Oncorhynchus tshawytscha) from western Alaska. Patterns of population structure were similar to those of past studies, but our ability to assign individuals back to their region of origin was greatly improved . We also calculated effective size with and without removing physically linked loci identified from a linkage map, a novel method for nonmodel organisms. Estimates of effective size were generally above 1000 and were biased downward when physically linked loci were not removed. Outlier tests based on genetic differentiation identified 733 loci and three genomic regions under putative selection. These markers and genomic regions are excellent candidates for future research and can be used to create high-resolution panels for genetic monitoring and population assignment. This work demonstrates the utility of genomic data to inform conservation in highly exploited species with shallow population structure.Recent advances in population genomics have made it possible to detect previously unidentified structure, obtain more accurate estimates of demographic parameters, and explore adaptive divergence, potentially revolutionizing the way genetic data are used to manage wild populations. Here, we identified 10 944 single-nucleotide polymorphisms using restriction-site-associated DNA (RAD) sequencing to explore population structure, demography, and adaptive divergence in five populations of Chinook salmon ( Oncorhynchus spp.) which exhibit extensive population structure  is an important parameter in conservation biology  from western Alaska represent an excellent system to explore the utility of genomics in a management context. Chinook salmon inhabit four major regions in western Alaska: Norton Sound, the Yukon River, the Kuskokwim River, and Bristol Bay, all of which vary significantly in size, hydrology, and climate  sequencing to investigate the population structure and demography of Chinook salmon from western Alaska. We identified over 10 000 SNPs in 270 individuals from five populations across western Alaska. Patterns of genetic variation were assessed using both population-and individual-based methods and validated with assignment tests. We then aligned our RAD markers to a linkage map to calculate Tissue samples from spawning Chinook salmon were available from four regions in coastal western Alaska and one in the upper Yukon River Fig. , Table 1SbfI following the methods of Baird et al. , and uninformative polymorphisms. First, we removed any putative SNP that failed to genotype in >80% of individuals. We then removed those with a minor allele frequency <0.05 in all populations. These polymorphisms are likely to be uninformative, are difficult to distinguish from sequencing errors, can distort signals of selection and drift in natural populations, and may bias tests for selection . We then removed individuals that were missing genotypes at > 15% of the SNPs. As a final filtration step, we used ML-Relate  implemented in the R package  Jombart . The sig Jombart . These famova) in Arlequin 3.5 to examine the variation within and among groups of genetically similar populations. The hierarchy for this analysis was chosen based on the clustering from the PCA: (i) Koktuli River, Kogrukluk River, and Anvik River, (ii) Tubutulik River, and (iii) Big Salmon River. Separate amovas were conducted for (i) the entire data set and (ii) all populations except the Big Salmon River. Finally, we calculated global and per-locus observed and expected heterozygosities for each population in GenAlEx 6.5 . This analysis was conducted separately for the 10 944 RAD SNPs and 39 of the 43 SNPs from Templin et al.  that werGeneClass2.0 . NeEstimator was used to calculate r2 values for each locus pair with the following parameters: a minimum allele frequency cutoff of 0.02 and a random mating model. We then implemented the methods described in Waples . We calculated Ne for three data sets: (i) all RAD SNPs that aligned to the linkage map, (ii) all RAD SNPs that aligned to the map with pairwise comparisons between markers on the same linkage group removed, and (iii) the 39 SNPs from Templin et al. (Estimates of hod Hill  updated hod Hill . This meage Park . Additioage Park  (see beln Waples  and Peeln Waples  in R to Ne/N) using Ne calculated with RAD-derived SNPs after removing physical linkage and estimates of total escapement obtained from aerial surveys  and weir counts . Multiple aerial surveys were used to estimate total run size for the Anvik River and Koktuli River populations, but only single aerial counts were available for the Tubutulik River population. Single aerial counts from a river near the Tubutulik River collection were approximately four times smaller than those taken from a counting tower; therefore, we multiplied the aerial count from our collection by four. We averaged the last ten years of data to obtain an approximate value of census size for each population (only last 3 years used for Koktuli River due to data availability).We calculated the ratio of effective size to census size  per year (Ne/N) and Ne divided by the total census size per generation (Ne/NG). Values of G for each population were obtained from the sources in Table Estimates of p Waples . Single-Arlequin 3.5. This program uses coalescent simulations to create a null distribution of F-statistics then generates P-values for each locus based on this distribution and observed heterozygosities across loci  genomic region to a null distribution created by bootstrapping over the complete data set . Alignments using BLAST annotation of these contigs yielded significant hits for 