamster populations is difficult to estimate. The pairwise Fst values between populations are usually quite high and statistically significant (Neumann et al. In conclusion, spatial and temporal variations in habitat quality serve as efficient barriers between the phylogeographic lineages of the common hamster in the Małopolska Upland. The strength of such a seemingly insubstantial barrier is an important clue for any conservation plans such as creating corridors for migration or agroreserves for hamster protection. It is important that any such conservation plans should be preceded by a scrupulous investigation of habitat quality. Placing agroreserves in sink habitats would result in a waste of efforts and funds and a population declining to extinction in low density years. Similarly, corridors not taking into account small natural barriers will result in no gene flow or, at most, some male dispersal with females effectively isolated.


Nm). We achieved this goal by studying the shape of the gene genealogy at several sampling levels and using spatial explicit simulations. Both the direction and the rate of migration differ between male and females, with an Nm estimated to be >6 times higher in the latter under many evolutionary scenarios. We finally highlight the importance of applying metapopulation models when analyzing the genetic diversity of a species.Mitochondrial DNA (mtDNA) and Y chromosome (NRY) genetic markers have been often contrasted to investigate sex-specific dynamics. Traditionally, isolation by distance, intrapopulation genetic diversity and population differentiation are estimated from both markers and compared. Two possible sources of bias are often neglected. First, kilometric distances are frequently used as predictor of the connectivity between groups, hiding the role played by environmental features at a microgeographic scale. Second, the comparison of intrapopulation diversity and population differentiation between mtDNA and NRY is hampered by their different mutational mechanisms and rates. Here, we show how to account for these biases by analyzing from a different perspective a published dataset of eight West New Guinea (WNG) populations for which mtDNA control region sequences and seven linked NRY microsatellites had been typed. First, we modeled the connectivity among sampled populations by computing the number of days required to travel between groups. Then, we investigated the differences between the two sexes accounting for the molecular characteristics of the markers examined to obtain estimates on the product of the effective population size and the migration rate among demes (We have applied the prediction of the sampling theory in a meta-population and we have corroborated our finding using spatial explicit simulations. Both approaches are fundamentally meant to deal with structured populations: we strongly believe in the importance of tacking structure into account when inferring the demographic history of a species. Sex-specific demographic patterns are traditionally investigated in humans by using uniparentally inherited markers, namely mitochondrial DNA (mtDNA) for females and Y chromosome (NRY) for males. Reports usually focus mainly on three classes of analysis, whose results are compared between the two markers: (i) isolation by distance (IBD)  groups . The shape of the genealogy does not depend on the neutral mutation rate  of WNG.Here, we took environmental conditions into account by recording the walking time between the eight groups. Furthermore, we compared the demography of males and females using the prediction of the sampling theory in a metapopulation  and NRY (Rst) were computed using Arlequin 3.5  and Rst in males (from NRY). The correlations among the four matrices were estimated by pairwise Mantel tests, with the significance assessed after 10,000 permutations. Isolation by distance was further tested at both genetic markers by means of the Mantel correlogram . The second is the distance in days of walk (DAY), which was computed by one of us (M. T.-P.) by traveling among all groups together with local guides. DAY implicitly reflects the environmental heterogeneities, as it represents the facility to move from one village to another. Pairwise genetic distances at mtDNA  the coalescent history of a sample of lineages can be divided into a scattering and a collecting phase . We used BEAST   in space and time. After the regulation step, migrants are sent to the four neighboring demes at rate m. The process is repeated for successive generations for each nonempty deme, resulting in a wave of advance of the whole population. At equilibrium, each deme will send Nm migrants per generation to its surrounding demes. At each generation, the demographic and migration histories of every deme are stored in a database, which is then used in the backward coalescent step. The second phase of the algorithm then starts at the present generation, proceeding backward in time. The effective number of individuals present in a deme is used to compute the probability of a coalescent event, and the migration rates determine the probability of each sampled gene to emigrate, backwards in time, to the surrounding demes. The coalescent process stops after all genes have coalesced.We performed a set of spatial explicit simulations of the demographic history of males and females in WNG using the software SPLATCHE , whereas the remaining five are hunter/fisher–gatherers (HG groups). Differences in Nm values between males (NmM) and females (NmF) can be due (among other factors) to different marital residence pattern and variance in reproductive success . It is not known when these cultural behaviors arose in human populations and if they differ markedly between AG and HG groups  Scenario 2: differences between NmM and NmF in AG, but not in HG. Postagriculture demographic expansion in AG; (iii) Scenario 3: reduction in NmM compared with NmF in both AG and HG. Postagriculture demographic expansion in AG; (iv) Scenario 4: differences between NmM and NmF in both AG and HG. No postagriculture demographic expansion in AG; (v) Scenario 5: reduction in NmM compared with NmF in both AG and HG. These differences arose in all demes only after the emergence of agriculture. Postagriculture demographic expansion in AG; (vi) Scenario 6: reduction in NmM compared with NmF in HG. These differences arose in HG only after the emergence of agriculture. Postagriculture demographic expansion in AG.Spatial simulations were used to investigate under which demographic conditions we could reproduce the  Marlowe . For thiNm thereafter. This choice was made for two reasons: first, to ensure the same colonization time for both sexes, and second, to cope with the earliest human remains in Australia. Human remains are as old as 46,000 years b.p. . Mutation rate for the hypervariable region of mtDNA was set according to Soares et al. , but not of FstF (RstM drop to 0.38 when excluding the Dani (P ≈ 0.20).Conversely, GEO is not correlated with any of the two genetic distance matrices (Table 2) and shows no clear trend when used in the Mantel correlogram analysis  and an FstF of 0.116 (P < 0.001) among the sampled populations. The RstM/FstF ratio is 4.2 which correspond approximately to an NmF/NmM ratio of 7.26 assuming the Wright  and an NmF at least 10 times higher in HG is compatible with observed data, independently from the Nm in AG. Generally, reduction in NmM must be at least sevenfold larger when compared with NmF . Similarly, when we assumed that sex-specific differences occurred in HG only after the Neolithic we found still that an NmM reduction 10 times higher is needed to generate the observed RstM/FstF ratio, independently from Nm in AG . In Scenario 2, we tested several combinations of NmM and NmF of AG for fixed Nm in HG. Under this hypothesis, we never found an RstM/FstF ratio higher than 2 (Nm HG of 1), independently on the Nm in HG . This result suggests that different behavior between males and females in West New Guinea are not (only) related to the emergence of agriculture as they must be present in HG as well in order to obtain such values of RstM and FstF. Scenario 3 and 5 assume that differences in the same magnitude between NmM and NmF are present both in HG and AG. In Scenario 5, such differences were established after the emergence of agriculture in all groups, whereas in Scenario 3 they have been always present. In Figures RstM/FstF for various NmF in HG and an NmF = 150 in AG for scenario 3 and 5, respectively. In both scenarios, we found that a decrease in NmM of at least sixfold is needed to generate an RstM/FstF ratio higher than 4, independently of the NmF set in AG . Finally, we presented in Fig. 9 the results of Scenario 4 where we assumed no difference in carrying capacity between HG and AG . We found a pattern quite similar to Scenario 1 for various values of Nm in AG. However, we note that the absence of a Neolithic expansion increases the RstM and FstF values rather than their ratio, leading to an average RstM of around 0.71 for NmM = 1. This implies that an NmM ≈ 2 (leading to an average RstM of 0.40) and an NmF at least seven times higher is more compatible with our data.We computed an e Wright  island mUniparental markers, namely mtDNA and NRY, have been used for a long time to characterize the influence of sex-specific