1576 (14%) of 10 944 SNPs (Table S2). Of these hits, over one-third aligned to transposable elements. Other common functional groups included DNA polymerases and transmembrane proteins.Paired-end assemblies produced 11 666 contigs with an average length of 268 bp . FST of the full data set was 0.041, and pairwise FST values ranged from 0.003 for the Koktuli River–Kogrukluk River comparison to 0.098 for the Big Salmon River–Tubutulik River comparison (Table P < 0.001). The results of these significance tests should, however, be interpreted with extreme caution due to the large number of loci, which may overestimate precision.Principal component analysis revealed that the Big Salmon River and Tubutulik River populations formed completely separate clusters, while the Koktuli River, Kogrukluk River, and Anvik River populations essentially formed a single cluster Fig. . The oveon Table . Geneticamova for the entire data set and for a data set without the Big Salmon River population , but because of the large number of axes, the actual proportion of variation explained was small.The relatively small amount of variation (1–5%) explained by the first and second principal components (PCs) in our PCAs Figs  and 3 caAssignment accuracy was much higher using >10 000 SNPs (≥ 89% assignment to correct population) compared to 39 SNPs . This proportion may seem small, but it is important to note that the map was constructed using a single Chinook salmon from Washington State. Chinook salmon from Washington State are substantially diverged from populations in western Alaska  that were significant outliers at the 5% level and 178 (1.6%) that were significant at the 1% level. BLAST annotation of the outliers at the 5% level revealed 96 significant hits (13% success rate). Transposable elements represented over one-third of the significant hits which is consistent with the pattern from the complete data set.Outlier tests in The number of genomic regions under putative selection for each population pair ranged from 20 to 25 and generally increased when the Big Salmon River population was included Fig. , Table 2Ne ranged from 516 to infinity and appeared to be biased downward when loci that were physically linked were not removed. Regions of putative adaptive divergence appeared to be randomly distributed across the genome with few shared areas of high divergence across populations, but we did find three genomic regions that displayed high divergence in multiple populations. Using genomic data, we were able to conduct individual assignment in populations where it was previously unfeasible, discover genomic regions under putative selection, and estimate Ne in populations with >1000 individuals. Our approach therefore represents a significant improvement over previous studies employing fewer markers and no linkage map.We used RAD sequencing to characterize the genetic structure, genomic divergence, and demography of five populations of Chinook salmon from western Alaska. Patterns of genetic differentiation were similar to but more identifiable than in past studies  were least divergent, displaying pairwise N = 1700, Ne=516). The Kogrukluk River and Koktuli River populations, on the other hand, are at least four times larger than the Anvik River population and may not have been affected as substantially by genetic drift. Individuals that did not fall within major clusters were found in the Kogrukluk River and Anvik River populations. These individuals may represent evidence of gene flow from genetically diverged upriver populations, but could have also resulted from within population variation. Individual-based PCA using 39 SNPs from Templin et al.  per year (N) and the census size per generation (NG) to account for both of these possibilities when comparing Ne to census size. The Ne/N and Ne/NG ratios were highly variable across our populations, indicating that effective and census size are not well correlated in our study system.Estimating and interpreting t Waples . Ne estiNe/N found a median value of 0.14 and also showed that Ne/N ratios are generally larger in smaller populations  with 10–100 genetic markers is extremely challenging due to the small amount of linkage disequilibrium caused by drift (Waples and Do Ne may be feasible (Allendorf et al. Ne from our study were infinite for three of five populations with 39 SNPs, but only infinite for one population with 1118 SNPs. Additionally, all estimates with 39 SNPs, but only two estimates with 1118 SNPs, displayed confidence intervals including infinity, and confidence intervals were consistently smaller with 1118 SNPs. Our results indicate that genomic data can improve the accuracy of Ne estimates in large populations, aiding management in many species.Estimating BLAST annotation of outlier loci revealed a high frequency of transposable elements, similar to the overall data set. These transposable elements are quite common in teleost fish and are generally assumed to behave as neutral markers (Radice et al. We identified 6.7% of SNPs in our data set as outliers, consistent with past studies identifying 5–10% of markers as candidates for directional selection (Nosil et al. Tests for genomic regions under putative selection revealed that these regions appeared to be spread randomly across the genome with few common ‘hot spots’ among populations. This pattern is consistent with Bourret et al. , who fouReturns of Chinook salmon to western Alaska have fallen dramatically over the last decade compared to their long-term average ADF&G . This prThis high-throughput SNP panel could also be used to investigate the