behaviors on the genetic structure and diversity of human populations  about seven times higher in females than in males. This value is based on equilibrium model (Wright island model) which also overlooks the molecular properties of the two markers and their effect on the estimation of the fixation indices. To compare Nm between the chosen markers without worrying about their different molecular properties, we investigated the shape of the gene genealogy in all demes and in datasets created by resampling lineages from each of them. Range expansion is still visible in mtDNA, where resampled datasets support a growth around 50,000 years b.p.  at various sampling intensities and/or using them as new and powerful summary statistics under an approximate bayesian computation inferential procedure. Coalescent approach coupled with sampling theory can be therefore applied to any structured species, provided that it is composed by a large (>100) number of demes.Not only the direction but also the intensity of gene flow differs between the two sexes. We found an ars b.p. , consistars b.p. . Contrarars b.p. , we founars b.p. . Therefo Wakeley ). This cNmM and NmF we also used spatial explicit simulations. The advantage of using simulations was threefold: first, we explicitly modeled the molecular dynamics of both mtDNA and NRY; second, we accounted for the genetic structure within WNG, including also the unsampled demes; third, we introduced in the population genetic model spatial and temporal heterogeneity . For these reasons, we could not only make quantitative statements about the NmF/NmM ratio, but also investigate when the differences arose. Independently of the scenario tested, we found that the observed RstM/FstF ratio is compatible only with a number of male migrants per generation at least six times lower than female migrants . Moreover, we determined by simulations not only a quantitative difference in the number of migrants but also when social behavior started affecting the genetic variability in West New Guinea. Importantly, we also showed that many but not all populations need to have sex-specific behavior in order to affect the whole metapopulations. As a consequence, even populations with no sex-specific patterns will show differences between NmM and NmF. Such a fine-scale characterization of metapopulation genetics could have not been possible using equilibrium based methods only.To further investigate differences between ts Figs. . Even thts Figs. . It is pts Figs. . Indeed,G groups  or that Here, we have shown how to characterize sex-specific dynamics using both qualitative and quantitative approaches. Even though the specific pattern we observed could have been also influenced by more local events occurred at the single population level , the main message of this work is to show the importance of analyzing a structured species under a metapopulation framework and of introducing as much as possible the ecological features of an environment in the population genetics modeling. We did this by both performing spatial and temporal explicit simulations of genetic diversity and by applying indirect metapopulation inferential models. We showed how we can exploit prediction of the metapopulation theory simply by an appropriate sampling of genetic data, gaining a wealth of information  which we would have missed under more traditional approaches. Moreover, the use of complex simulation schemes gave us the opportunity, not only of directly considering the connectivity among the sampled populations but also of introducing temporal and environmental heterogeneity and test their effects on genetic diversity. An extension to these analyses would be to implement approximate Bayesian computation (Beaumont et al.


Alexandrium fundyense that occurred in the Gulf of Maine in 2005. This bloom was notable for its intensity and duration, covering hundreds of kilometers and persisting for almost two months. Genotypic analyses based on microsatellite marker data indicate that the open waters of the northeastern U.S. harbor a single regional population of A. fundyense comprising two genetically distinct sub-populations. These subpopulations were characteristic of early- and late-bloom samples and were derived from the northern and southern areas of the bloom, respectively. The temporal changes observed during this study provide clear evidence of succession during a continuous bloom and show that selection can act on the timescale of weeks to significantly alter the representation of genotypes within a population. The effects of selection on population composition and turnover would be magnified if sexual reproduction were likewise influenced by environmental conditions. We hypothesize that the combined effects of differential growth and reproduction rates serves to reduce gene flow between the sub-populations, reinforcing population structure while maintaining the diversity of the overall regional population.Historically, cosmopolitan phytoplankton species were presumed to represent largely unstructured populations. However, the recent development of molecular tools to examine genetic diversity have revealed differences in phytoplankton taxa across geographic scales and provided insight into the physiology and ecology of blooms. Here we describe the genetic analysis of an extensive bloom of the toxic dinoflagellate  Skeletonema costatum, a marine diatom. In the intervening thirty years, a variety of studies have used different molecular markers to examine the diversity of microalgal populations over a range of temporal and spatial scales. Population substructure has been detected within a single sample Because of the apparent lack of barriers to dispersal and therefore gene flow in the marine pelagic environment, it would be reasonable to hypothesize that phytoplankton species comprise a single, largely unstructured population. This assumption was first challenged by Gallagher (1980) Alexandrium fundyense is an ideal system for studying the diversity and dynamics of algal blooms. It is a cosmopolitan genus catenella/fundyense/tamarense species complex are found in temperate coastal waters worldwide. Because of its ability to produce potent neurotoxins and therefore cause harmful algal blooms, it is subject to intense monitoring with respect to population dynamics and accompanying environmental conditions  populations? Second, is the diversity and composition of the bloom static, or does it change over time? If so, on what scale? Lastly, what can the diversity and dynamics of this bloom tell us about how A. fundyense populations are maintained year-to-year, given their annual alternation in life cycle? Our results show that the beginning of the 2005 A. fundyense bloom was characterized by the presence of a common and diverse sub-population at three geographically separated sampling sites. Samples collected later in the bloom were genetically differentiated from these early samples, with the change in population composition occurring on the timescale of approximately four weeks. The temporal changes observed during this study provide clear evidence of succession during a single bloom and show that selection can act on the timescale of weeks to significantly alter the representation of genotypes within a population. If environmental conditions drive both the changes in population composition and the induction of sexual reproduction, the resulting interactions between succession and reproduction will reinforce the genetic differentiation between sub-populations while maintaining the overall diversity of the regional population.Here we describe the temporal and spatial changes in the genetic composition of a continuous bloom of No permits are required for collection of water samples in U.S. coastal waters.A. fundyense cell to 200 µL of f/2-Si medium A total of 171 clonal strains were used in this study. Strains were isolated from six different locations throughout the Gulf of Maine; isolation dates and locations are shown in −2•s−1. When cultures reached mid-exponential phase, approximately 20 mL was harvested by centrifugation (3000× g for 5 min). DNA was extracted from cell pellets using the Qiagen DNeasy Tissue Kit . The protocol was modified by the addition of a mechanical breakage step, in which a double volume of lysis buffer and 0.5 mm silica-zirconium beads  were added to the cell pellets followed by vortex mixing for 1 minute at maximum speed. All subsequent buffer volumes were also doubled, prior to application of the lysate to the Qiagen column. Wash buffer volumes were not increased, and DNA was eluted in 2×100 µL of elution buffer. Whole genomic DNA was stored at −20°C until used for PCR amplification.For DNA extraction, strains were grown in medium consisting of 0.45 µm-filtered and autoclaved natural seawater  enriched with modified f/2-Si nutrients. Cultures were grown at 15°C on a 14∶10 light∶dark cycle with cool-white fluorescent illumination of approximately 100 µmol photons•mA. fundyense, to determine the most suitable markers (data not shown). Five loci were chosen based upon the number of alleles observed and the percent of amplifying samples, and these five primer sets were used to determine the genotype of all of the strains used in this study. PCR amplification reactions contained 5 ng of template DNA, 0.5 mM of each dNTP, 0.25 µM of each designed