migration and distribution patterns of Chinook salmon on the high seas (e.g., Murphy et al. Our study demonstrates the utility of genomic data when attempting to differentiate closely related populations and estimate demographic parameters. The methods we employed will be especially applicable in marine species, which are often characterized by low genetic differentiation and large population sizes Waples . For exaNe in large marine populations can also be improved using approaches similar to ours (e.g., Gruenthal et al. Ne and Ne/N in many economically important marine species. These estimates can provide important insights into the adaptive potential of marine populations and can be used to inform management (Hare et al. Estimates of Ne for each population using a novel method for nonmodel organisms. Estimates were generally large and provided some evidence that metapopulation dynamics influence demography in this region. Investigation of loci and genomic regions under putative selection found three potential regions of adaptive divergence. The methods described in our study will be particularly applicable to marine species or any species where large population size and shallow structure are common.Our results demonstrated fine-scale structure between regions in western Alaska. This structure allowed us to assign fish back to their region of origin with greater than 90% accuracy, representing a significant improvement over past studies. We also estimated


Pinus massoniana, an ecologically and economically important conifer, is widespread across central and southern mainland China and Taiwan. In this study, we tested the central–marginal paradigm that predicts that the marginal populations tend to be less polymorphic than the central ones in their genetic composition, and examined a founders' effect in the island population.P. massoniana based on nucleotide sequences of cpDNA atpB-rbcL intergenic spacer, intron regions of the AdhC2 locus, and microsatellite fingerprints. SAMOVA analysis of nucleotide sequences indicated that most genetic variants resided among geographical regions. High levels of genetic diversity in the marginal populations in the south region, a pattern seemingly contradicting the central–marginal paradigm, and the fixation of private haplotypes in most populations indicate that multiple refugia may have existed over the glacial maxima. STRUCTURE analyses on microsatellites revealed that genetic structure of mainland populations was mediated with recent genetic exchanges mostly via pollen flow, and that the genetic composition in east region was intermixed between south and west regions, a pattern likely shaped by gene introgression and maintenance of ancestral polymorphisms. As expected, the small island population in Taiwan was genetically differentiated from mainland populations.We examined the phylogeography and population structuring of the The marginal populations in south region possessed divergent gene pools, suggesting that the past glaciations might have low impacts on these populations at low latitudes. Estimates of ancestral population sizes interestingly reflect a recent expansion in mainland from a rather smaller population, a pattern that seemingly agrees with the pollen record. Climate changes over the past two million years are often interpreted as the primary driver of range fragmentation and the speciation of animals and plants in the northern hemisphere Pinus luchuensis and P. taiwanensisCycas taitungensis and C. revolutaBecause a species is spatially structured into populations, the population size and extent of interpopulation gene flow would determine the levels of genetic diversity within populations/species. A central–marginal paradigm predicts that populations around the distributional center tend to possess higher levels of genetic variation than the peripheral ones because genetic variations would be lost through stochastic drift faster in marginal populations than in the central ones Pinus massoniana Lamb., a species of sect. Pinus, is distributed in central and southern mainland China and Taiwan P. massoniana acts as an ecological pioneer, colonizing mesic, harsh sites and competing little with other woody plants Cunninghamia lanceolataPinus massonianaP. massoniana, enabling one to recover the demographic histories P. massoniana also survived glacial cycles Pinus has generally been an important component of the regional vegetation. Many Chinese subtropical Pinus dominated the vegetation during the late Pleistocene and Holocene with only minor population changes As a common species, mainland populations are likely mediated by some degree of gene flow, especially via wind-dispersed pollen P. massoniana based on sequence variation in the atpB-rbcL intergenic spacer of cpDNA, the introns 4 to 8 of AdhC2 gene of nuclear DNA, and 11 microsatellite loci. Both nuclear and cpDNA markers can be dispersed via pollen and seed, as well as nuclear microsatellite loci which are biparentally inherited. The cpDNA of Pinus is exclusively carried by pollen, a pattern unlike most other plants. AdhC is one member of the Adh gene family, which encodes a key enzyme, alcohol dehydrogenase, in the glycolytic pathway. Fast molecular evolution and near neutrality make its intron regions suitable for population study Pinus populations less polymorphic than the central ones in their genetic composition? (2) Did the marginal populations in south region act some refugia over the glacial maxima? And (3) how and when was the island population colonized by migrants from the adjacent mainland? Has the Taiwan Strait