primer pair, with one primer labelled with D4-WellRED , 1× PCR buffer , and 0.25 U of Ampli Taq Gold (Applied Biosytems Inc.) to a total volume of 10 µL. The PCR cycling conditions were as follows: 5 min at 96°C, 45 cycles of 45 s at 95°C, 45 s at 54°C  or 58°C (Atama39), and 1 min at 72°C, and a final elongation for 1 min at 72°C. PCR products were diluted with nuclease-free water and 1 µL of diluted product was mixed with 500 LIZ Size Standard and Hi-Di Formamide, and then analyzed using an ABI 3730xl DNA Analyzer. Allele sizes were determined using the program FPMiner .Of the microsatellite loci isolated by Nagai et al. (2004) ST) and genetic distance between samples based on allele frequencies, and further tested using an exact G test. Lastly, the membership of individuals and samples in the different sub-populations was determined using Bayesian assignment tests, and their similarity was visualized by Principal Components Analysis.We performed a variety of tests to characterize the diversity and potential structure present in the bloom assemblage. The diversity was characterized by computing the molecular diversity and proportion of unique haplotypes within the samples. The presence of linked loci, a common consequence of predominantly clonal reproduction, was also assessed. A primary question in this study concerned the potential presence of genetic structure within the bloom. Genetic distances between samples were quantified by calculating the pairwise Fixation Index  was investigated using the Monte Carlo method in LIAN v 3.5  for genic differentiation (null hypothesis = no differentiation) was performed using GenePop v4.0.10  maintained by Jim Manning at the NOAA Northeast Fisheries Science Center Oceanography Branch Temperature, salinity, and nutrient conditions at our sampling sites are listed in A. tamarense strains of the NorthAmerican/Group I ribotype from Japan Alexandrium fundyense in the northeastern U.S. belong to this same clade, and so it was expected that the microsatellite markers would be generally applicable to strains from the North American/Group I ribotype A. fundyense cells. All five loci were polymorphic in all samples, except for locus Atama27 in sample MV1 . The overall amplification success was 95% of individuals, with nine strains having missing data at a single locus each. Of the 171 strains analyzed, 119 (70%) had unique five-locus genotypes. The proportion of unique multilocus genotypes per sample ranged from 83 to 92%  was found within samples . When thFST and Fisher's exact test - were performed to determine the presence of genetic differentiation between samples, with somewhat different results  was significant. The genetic similarity between the samples was assessed using Nei's (1972) genetic distance to construct a dendrogram of the six samples  is lower than other studies of Alexandrium that have used more loci The number of loci used to genotype strains in this study was constrained by our ability to successfully amplify the published A. fundyense that occurred along the coast of New England, USA (for a detailed description of the bloom see −1). The next sample, CA, was collected 12 days later and is considered to represent the “mid-bloom”, both geographically and temporally. At that time, cell concentrations were very high (thousands per liter) in that region. The last two samples, MV1 and MV2, were both collected south of the island of Martha's Vineyard, which was the southernmost part of the bloom. Cell concentrations were generally high in this region during June (often >1000 cells L−1), even though they were decreasing in areas to the north. These samples were collected eight days (MV1) and ten days (MV2) after the previous samples, and in the weeks following the MV2 sample, cell concentrations across the region declined rapidly until the bloom was considered to be over about four weeks later. Thus, samples MV1 and MV2 are considered to be representative of “late-bloom” conditions, geographically and temporally.Samples were collected over a 37-day period during an extensive bloom of It is important to keep in mind that while we consider the 2005 event to be a single bloom, we are working in an open basin where water is constantly moving, transporting and potentially mixing cells from different regions. The general circulation pattern in the Gulf of Maine is from north to south along the coast e.g. see , with anFST values were found in all pairwise comparisons of early- and late-bloom samples  Alexandrium catenella populations from France and Japan, and Alpermann et al. (2009) FST of 0.06 between Alexandrium tamarense subgroups (delineated using AFLP) of a single net tow sample. By comparison, the early- and late-bloom samples show greater differentiation than subgroups of a single sample, but less than samples derived from different continents. Because of their hydrographic connection and the level of gene flow observed, we do not consider the early- and late-bloom clusters to represent separate populations, but rather different sub-populations within the regional Gulf of Maine A. fundyense population. This designation is supported by the results of the AMOVA analysis  and the second containing only the late-bloom samples (MV1 and MV2). Significant  samples , ranginganalysis , which sanalysis ; at thisFST distinguishes samples CA and MV1, but the FST between CA and MV2 is significant, whereas the Fishers' test does not reject panmixia between the two samples. At locus Atama15, sample CA shares one unique allele with the early bloom samples  and another with the late-bloom samples (MV1 and MV2). The intermediate position of sample CA is exemplified by its location in the PCA analysis  having higher toxin content and different toxin profiles than isolates from the south, including Cape Cod The early- and late-bloom sub-populations observed in 2005 may be representative of “northern” and “southern” populations identified in previous studies of A. fundyense population dynamics. In this scenario, the regional population would comprise a large number of different clonal lineages, which have different growth responses to environmental conditions . All of these lineages are present in each sample, but their proportional representation would be determined by environmental conditions. Because we sampled only 18–42 individuals from each location, we are analyzing only the most abundant lineages in the sample. The genetic differentiation observed would result from selection of different lineages (ecotypes) that were most successful under the conditions at a particular site. Given the general north-to-south coastal circulation pattern in the Gulf of Maine, this scenario requires only a genetically heterogeneous source of cells in the northern part of the region.Differences in the growth response to environmental conditions appear to play a role in structuring marine diatom populations Alternatively, there could be geographically separate populations, as suggested by Anderson et al. (1994) While the clear differentiation between early- and late-bloom samples could be explained by either selection  or geography, the “transitional” nature of sample CA argues for selection as the primary factor driving the differentiation of the two sub-populations. Our results show that CA is not significantly differentiated from the early-bloom samples or MV1, yet it shares some affiliation with MV2. Given that CA is located upstream of MV2 in the transport pathway, the presence of both early- and late-bloom genotypes in sample CA could not result from the mixing of water masses; they must exist in sample CA before it is transported south. The change in the proportions of early- and late-bloom genotypes is presumably driven by selection, although the selective factor is not evident in the limited environmental dataset that we have . Thus, wDitylum brightwellii and found no significant differentiation between any of their daily samples. An important difference between these two studies is the temporal scale of the bloom. The Ditylum bloom lasted only 11 days whereas the 2005 Alexandrium bloom persisted for almost 8 weeks, and was tracked for 39 days. While water movement in the Gulf of Maine makes it generally difficult to track a single water mass, or “piece” of the bloom, we do have two samples, CA and MV2, where the sampling interval was roughly equal to the water transit time between the sites. This provides an estimate of the timescale on which genetic differentiation can occur in a planktonic bloom. In the 18-day interval between samples CA and MV2, the samples became sufficiently differentiated that their pairwise FST  was significant. The lack of differentiation observed from FST and Fisher's exact test comparisons of CA and MV1 , and MV1 and MV2  suggest that the transition from the early-bloom to late-bloom genotypes was in progress, but not yet complete, in samples CA and MV1. The rate of population differentiation should be affected by the relative growth rates of different clonal lineages, i.e. how quickly one lineage can outgrow another, and also the range and rate of change of environmental conditions during a bloom. In the comparison of the Ditylum and A. fundyense blooms, turnover should be faster in Ditylum because of their greater maximum growth rates. However, the A. fundyense bloom likely experienced a wider range of