acted as a barrier and caused genetic differentiation between the populations of mainland and Taiwan, especially given effects of founders' effects in the latter?The distribution of a species across mainland and island provides a perfect system for testing the central–marginal paradigm Pinus massoniana. Eight populations of Pinus massoniana throughout its whole geographical range were surveyed  were identified , Table 2AdhC2 introns were identified. They were clustered into three types, α, β, and γ   . Four maE972272) . To undeE972272) , based oAdhC2 introns was reconstructed, rooted at P. taiwanensis individuals (accession numbers: HE972269-HE972270)  . Three mstructed . Within structed .OH) and expected heterozygosity (eH) varied with a range of 0.09091–0.29032 and 0.18989–0.47385, respectively , indicating deficiency of heterozygosity.Estimates of diversity varied among populations based on 11 microsatellite loci . The aveectively . The Daiet al.An un-rooted phylogram was constructed from microsatellite frequencies using Nei's D to assess the genetic relationships . Three gP. massoniana, a SAMOVA analysis (K = 2) displayed the greatest value of FCT and a maximal variance  . Microsatellite data indicated the different pattern based on SAMOVA, HS I, and DA population were grouping together, and others formed another group   . The gro P<0.05) .P. massoniana populations, cpDNA displayed a bimodal or multimodal mismatch distribution, except for population Huangshan I  and Taiwan (TAI)  . The oveibutions . Fu and negative . Howeverssoniana . Based o in nDNA .Pinus massoniana represents a species of typical evergreen forests in central and southern China. As a dominant species at mesic and harsh habitats with a wide distribution, P. massoniana is expected to have high levels of genetic diversity. Nevertheless, the level of nucleotide diversity of cpDNA (0.00596) detected from 320 individuals across eight natural populations was not high. The genetic diversity approximated that of P. hwangshanensis (0.00605), while much lower than that of P. luchuensis (0.05014) and P. taiwanensis (0.07051) at the same locus et al.P. massoniana, identified 6 haplotypes with 20 polymorphic sites based on three cpDNA genes, which were relatively lower than 14 chlorotypes with 24 polymorphic sites detected in this study. The difference may simply come from the sampling difference. Besides, the level of nucleotide diversity of AdhC2 introns (0.01142) in P. massoniana was lower than that of other nDNA loci, such as lp3-3 (0.0370), dhn1(0.0144), and dhn3(0.0198), whereas higher than that of lp3-1(0.0095), chcs (0.0075), and dhn2(0.0074) in P. sylvestriP. massoniana populations had lower allele number (1.636–2.545) and expected heterozygosity (0.18989–0.45709)  P. contorta  P. halepensis  0.45709)  than thoP. hwangshanensis, reflect the effects of a common geological history across the mainland, likely associated with the Quaternary glacial maxima P. massoniana, the existence of the higher numbers of haplotypes and nucleotide diversities at both loci in populations in the south and in Hunan (HN) of central China, and highest averaged allele numbers and observed heterozygosity (Ho) at micorsatellite loci in populations in the south, highlights such impacts of the climatic oscillations Furthermore, approximating levels of genetic diversity in another sympatrically distributed species, P. massoniana, are often a consequence of past climatic changes P. massoniana populations, as indicated by the SAMOVA based on cp-and nDNAs, agreed with the scenario (mainland China vs. Taiwan). In contrast, microsatellite dataset revealed a different geographical pattern likely due to the existence of numerous ancestral polymorphisms shared between populations. Such population structuring was likely a result of historical fragmentations, while recent demographic decline must have worsened the loss in genetic diversity.Range disjunctions for plant species, a pattern also occurring for P. massoniana, although the two populations in Huangshan had the lowest levels of genetic diversity at cp- and nDNA loci likely agreeing with the paradigm prediction, populations of the southernmost populations (DA and HO) were highly polymorphic in their genetic composition of genes and microsatellite fingerprints  and great genetic differentiation between populations , and lineages β and γ (nuclear DNA intron).Likewise, Taiwan, as a subtropical continental island, also provided a refugium for the southward migrants P. massoniana within mainland and island were affected by population expansion after the Last Glacial Maximum (LGM), approximately 18000–20000 years ago  were likely to be intermixed between populations of the south and west . FurtherP. massoniana is expected to have had a large ancestral population. In this study, the BEAST program was used to estimate fluctuations of the ancestral effective population size in mainland populations and in the island population based on Bayesian skyline plots. Larger effective population sizes in the mainland than in Taiwan were detected with cp- and nDNAs. Based on both loci, a long history of constant effective population size of P. massoniana was detected across mainland populations, followed by subsequent demographic expansion. Likewise, Taiwan population of P. massoniana almost remained nearly constant before a recent population expansion in the nuclear DNA, whereas, a constant and stable population size was detected in cpDNA. Following the above, mainland and Taiwan populations did not undergo large fluctuations in population size until its recent population