environmental conditions, as it transited roughly four degrees of latitude in the course of six weeks, whereas the Ditylum bloom occurred in a single coastal embayment. These two factors may balance each other to result in the finding of a common lack of genetic differentiation over ∼10 days in both a diatom and dinoflagellate bloom; however, more studies are needed to determine if this represents a minimum bound for population turnover in marine planktonic algae.The changes in genetic composition that we observed during this study show that phytoplankton blooms are not static; they comprise a tremendous diversity of strains whose representation shifts over time, presumably in response to changing environmental conditions. Our results contrast somewhat with the findings of Rynearson and Armbrust (2005) A. fundyense is characterized by an annual alternation between asexual haploid cells that reproduce by binary fission in the water column, and a resting stage (cyst) that results from the fusion of two haploid gametes. Sexual reproduction requires different mating types and, in the laboratory, is triggered by adverse environmental conditions A. fundyense cells do not overwinter in the water column, but rather in the sediments as cysts. These resting cysts can remain viable in the sediments for years, and after a mandatory dormancy period, they germinate in response to favorable conditions in the early spring. Because sexual reproduction, and therefore recombination, happens only once a year, changes in the composition of planktonic populations have the potential to affect the diversity of the resting cyst reservoirs that are the source populations of later blooms.The life cycle of A. fundyense. If it is triggered by unfavorable environmental conditions, as it is in the laboratory, then it has implications for the genetic composition of both the planktonic and resting cyst populations. In the plankton, environmentally-induced encystment could serve to magnify the effects of selection on the proportions of different genotypes by reducing or removing lineages that are less successful under the prevailing conditions. As a result, encystment would occur throughout the bloom, not just at the end, thereby contributing to the diversity of the cyst seedbeds via the asynchronous input of cysts derived from different sub-populations. Induction of sexual reproduction by unfavorable conditions might also serve to reinforce the genetic differences between sub-populations. For example, if the early-bloom sub-population thrives at lower salinities than the late-bloom sub-population, then as a mixed population encounters a region of higher salinity, early-bloom genotypes will be more likely to participate in mating and encystment, while late-bloom genotypes will be favored and remain in the plankton to continue vegetative growth. This would increase the likelihood of genetically similar cells reproducing, effectively reducing gene flow between sub-populations. Thus, sexual reproduction in response to adverse environmental conditions may serve to both reinforce the genetic differentiation between sub-populations while maintaining the overall diversity of the regional population.There is little data on the triggers, timing, or magnitude of encystment in natural populations of Alexandrium fundyense, covering hundreds of kilometers and persisting for almost two months. Practical considerations limited the temporal and geographic density of our samples as well as the number of loci analyzed, thus we consider our results to be conservative estimates of the diversity and differentiation present in this bloom. Genotypic analyses based on microsatellite marker data indicate that the open waters of the northeastern U.S. harbor a single regional population of A. fundyense comprising at least two genetically distinct sub-populations. These subpopulations were characteristic of early- and late-bloom samples and were derived from the northern and southern areas of the bloom, respectively. We hypothesize that the observed populations structure results from differential environmental selection on the two sub-populations. The effects of selection on population composition would be magnified if sexual reproduction were likewise differentially influenced by environmental conditions. The combined effects of differential growth and encystment would serve to reduce gene flow between the sub-populations, reinforcing population structure while maintaining the diversity of the overall regional population.We examined the genetic composition of an extensive bloom of the toxic dinoflagellate Figure S1Population structure determined by Bayesian cluster analysis for K = 4. Plots of the statistic ΔK  vs. K showed two modes, at K = 2 and K = 4. Results for K = 2 are included in the text, as the presence of two sub-populations is supported by the results of other analyses, whereas the possibility of four sub-populations is inconsistent with the other data. The bar plot shows the proportion of membership in Cluster 1 (red), Cluster 2 (green), Cluster 3 (blue) or Cluster 4 (yellow) for each individual. Samples are delineated by vertical black lines, and the average proportion of membership in each cluster by sample is listed underneath the sample names.(TIF)Click here for additional data file.Table S1Summary of molecular diversity indices for the six samples analyzed, both per locus and across all loci. The number of unique haplotypes per sample is listed beside the sample name.(DOC)Click here for additional data file.


Dendrocalamus membranaceus Munro is a woody bamboo with a high economic and ecological value that often occurs as natural stands, such as in the large-scale forested areas of China’s Yunnan Province. Due to its overexploitation, the habitat of D. membranaceus in Yunnan has been dramatically reduced, and the quality of the stands has declined. As a preliminary analysis in considering the effective protection for these germplasm resources, we assessed the genetic diversity of 12 natural populations in Yunnan, using inter-simple sequence repeat (ISSR) markers. From 10 ISSR primers, we generated 155 bands, of which 153 were polymorphic (98.71%). Compared with other species in the genus, this species demonstrated a greater genetic diversity (S = 0.349) and lower genetic differentiation (GST = 0.252). Our analysis of molecular variance revealed that the genetic differentiation among the populations is significant. A large proportion of the genetic variation (78.95%) resides among the individuals within populations, whereas only 21.05% are found among populations. Mantel tests indicated no significant correlation between genetic and geographic distances among the populations. Given the low sexual reproducibility and characteristics of monocarpic plants, we recommend implementing in situ conservation measures for all of the D. membranaceus populations in Yunnan and collecting sufficient samples for ex situ conservation. Furthermore, the conservation area should be extended to its main natural habitats, the Lancang-Mekong River Valley. Dendrocalamus membranaceus Munro is one of the most frequently occurring, clump-forming woody bamboos (with pachymorph rhizomes) in Southeast Asia. It is naturally distributed in Laos, Myanmar, Northern Vietnam and Northern Thailand [Thailand –3 in addThailand . This spThailand .2 of natural D. membranaceus forest, which provided important support for the local ecosystem, including uses as a food resource (bamboo shoots and young culms) and as habitat for the wild Asian elephant [D. membranaceus has been dramatically reduced, to less than 30,000 hm2 in 2008, with obvious degradation of the quality of the remaining forest [In 1995, Yunnan Province had approx. 70,000 hmelephant ; howeverg forest ,7. ThereDendrosalamus is regarded as hexaploid [D. membranaceus collected from Yunnan is 2n = 70. However, the chromosome number of paleotropical woody bamboo is generally variable, and 2n = 70 ± 2 is the most frequently found in Dendrocalamus [D. membranaceus is most likely hexaploid. Recent work using dominant genetic markers has shown that plant polyploids possess a high genetic diversity [D. membranaceus has been subjected to a severe reduction in its natural distribution, and, in general, woody bamboos reproduce clonally, two conditions that can decrease the levels of genetic variation. Thus, D. membranaceus is a good model organism for understanding how bamboos genetically change under declining situations.Woody bamboos are presumably ancient polyploids, and exaploid . Accordiexaploid , the chrocalamus . If x = iversity ,13. HoweD. membranaceus in Yunnan Province, China , whereas GL had the lowest . By comparison, the mean within-population gene diversity among monocotyledons is 0.144 for Nei’s expected heterozygosity [Furthermore, a high level of genetic diversity was found for this species  the existing population retains the genetic diversity from their ancestors; and (3) the habitat fragmentation caused by human activities does not perturb the genetic base of the existing D. membranaceus populations.The high genetic diversity detected for exaploid ,11, and exaploid . Howeverexaploid . Moreoveexaploid ,6. Therep < 0.001) (GST = 0.252). Furthermore, 24 private bands were found in eight populations  showed that a large proportion of the genetic variation (78.95%) existed