expansions  for one cycle of denaturation at 95°C for 4 min; 30 cycles of 45 s denaturation at 94°C, 75 s annealing at 52°C, and 90 s extension at 72°C; then 10 min extension at 72°C. Template DNA was denatured with reaction buffer, MgCl2, NP-40 and ddH2O for 4 min (first cycle), and cooled on ice immediately. Primers, dNTP, and Taq polymerase  were added to the ice-cold mixture. Primers were synthesized for amplifying the atpB-rbcL intergenic spacer (atpB-1: 5′-ACATCKARTACKGGACC AATAA-3′ and rbcL-1: 5′-AACACCAGCTTTRAATCCAA- 3′) AdhC2 introns (Pinus-AdhC2 exon4-F5′-TAC ATC GAC TTT CAG CGA GTA C-3′ and Pinus-AdhC2 exon 9-R 5′-TATG ATA GAA GCC TTA CCT TAG C-3′) Variation within and between populations was analyzed for the organelle DNAs and AdhC2 introns, purified DNAs were sequenced directly or ligated to a pGEM-T easy vector  if the heterozygote was directly sequenced. Five randomly selected clones were purified to obtain DNA plasmids using the Plasmid DNA Extraction Miniprep System . Eluted PCR products and DNA plasmids were sequenced directly in both directions by standard methods of the Taq dye deoxy terminator cycle sequencing kit . Products of the cycle sequencing reactions were run on an ABI 377XL automated sequencer .PCR products were electrophoresed on agarose gels, and the desired fragments were excised from the gel and purified. For the 2, 200 µM dNTP and 0.4 µM (each) primer; the forward primers Eleven variable microsatellite regions Nucleotide sequences were aligned with the program BlastAlign A network analysis complementary to conventional cladistics provides the power to discriminate between phylogeographical patterns that result from restricted gene flow and those that arise from historical events at the population level P. massoniana, we employed both mismatch distributions and statistical tests of neutrality. We calculated Tajima's DD* statistic Summary statistics such as the number of haplotypes (h) and minimum number of recombinations (Rm) were determined. The level of genetic diversity within populations was quantified by measures of nucleotide diversity θ FCT values) when the program was assigned to identify K = 2–6 partitions of populations.SAMOVA  was applied to identify groups of geographically adjacent populations that were maximally differentiated based on sequence data We also investigated historical demographics of populations by plotting mismatch distributions Ho), and expected (He) heterozygosities by using the Arlequin program version 3.1 st), to analyze molecular variance (AMOVA), and to test Hardy-Weinberg equilibrium (HWE). SAMOVA  was performed to identify groups of geographically adjacent populations based on microsatellite data et al.et al.et al.For microsatellite loci, genetic diversity within populations was assessed by calculating allele number (A), observed Click here for additional data file.Figure S2Mismatch distribution of nDNA haplotypes based on pairwise sequence differences against the frequencies of occurrence for seven populations of Pinus massoniana from mainland China and one from Taiwan. The number of pairwise nucleotide differences between haplotypes is represented on the X axis; their frequencies are represented on the Y axis.(TIF)Click here for additional data file.Table S1ST among populations deduced from sequences of cpDNA  and nDNA  for Pairwise FPinus massoniana.(DOC)Click here for additional data file.Table S2ST among populations deduced from microsatellites dataset for Pairwise FPinus massoniana.(DOC)Click here for additional data file.


Advances in understanding the process of species formation require an integrated perspective that includes the evaluation of spatial, ecological and genetic components. One approach is to focus on multiple stages of divergence within the same species. Species that comprise phenotypically different populations segregated in apparently distinct habitats, in which range is presently continuous but was putatively geographically isolated provide an interesting system to study the mechanisms of population divergence. Here, we attempt to elucidate the role of ecology and geography in explaining observed morphological and genetic variation in an understorey-dwelling bird endemic to southeastern Africa, where two subspecies are recognized according to phenotype and habitat affinity. We carried out a range-wide analysis of climatic requirements, morphological and genetic variation across southeast Africa to test the hypothesis that the extent of gene flow among populations of the brown scrub-robin are influenced by their distinct climatic niches. We recovered two distinct trends depending on whether our analyses were hierarchically structured at the subspecies or at the within subspecies level. Between subspecies we found pronounced morphological differentiation associated with strong reproductive isolation (no gene flow) between populations occupying divergent climatic niches characterized by changes in the temperature of the warmest and wettest month. In contrast, within subspecies, we recovered continuous morphological variation with extensive gene flow among populations inhabiting the temperate and sub-tropical forests of southern Africa, despite divergence along the climate axis that is mainly determined by minimum temperature and precipitation of the coldest months. Our results highlight the role of niche divergence as a diversifying force that can promote reproductive isolation in vertebrates. Understanding the factors affecting the process of speciation has been a topic of enthusiastic debate in evolutionary biology for decades