among individuals within the populations, and that 21.05% resided among the populations. The genetic differentiation among the populations was significant (< 0.001) . Consistulations , implyinD. membranaceus can reproduce via seed in the wild, although this phenomenon is rare, and the rate of seed set is low [D. membranaceus is likely anemophilous and prone to be an out-crosser [GST = 0.252) that was similar to the average of out-crossing species (GST = 0.22) [Many factors can determine the genetic structure of plant populations, including the reproductive biology , and gent is low ,16. We f = 0.22) .D. membranaceus in Yunnan.Therefore, extensive longevity and predominant out-crossing may be two important factors for the genetic structure of the existing D, revealed that the 12 populations could be separated into two clusters: XP plus Mengman (MM) and the other 10. We found no agreement with the geographic distance, which was supported by our Mantel test results, demonstrating that D was not significantly correlated with the geographic distance  .Our UPGMA tree , based o= 0.184) . This ou bamboos ,30,31. AD. membranaceus in Yunnan Province have been overexploited for many years, leading to the deterioration of its habitat [The natural resources of  habitat ,8. The s habitat . Althoug habitat ,6.D. membranaceus in Yunnan, China. However, the genetic differentiation among the populations and the limited reproductive capability of D. membranaceus in the wild indicate that it is still necessary for us to protect all of the existing natural populations and their habitat in Yunnan, China. At the same time, germplasm collection and conservation in the Lancang-Mekong River Valley, the distributional center of this species, should be placed at the first and foremost position.This new information about the diversity in its population genetics demonstrates the degree of success these plants have had in adapting to their altered environments over time. Therefore, it is of critical importance that this knowledge be applied in assessing their conservation value and the status of particular populations when developing management plans ,33. DespDendrocalamus membranaceus in Yunnan Province, China , 2% formamide, 100 nM of each primer, 1.5 units of Taq polymerase (Fermentas), and double-distilled water. The cycling conditions included an initial denaturation at 95 °C for 3 min; then 40 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 2 min; and a final extension at 72 °C for 10 min. The PCR products were electrophoretically separated using 2.0% agarose gels buffered with 0.5× TBE. A 100-bp DNA ladder (Fermentas) was used as a size marker. The PCR reactions were repeated twice to ensure reproducibility. After staining with ethidium bromide (1 μg·mL−1), the DNA fragments were identified by software Lab Works Software 3.0 [Genomic DNA was PCR-amplified using ISSR primers obtained from the Biotechnology Laboratory, University of British Columbia . Amplifications were performed using an ABI 2720 Thermal Cycler . The 20 μL mixture contained 10 ng of template DNA, 2.0 μL of 10 × PCR buffer, 1.8 mM MgClware 3.0  for gel HE), coefficient of gene differentiation (GST), genetic distance (D), and Shannon diversity index (S). An analysis of Molecular Variance (AMOVA program Version 1.55) [Only distinct, reproducible, well-resolved fragments were scored as present (1) or absent (0) for each marker, and were displayed as part of a binary matrix. The data matrices were analyzed using POPGENE version 1.31 . The folon 1.55)  was perfon 1.55) .D, implementing an unweighted pair-group method of cluster analysis that used arithmetic averages (UPGMA) and the software program NTSYS-PC V2.1 [TFPGA software [To visualize the genetic relationships among the populations, we constructed a dendrogram based on Nei’s  genetic -PC V2.1 . To testsoftware , computiDendrocalamus membranaceus populations in Yunnan. A large proportion of the genetic variation (78.95%) resides among individuals within the populations, whereas only 21.05% exist among the populations. No significant correlation was found between the genetic and geographic distances among the populations. Considering that D. membranaceus is a monocarpic and has prominently low sexual reproduction, we propose to implement in situ conservation measures for all of the populations and collect sufficient samples for ex situ conservation. Our results are also of important significance for the resource conservation of D. membranaceus in its main natural habitat, the Lancang-Mekong River Valley.In summary, high genetic diversity and lower genetic differentiation were detected among the


Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa.The all-female Caucasian rock lizard species  Naturally occurring unisexual reproduction is documented in less than 0.1% of all vertebrate species In 1989, Parker et al. summarized the allozyme variation patterns observed among parthenogenetic species of several genera of lizards and proposed a hierarchical model for their origin, to predict whether they arose from single or multiple interspecific hybridization Darevskia is of special significance, because these lizards have been the subject of extensive ecological and biogeographical studies Darevskia species are generally in accordance with Parker et al.’s model. Among these species, Darevskia dahli has a single origin The genus D. dahli, which is one of the seven unisexual species, occurs in the southern Caucasus Mountains of Armenia and Georgia D. dahli as D. mixta and D. portschinskiiD. mixta occurs only in Georgia, and D. portschinskii is distributed in southern Georgia, northern Armenia, and western Azerbaijan et al. (1967), parental species are mainly allopatric D. dahli is typically distributed in forest regions at altitudes ranging from 700 to 1500 m D. dahli populations are rather numerous, they are usually isolated from one another. However, some of them can be sympatric with bisexual and/or parthenogenetic lizards of the genus Darevskia. For instance, in the Dzoraget region (near Stepanavan), D. dahli is sympatric with the bisexual parental species D. portschinskii and with parthenogenetic D. armeniaca; near Sevkar village, D. dahli is sympatric with parthenogenetic lizards D. rostombekovi and D. armeniaca, and with bisexual lizards D. raddei. The interspecific hybrids identified in these zones are infertile triploids D. dahli are known D. dahli revealed the existence of five clones; however, unlike what was observed for the color varieties, one clone was widespread and abundant, and four other clones were rare and geographically restricted D. dahli species, and that the initial successful clone was formed only once D. dahli, a rather high level of variation among individual fingerprint patterns was demonstrated when various microsatellite probes were used for blot hybridization D. dahli can be obtained via molecular and genetic studies of individual genomic loci.In our DNA fingerprinting studies of D. dahli species originated from a single or multiple interspecific hybridization events. For this, we sequenced all allelic variants of three microsatellite loci found among 111 D. dahli specimens from five Armenian populations, identified nucleotide variations among sequenced alleles, and established a distinct genotype for each individual. We hypothesized that the nucleotide variations found among alleles in the hybrid genomes of D. dahli were inherited from two parental species, and that their specific combinations formed by two alleles in genotypes may reflect independent hybridization events between bisexual individuals of parental ancestors. In this way, we detected 11 genotypes and identified at least three hybridization founder events in the D. dahli populations sampled. These data provide new knowledge regarding the evolutionary origin of D. dahli that suggest its multiple hybridization origin. Our approach based on the detection of genotype-specific markers formed by combinations of parental-specific nucleotide markers can be used to address similar problems in other unisexual taxa.The main objective of this study was to examine whether the Darevskia was not counted as an endangered species in that period D. dahli individuals of five isolated populations in Armenia that were available to us: “Papanino” (n = 69), “Fioletovo” (n = 9), “Vaagni” (n = 17), “Dzoraget” (n = 6), and “Dendropark” (n = 10)  and 1 µM of each primer. The reaction conditions were: one cycle of 3 min at 94°C; 30 cycles of 1 min at 94°C; 40 s at the annealing temperature ; and 40 s at 72°C, followed by one cycle of 5 min at 72°C. PCR products (15 µL) were loaded onto an 8% native polyacrylamide gel  and run for 12 h at 60 V. A 50 bp ladder (Fermentas) was used as a size marker. The amplification products were visualized by staining the DNA in the gel with ethidium bromide. The well-resolved individual PCR products, which corresponded to the two individual alleles of the locus, were excised from the gel, purified by ethanol reprecipitation, and sequenced directly in both directions using a chain termination reaction with an ABI PRISM BigDye Terminator ver. 3.1 on an Applied Biosystems 3730 DNA analyzer. This procedure was carried out twice for genotypes 5–11, because each of them was represented by only one individual. In all of these cases, allelic identity was checked