e.g., . AlthougRecently, an alternate conceptual approach to the study of speciation has been proposed. It incorporates micro-evolutionary concepts into the framework to address how geography, ecology and genetics interact to restrict gene flow between populations Cercotrichas signata that inhabits forest understorey habitats in southern Africa is one such species. It comprises two subspecies that occupy adjoining ranges: i) the dark olive-brown C. s. signata spans the temperate and subtropical forests of coastal Pondoland and the highlands of Swaziland, Mpumalanga and Limpopo; and ii) the paler, smaller and shorter-billed C. s. tongensis is restricted to the lowland tropical forests of the Maputaland region Species comprising phenotypically different populations occupying distinct habitats, despite an overall apparently contiguous distribution, provide intriguing systems in which to study the underlying population-level mechanisms that promote divergence. The elusive brown scrub-robin In this study, we initially modelled climatic niches and quantified the differences to then test for any relationship with morphological divergence and whether increased differences in climatic niche leads to reduced gene flow. Niche differences can be estimated using GIS data and spatial statistics We demonstrate that our analytic framework provides a means to understand where along the divergence continuum see  brown scThe South African National Parks and the South African Provincial Departments granted research and collecting permits to capture birds using mist-nets and flap traps. The capture and sampling were conducted with approval from the Animal Use committees of the University of California Berkeley, the University of Cape Town, and the South African bird-banding scheme (SAFRING).Characterizing geographic distributions and testing whether climatic factors influence subspecies ranges serve as a proxy to assess the ecological similarities or differences among subspecies.signata and tongensis, we constructed environmental niche models using MaxEnt v.3.3.3. r <0.80. The presence-only point locality data used in these models were primarily obtained from our field expeditions and supplemented with museum specimen records of adult birds as well as ringing records kindly provided by Craig Symes. We modelled the climatic niche supporting the occurrence of the species  and also the two subspecies independently . The model performance was assessed by estimating the “area under the receiver operating curve” – AUC. The predicted probability of occurrence obtained was spatially projected using ArcMap 9.3 In order to assess for equivalency, i.e. overlap, in the climatic niches of Di observed (signata – tongensis). The null distribution for Di was generated by randomly reshuffling the data. All occurrences were pooled regardless of their origin, randomly allocated into two groups (subspecies) with the same number of samples as the original data set and Di null (signata – tongensis) estimated. This procedure was repeated 9999 times to generate the null distribution. The observed test statistic (Di observed) was then compared to the distribution of the simulated values (Di null) to examine whether the observed value was significantly different from zero. The estimated probability of obtaining a result that exceeded the observed value under the null hypotheses was estimated as p = [(number Di null≥Di observed)/total number randomizations]. The same procedure was implemented to estimate niche differentiation between i) contiguous populations across the subspecies boundary: populations from KwaZulu-Natal scarp (KZN) versus populations from the St. Lucia Region (SLR); and ii) populations within subspecies signata: populations from KwaZulu-Natal scarp (KZN) versus populations from Port St. Johns coastal scarp (PSJ). KZN and PSJ are thought to have supported forest remnants during the Last Glacial Maximum . All statistical analyses were performed using R 3.0 Assessing niche overlap as the graphical output of the niche models projected onto geographical space has an inherent problem: spatial autocorrelation in environmental variables. To account for the possible influence of autocorrelation in the GIS data used, while testing for niche equivalency, i.e. whether there is a difference in the climatic niche occupied by the two subspecies, we implemented a multivariate approach as follows. We used a principal components analysis (PCA) to reduce the 19 climatic variables mentioned in the previous section into a small and uncorrelated set of axes. Differentiation in climatic niche was then quantified as the difference of means along each orthogonal PC axis: C. c. signata from ten localities and 26 C. c. tongensis from four localities . Tarsus-length was measured with a digital calliper to an accuracy of 0.1 mm, wing-length  was measured with a wing-rule to an accuracy of 0.5 mm, and body mass was measured with a 50 g Pesola spring balance to an accuracy of 0.5 g.Morphological data were collected from 81 individuals from 14 localities across the range of the species: 55 calities . As samp10 transformed to meet assumptions of normality. We used the residual values from the regression of log-transformed wing on tarsus as a proxy of structural body size. Exploratory scatterplots indicated morphological variation within the range of the species and between sexes (Body mass (g), wing-length (mm) and tarsus-length (mm) of adult birds  were used to quantify morphological variation across the entire range of the species. The sex of the birds was determined using a PCR-based assay en sexes . Therefoen sexes  between Gallus gallus chromosome 1, G. gallus chromosome 3, 26438  and then analyzed on an ABI 3730 automated sequencer. Sequences were edited and aligned using CodonCode Aligner v3.5.2 (CodonCode Corporation 2009) and Geneious Pro v5.4 (Biomatters Ltd 2011). Length polymorphisms were found in two nuclear loci: TGFb2 and 26438. For TGFb2, we chose to remove them from the alignments prior to further analysis. For 26438, because length polymorphism was located at the very end of the fragment we truncate the sequence data at the indel in the forward direction. All sequences have been submitted to GenBank (KF873022–KF873472).Blood or muscle samples collected in the field were used to quantify genetic variation. We extracted genomic DNA from 74 blood/tissue samples using DNeasy kits . We sequenced two mitochondrial protein-coding genes  and the alleles with greatest probability  were used for subsequent analyses θst; a haplotype based statistic similar to Fst) D  D TD; .admixture model with correlated allele frequencies. Because the algorithm implemented in STRUCTURE may have difficulty to detect hierarchical genetic subdivision we implemented the analysis within the subspecies C. c. signata to evaluate the extent of current subdivision. We ran five pseudo-replicates with 106 Markov-Chain-Monte-Carlo iterations following a burn-in of 105. Because the model implemented in STRUCTURE assumes no linkage disequilibrium, it was only run with the nuclear data. To test whether greater differentiation in the mtDNA was coincident with the groups recovered with nuclear data only, we used an analysis of molecular variance  and PSJ (n = 16). Two separate analyses were performed: i) six nuclear loci and mtDNA locus and, ii) only six nuclear loci. After preliminary runs to determine the appropriate range of priors and confirm proper MCMC mixing, the final runs were performed with 30 coupled chains with a geometric heating scheme (g1 = 0.9 and g2 = 0.8) and allowed to continue for at least 3×106 steps. The first 8×105 steps were discarded as the burn-in. Parameter trend plots and ESS were used to assess convergence. Because the three types of marker are characterized by different effective population sizes, we included an inheritance scalar to adjust the parameters in the model: 0.25 for mitochondrial, 0.75 for Z-linked and 1.0 for autosomal loci. Time of divergence was obtained assuming a generation time of one year  we used the coalescent-based The first three principal components accounted for 91% of the variance in the climatic data that characterize the range of the brown scrub-robin: PC1, PC2 and PC3 explained 53%, 26% and 12% , respectsignata was predicted as climatically suitable for tongensis and vice-versa . When comparing the niche differentiation within signata only the Di observed for PC1 was significantly larger than the Di null (p<0.01).The geographical projection of the climatic niches of each subspecies revealed areas of reciprocal suitability, i.e. niche overlap. Some portion of the geographical range of ce-versa . The mod p<0.01;  C1–C3. AF = 8.025, p = 0.006; wing: F = 49.072, p<0.001; tarsus: F = 17.690, p<0.001; size: F = 16.3728, p<0.001). Furthermore, the ANOVA revealed significant differences in all traits between birds from different geographical areas , wing-length  and structural size , but not in tarsus-length .We found sexual dimorphism for all morphological variables quantified  were smaller than birds from the subtropical and temperate portion of the range  and divergence (Kst) for mtDNA and nDNA sequence data are summarized in S varied from 0.001 to 0.01. Genetic differentiation at the mtDNA genes among localities, Kst, was high . For the six nuclear introns, significant differentiation was also recovered: Kst values varied from 21% to 69%. Overall, we found deviations from neutral expectations of evolution for mtDNA  =  −663.5±53.72) with strong geographical affinities. Members of cluster I were all from subtropical and temperate areas (signata) and members of cluster II were all from tropical Maputaland (tongensis) with no evidence of recent introgression having occurred  =  −342.12, Var (LnP) = 13.64 vs. LnP (K = 2) =  −341.86, Var (LnP) = 55.38). Genetic differentiation along the costal transect peaked between populations in KZN and those south of SLR (Kst nuclear = 0.539), and between PSJ and KZN (Kst nuclear = 0.292).The SPB and GARD algorithms did not detect any evidence of recombination events. Thus, all the subsequent analyses were performed using full sequences. The Bayesian clustering analysis of nuclear genotypes revealed two distinct nuclear clusters (LnP (K = 1) =  −1043.84±30.78 occurred . All butCT = 0.962, p<0.001) with only a smaller portion attributed to among populations within subspecies . Because of this strikingly high differentiation between groups living in tropical versus sub-tropical-temperate forests  and the fact that the mtDNA loci examined are protein coding, we also analysed the amino acid profiles. This revealed that of the seven non-synonymous mutations found at ATP synthase subunit 6, five amino acids segregate between the two nuclear genetic clusters and coincide with the currently defined subspecies boundaries , as well as that the estimates of gene flow vary in response to whether or not the mtDNA data was included in the analyses (KZN − SLR = m0.0015 (90% High Posterior Density: 0.0015–1.4535) and SLR – KZN = m0.0015 (90% HPD: 0.0015–0.4275). Note that 0.0015 was the first bin of the prior and therefore corresponds to zero in both directions. When removing the mtDNA from the