and confirmed via the comparison of sequences obtained independently. In a separate experiment, the PCR product of the Du215 locus in genotype 11 was excised from the polyacrylamide gel, purified, and cloned into the pMos blue vector according to standard procedures . The clones were amplified in MOSBlue competent cells grown at 37°C, and sequenced as described previously Parthenogenetic lizards were collected between 1997 and 2006 from their natural habitats in Armenia. All territories in which lizard samples were collected are governmental. There are no protected areas or private land on these territories. Therefore, no specific permissions were required for our sample collections in these locations. Our field studies did not involve endangered or protected species, and according to the Red Book of Armenia, the genus (n = 10) . The locThe nucleotide sequences were compared using the MEGA ver. 5.1 software The number of alleles, allelic richness, as a measure of allele counts adjusted for sample size, and expected heterozygosity, as a measure of gene diversity, were calculated per locus and per population using the Fstat software ver. 2.9.3.2 D. dahli genotypes using the method of Templeton et al. (1992) The TCS program implements a statistical parsimony approach for estimating genealogical relationships between D. dahli from five populations of Armenia were analyzed using locus-specific PCR and DNA sequencing of PCR amplificants. It was shown that all D. dahli individuals but one were heterozygous at the three loci analyzed, and contained two alleles that differed from each other regarding the length and structure of microsatellite clusters, and regarding single nucleotide variations (SNVs) in fixed positions of the flanking regions  . The higD. dahli species originated from single or multiple interspecies hybridization events, genotype-specific nucleotide markers formed by combinations of parental-specific SNVs of three loci were established and compared. D. dahli result from at least three independent interspecies hybridization events, rather than from a single origin. In addition to having the same combination of parental-specific SNVs, genotypes 1–9 also exhibited a similar organization of allelic microsatellite clusters. Different situations occurred in individuals with genotypes 10 and 11. The microsatellite cluster of the ACT allele of genotype 10, unlike what was observed for all other alleles, had no (GCAA)4 nucleotide group. The individual with genotype 11, unlike all other individuals, was homozygous at the Du215 locus.To clarify whether the An unresolved question remains regarding the relationships between genotypes 1–9. These genotypes differed from each other only by microsatellite sequences. According to D. dahli, which resulted in the formation of three initial clones and eight clones that probably arose through postformation microsatellite mutations of the initial clones. Previously, an allozyme analysis conducted at 35 gene loci in three Armenian and three Georgian sampled populations of D. dahli revealed one major widespread clone and four rare clones detected only in restricted populations D. dahli generally followed Parker et al.’s model D. dahli should be investigated further using more effective genomic markers Darevskia, D. rostombekovi was also suggested to be of single origin D. armeniaca, four allozyme clones were detected D. armeniaca. However, the allozyme data failed to distinguish multiple origins and mutation events. Fu et al. (2000) noted that many studies that were performed at the species level examined various population characteristics, but few did this at the genotype level The main finding of this study was the presence of at least three interspecific hybridization founder events in D. dahli genotypes formed by independent hybridization between bisexual parental individuals was developed. This approach included microsatellite genotyping of the sampled individuals, and the identification of genotype-specific nucleotide markers formed by combinations of parental-specific nucleotide markers (SNVs). The examination of the allelic combinations of three microsatellite loci in 111 D. dahli individuals revealed 11 clones, two abundant (major and intermediate) and nine rare and geographically restricted. This pattern of microsatellite-clone distribution raises questions about the scenario of the D. dahli species formation, and about the relationship between major, intermediate, and rare clones. The comparison of genotype-specific markers showed that all individuals with genotypes 1–9 exhibited the same combination of parental-specific SNVs and, therefore, could have a common origin. Two individuals with genotypes 10 and 11, respectively, possessed a unique combination of parental-specific SNVs, and thus were likely to have independent origins. Our data do not rule out further hybridization events. First, the variation observed among clones 1−9 could actually be a polymorphism that was inherited from the parental populations, and there were differences between individuals with similar genotypes; that is one potential source of underestimation of the frequency of hybridization. Second, in the case of low variation in parental populations, some hybridization events could have led to identical unisexual genotypes with fewer hybridization events. Nevertheless, some of the clones 1−9 may indeed have originated via postformation microsatellite mutations of the initial clones. Consideration of the SP network, which reflects the relationships between genotypes 1–9, does not allow the disclosure of the initial clone. According to Parker et al.’s model n-containing locus in parthenogenetic D. unisexualis. We identified and sequenced mutant alleles in some offspring of the first generation and showed that de novo mutations occurred via deletion or insertion of a single microsatellite repeat In our study, a new approach for the detection of Cnemidophorus and HeteronotiaHeteronotia binoei; Cnemidophorus tesselatus; Darevskia parthenogens; D. dahliMultiple hybridization events leading to multiple allozyme clones have been described in D. dahli. Of the rare clones, six, including those with genotypes 10 and 11, occurred in the Vaagni population. This population, together with the Dendropark population, has an enhanced level of clonal diversity  overlaps with the bisexual parental species D. portschinskii, but the upper altitudinal limit of the unisexual species is higher D. dahli and D. portschinskii is restricted to competition for food or cover and, as noted above, the interspecific hybrids observed were infertile triploids Nine relatively rare clones, each represented by one or several individuals, were found in four sampled populations of iversity . InterpoThe greater clonal diversity of the Vaagni and Dendropark populations could be also connected with their geographical location in the seismically active region of Armenia, where a casting of chemical mutagens has sometimes occurred The lack of statistical approaches for the evaluation of population diversity in parthenogenetic species has forced us to use parsimony networks, which are well known in the field of phylogeography, to demonstrate the peculiarities of the geographical distributions of individual clones. Despite the formal similarity of the two types of molecular markers, the mechanisms of accumulation of nucleotide substitutions (SNPs) in mitochondrial DNA molecules are rather different. Here, we used many simplifications and assumptions, such as the absence of linkage among the three loci, the independence of replication events at each of the three loci, the equal rate of microsatellite repeat deletions or insertions, and the lack of intergenic recombination and genic and chromosomal interference Darevskia species et al. (1985) estimated that the parthenogenetic Caucasian rock lizards originated after the Pleistocene glaciation of the Caucasian mountains, 5000 to 7000 years ago D. dahli and two parental species, Murphy et al. D. dahli arose in Georgia and dispersed into Armenia. This is correlated with today’s Georgian localization of the maternal species D. mixta and the strong allopatric distribution of parental species. It can also mean that the initial microsatellite clones detected by us originated in Georgia, and then dispersed to Armenia. At the same time, the possibility that these clones originated in Armenia could not be excluded, because many climatic and vegetative changes have occurred postglacially in the Caucasus region, providing an abundance of microhabitats for lizard species. An alternative scenario of the origin of D. dahli can also be considered. Two populations, Vaagni and Dendropark, were characterized by the highest estimates of genetic diversity  was detected in this population . GATA repeat is designated by the letter A. Numbers before the letter A designate the number of the repeat on each of the two chromosomes that are separated in the Table by forward slash “/”. In concatenated sequences used for parsimonies network reconstruction, the absence of a GATA repeat is designated by dash (−).(DOC)Click here for additional data file.Table S2The population indices of gene diversity for three studied loci in five sampled populations. N – number of alleles, Rs – allelic richness, HE – expected heterozygosity.(DOC)Click here for additional data file.


Crocodylus suchus, which occur in isolated groups, usually less than five individuals, along the mountains of Mauritania (West Africa). We used both invasive and non-invasive sampling methods and a combination of mitochondrial DNA (12 S and ND4) and microsatellite markers (32 loci and a subset of 12 loci). Our results showed high genetic differentiation and geographic structure in Mauritanian populations of C. suchus. We identified a metapopulation system acting within four river sub-basins (high gene flow and absence of genetic structure) and considerable genetic differentiation between sub-basins (FST range: 0.12–0.24) with rare dispersal events. Effective population sizes tend to be low within sub-basins while genetic diversity is maintained. Our study suggests that hydrographic networks  allow C. suchus to disperse and maintain metapopulation dynamics within sub-basins, which attenuate the loss of genetic diversity and the risk of extinction. We highlight the need of hydrographic conservation to protect vulnerable crocodiles isolated in small water bodies. We propose C. suchus as an umbrella species in Mauritania based on ecological affinities shared with other water-dependent species in desert environments.The maintenance of both spatial and genetic connectivity is paramount to the long-term persistence of small, isolated populations living in environments with extreme climates. We aim to identify the distribution of genetic diversity and assess population sub-structuring and dispersal across dwarfed desert populations of  Crocodylus suchusHabitat connectivity is important for populations living in climate-extreme environments, which are naturally patchy and have temporally variable resources gueltas), located upstream of mountain valleys, and floodplains , located at the foothills of the mountains gueltas (0.001–1.0 ha) restricts their carrying capacity, thus they usually harbour less than five adult individuals gueltas and tâmoûrtsC. suchus may constitute a metapopulation. Analyses of molecular markers are needed to corroborate field observations and interpretations about the potential connectivity, possibly enabling gene flow between crocodile populations and confirming the existence of a dynamical metapopulation. Assessing genetic structure and gene flow among populations, as well as identifying landscape features that have an impact on gene flow are key to identify dispersal corridors as targets for conservation efforts. The genetic patterns of these relict crocodile populations may help understand possible metapopulations dynamics in desert landscapes and contribute to the prioritization of conservation actions on a species that could serve as a model for connectivity conservation, i.e. strategies for protecting and restoring dispersal corridors In Mauritania, crocodile populations are known from 81 small rocky pools  detect the occurrence of contemporary gene flow among gueltas/tâmoûrts and between sub-basins; and 3) quantify genetic diversity within each sub-basin. Taking into account isolated populations and small group size within each locality; we predict genetic structuring, reduced gene flow, low levels of genetic diversity and signs of inbreeding. However, the dispersal ability of crocodiles during the rainy season might counteract the negative effects of their isolation.Using a combination of mitochondrial DNA (mtDNA) and microsatellite markers, we aim to: 1) assess population sub-structuring across isolated populations of Fieldwork was developed with permission from the Ministére Délégué auprès du Premier Ministre Chargé de l'Environnement, Nouakchott (Permit: 460/MDE/PNBA). This permit was valid for the entire country and no specific permissions were required for any specific locality. Analyses were done at a CITES registered laboratory: 13PT0065/S. Field collection and handling practices were approved by the Committee of Animal Experimentation of the University of Porto  under the Directive 2010/63/EU of the European Parliament. No animal was sacrificed and there were no animal husbandry, experimentation and care/welfare concerns.gueltas; 2) the Gorgol el Abiod that results from water draining from the southern Tagant and northern Assaba; 3) the Gorgol el Akhdar and 4) Garfa sub-basins that are formed through water run-offs from the western Assaba; 5) the Karakoro that flows between the Assaba and the Afollé, although it is mostly fed by run-offs from the later mountain, and is characterized by low water availability; and 6) the Kolimbiné that flows east-ward of the Afollé. With the exception of the Gabbou, the other five sub-basins flow to the Senegal River and are composed of gueltas in mountain slopes and seasonal tâmoûrts at foothills of the mountains The study area encompasses three southern Mauritanian mountains: Tagant, Assaba and Afollé . These mC. suchus from fieldwork expeditions from 2007 to 2011 ca. 25% and 5% of the total population of crocodiles in the Tagant/Assaba and Affolé mountains respectively. We captured live crocodiles by hand or using hand nets, and then immediately released them at the point of capture after clipping a 5 mm piece of tail tissue for genetic analysis. Team members obtained the tissue samples following ethical guidelines for use of live reptiles. We used samples obtained from carcasses of C. suchus from Guinea-Bissau and C. niloticus from Malawi as outgroups in the analyses . We followed Frantz et al., We amplified two mtDNA fragments of 421 bp and 878 bp from 12 S and ND4, respectively, for fresh and near fresh samples in 10 µl reaction volumes, containing 5 µl of master mix , 0.5 µM of each primer and approximately 1 µl of genomic DNA , thus we excluded one from the dataset. We performed four PCR replicates for each sample (discarding samples that failed to have a good amplification rate) after a maximum likelihood evaluation of error rates  using Pedant 1.0 Na), and observed (Ho) and expected (He) heterozygosity values) within each geographic groups (sub-basins) identified in population structure analyses (see below) and using the 12 loci dataset to maximize the number of samples. We calculated allelic richness per sub-basin using the rarefaction algorithm implemented in ADZE 1.0 xyr implemented in IDENTIX 1.1 rxy for each river sub-basin and compared to their expected distribution, generated by 1,000 permutations of genotypes among individuals, under the null hypothesis of no relatedness.Two out of 35 samples that we successfully analyzed exhibited identical genotypes  ranging from 1 to 7, a burn-in period of 1×106 and a run length of 5×106 iterations. We implemented an admixture model with uncorrelated allele frequencies and without prior information on sample population membership. We used Structure Harvester 0.6.93 K by the highest value of log probability of data L(K) K) K-value. We calculated pairwise FST values P - values with the sequential Bonferroni correction.We used the Bayesian genotype clustering method implemented in STRUCTURE 2.3.4 To identify spatial genetic patterns we also performed multivariate analyses using the spatial Principal Component Analyses (sPCA) m) between sub-basins. BIMR uses assignment tests to estimate the proportion of genes derived from migrants within the last generation and assuming linkage equilibrium and allowing for deviation from Hardy-Weinberg equilibrium. We used a burn-in period of 1×105 and a run length of 5×105 iterations for each of the 10 replicate runs, and then averaged pairwise migration rates between and within sub-basins across runs.We used BIMR C. suchus haplotypes and diverged by 53 mutational steps from the second network containing all C. niloticus haplotypes, whereas a third network represented by a single haplotype from Malawi differed in 19 mutational steps from the C. niloticus main network. Mauritanian C. suchus exhibited low genetic diversity (only three haplotypes), with a common haplotype (Mauritania A) widespread across the whole study area (three mountains and main river sub-basins) and a second haplotype  that is restricted to four samples from the Karakoro-Kolimbiné sub-basin . Haplotype network reconstruction using statistical parsimony in TCS resulted in three haplotype networks not connected under the 95% connection probability criteria . One netub-basin . A thirdP<0.05 after Bonferroni correction) for the 12 loci dataset. As this deviation occurred only at Karakoro-Kolimbiné and was not observed for the 32 loci dataset, all loci were retained in the analyses. No evidence of linkage disequilibrium was apparent in any pair of loci/population after adjusting the significance level for multiple comparisons. Mean values of allelic dropout and false alleles amplification across loci  and across samples (7.7% and 0.0%) were low, supporting the robustness of the selected loci.All loci showed low to moderate polymorphism when considering the entire dataset. The number of alleles per locus ranged from three (CUD68) to seven (CpP302). One locus deviated from Hardy–Weinberg equilibrium at Karakoro-Kolimbiné sub-basin  with the exception of the Gorgol el Akhdar-Garfa sub-basin that showed the lowest level of inbreeding (xyr≈0.5) were found within three sub-basins at isolated gueltas  as the most probable number of groups within our dataset (estimated at both L(K) and Δ(K) methods). These genetic groups correspond to independent hydrographic sub-basins, Gabbou and Gorgol el Abiod in the Tagant mountain, and to combinations of neighbor sub-basins within mountains, Gorgol el Akhdar-Garfa in Assaba and Karakoro-Kolimbiné in Afollé (K = 2) clearly shows a partition between sub-basins in the Tagant and Assaba mountains  from sub-basins in the Karakoro-Kolimbiné in the Afollé mountains (K), suggesting that increasing the number of markers slightly improve the resolution of population structure (K = 3), sub-basins within Tagant and Assaba mountains are split (results not shown).STRUCTURE analyses identified four genetic demes , but no local structure  has been detected (vs. Afollé mountains). To investigate deeper genetic structuring in these two groups, we selected the second best number of clusters (K = 5) in which DAPC recovered the four clusters obtained in STRUCTURE and further subdivided the Karakoro-Kolimbiné in two clusters  to 0.24 (between Gorgol el Abiod and Karakoro-Kolimbiné) (Pairwise limbiné) . Estimatlimbiné)  and consC. suchus in Mauritania is consistent with the recent findings of Hekkala et al. C. suchus. However, relict populations or individuals of C. niloticus present in West Africa cannot be excluded since the sample sizes and geographic areas surveyed are still limited The presence of mtDNA haplotypes of FST) across relict populations in Mauritania, suggesting that geographic isolation might have started since the beginning of the arid phase. In addition to recent geographic isolation, we also note that demographic factors and drift FST values (0–15–0.24) between sub-basins of Afollé and sub-basins from both Tagant and Assaba together with the first split of these geographic groups identified with STRUCTURE, suggest that Afollé population may have experienced a longer isolation from the remaining populations. Genetic differentiation and genetic structure of these populations also reflect ecological constraints on dispersal in C. suchus. Pairwise FST values between sub-basins are much higher in comparison to those found in other studies of crocodilians C. suchus can be explained by a combination of geographic isolation, low population sizes and genetic drift. In addition, genetic demes correspond mainly to sub-basins  supporting previously hypothesized metapopulation dynamics in the region gueltas and tâmoûrts during the rainy seasons, while these water bodies act as refugia to relict populations during the dry seasons when they remain isolated by sandy areas. However, individuals from relatively close localities but different sub-basins  and genetic differentiation  considered for demographic independence K = 2; The results obtained from the combination of genetic markers unveiled the development of a metapopulation system of gueltasgueltas might act as dispersers, colonizing other water bodies by overland dispersals. Our study shows evidence of long dispersal between sub-basins for one individual (sample 2642), which leads us to question why and how do crocodiles disperse. Two main benefits of dispersal are the avoidance of kin competition (to limit inbreeding) and the access to better habitats gueltas are unsuitable and individuals would have to move long distances among gueltas, thus risking successful dispersal and eventually their lives. Permanent and temporal rivers are thought to be the main way to disperse for crocodiles, as for other semi-aquatic organisms The reduction of connectivity among fragmented populations is negatively associated with migration rates and gene flow, decreasing the colonization capacity and the rescue effect that ultimately impact both genetic diversity and structure C. suchus, highlighting that individual's dispersal abilities help maintain current populations by diminishing the negative effects associated with stochastic events and genetic drift, and thus preventing local extinctions and increasing the populations' recovery in this harsh and mostly unsuitable environment.Overall, our study confirms genetic connectivity among isolated water bodies within main sub-basins for C. suchus genetic demes , make them vulnerable to demographic and environmental events that may reduce genetic variability. However, our results do not show drastically low genetic diversity for any sub-basin. In fact, heterozygosities in C. suchus in isolated localities as complements to molecular approaches will contribute to understanding population dynamics and dispersal rates. In addition to the use of remote sensing techniques to quantify water availability patterns Future efforts should be focus on identifying landscape features shaping genetic structure and distribution of genetic diversity across crocodile populations in the region. Further fieldwork efforts are needed within these mountains along with integrative spatial/genetic analyses, such as those employed in landscape genetics approaches gueltas are presently overexploited by humans, resulting in water shortage during the dry season, faecal contamination, excessive eutrophication, and increased activities for excavating pools or pumping water C. suchus occur, as these may be considered independent conservation units. Identifying and protecting the species' refugia in temporary freshwater ecosystems can contribute to conservation plans designated to enhance the resilience of freshwater biodiversity To curb local population extinctions it is advisable to increase both the carrying capacity of populations and the facilitation of dispersal among populations. Increasing water quality is needed as many gueltasC. suchus in Mauritania constitutes a good candidate umbrella species, since it's conservation is tied to other aquatic or semi-aquatic organisms , which also live in isolated spots across the Sahara-Sahel. The use of genetic tools to study dispersal in umbrella species has been considered as a realistic approach to implement ecological networks that could assist species protection at the regional level C. suchus provides an initial framework to manage and protect threatened desert populations that depend on permanent or seasonal water bodies.Crocodiles are usually viewed as a conservation flagship species and considered a valuable indicator in ecosystem monitoring and restoration programs Table S1Crocodylus suchus samples analysed in this study. For each sample (ID), we list the country, mountain chain, basin, sub-basin, locality, GPS coordinates of collection sites and their genetic assignment at both mtDNA (haplotype) and microsatellite level (genetic deme).(PDF)Click here for additional data file.Table S2Loci used on this study with primer information, allele ranges, PCR conditions and references for description of primers used both for mtDNA and microsatellite markers.(PDF)Click here for additional data file.Table S3Pairwise relatedness (xyr) among all crocodile individuals.(PDF)Click here for additional data file.Input File S1Input file (Structure format) containing genotype information for 32 microsatellite markers.(TXT)Click here for additional data file.Input File S2Input file (Phylip format) containing sequences of the 12 s mtDNA fragment.(TXT)Click here for additional data file.Input File S3Input file (Phylip format) containing sequences of the ND4 mtDNA fragment.(TXT)Click here for additional data file.