data set, the estimates of migration were slightly greater . In contrast, migration between populations within subspecies signata was nearly 13 times larger than between subspecies populations  indicating that nuclear gene flow between these two populations occurs in both directions  strong multidimensional differentiation with marked reproductive isolation between signata and tongensis across divergent environments  continuous variation without reproductive isolation in populations of tongensis and signata might have been initiated in different environments with natural selection promoting adaptive divergence and thereby impeding gene flow; or it might have started via the reduction of migration and consequently gene flow between populations in similar environments, after which natural selection increased the frequency of phenotypes with greater fitness. In contrast, the lack of morphological and genetic divergence, and the evidence of extensive gene flow estimated between populations of signata currently occupying central and southern forests that were historically isolated  but not PC1 (gradient of winter mean temperature and precipitation), is associated with a lack of gene flow as well as with morphological divergence further strengthens the previous argument and highlights the role of niche divergence as a diversifying force that has promoted reproductive isolation in this system.Since the early 20The significant difference in structural body size and body mass found along the climatic axis representing the temperate to tropical gradient, with smaller birds occupying warmer climates, suggests these traits have diverged as a consequence of physiological adaptation to varying climates across the species range. Variation in body size can be explained by selection on thermoregulatory capabilities: larger-bodied individuals loose heat more slowly, and thus have greater fitness in cooler climates signata being larger and darker with narrower white markings than tongensis. Therefore, it is possible that a trait  involved in social interactions, such as mate attraction or competition with same-sex conspecifics, increased in frequency as gene flow progressively diminished. This sub-component of natural selection can promote assortative mating which, in turn, may lead to irreversible reproductive isolation. Song is another trait that could have facilitated the process of divergence Morphological divergence can also be explained by factors other than abiotic variables. Divergence can occur between populations, if traits involved in social interactions increase the mating success of some individuals Poynton 1961;  first detongensis and signata is centred on the late Pliocene , which suggests that divergence in the brown scrub-robin was not triggered by the formation of an estuarine barrier. Populations currently inhabiting the scarp forests of KwaZulu-Natal (signata) and the coastal Indian Ocean forest of the St. Lucia region (tongensis) have been following distinct evolutionary trajectories, with no current exchange of migrants between them. In contrast, there is a lack of current population genetic structure and extensive gene flow between populations of signata occupying the coastal scarp forest of the Eastern Cape (PSJ) and the southern coastal and northern interior scarp forests of KwaZulu-Natal (KZN). Furthermore, gene flow predominantly occurs from south to north (KZN – PSJ = m2.782 vs PSJ – KZN = m20.475) suggesting that the brown scrub-robin populations from the temperate forests might have a broader climatic niche and thus be more tolerant of subtropical conditions.Our estimate of the time of divergence between Although it is difficult to demonstrate a causative effect of the present morphological differences observed between the two subspecies as the driver of the divergence process, morphology likely co-varies with causal traits that maintain reproductive isolation. However, only by concentrating future work in the archipelago of small forest patches at the boundary between the two subspecies will we be able to fully understand whether reproductive isolation is a consequence of divergent selection on physiological tolerances and/or sexual selection on plumage, size or song.Gaining a population-level perspective is essential if we are to understand how ecological and micro-evolutionary processes interact to affect the continuous process of species formation. In turn, this basic knowledge about the origin of species is relevant to our understanding of why some areas, such as the understudied southern African Maputaland-Pondoland-Albany region, harbour such high species richness.Figure S1Dimorphism in quantitative traits measured across in the brown scrub-robin range. Triangles represent C. s. tongensis and circles C. s. signata. Sex is colour coded.(TIFF)Click here for additional data file.Figure S2Amino acid changes in the ATP synthase subunit 6, which is encoded in the mtDNA genome. Each individual vertical line represents one individual and the colour code (black vs red) one of the two amino acids observed. E.g., at position 14 all individuals of subspecies tongensis have a Leucine whereas all individuals from signata have a phenylalanine. Five of the seven amino acid changes segregate in concert with morphology, climate and nuclear genotypes. L: Leucine, F: Phenylalanine, M: Methionine, T: Threonine, N: Aspargine, S: Serine, A: Alanine, V: Valine, I: Isoleucine.(TIFF)Click here for additional data file.Table S1Point locality data with details whether morphological and genetic data was collected and the respective samples sizes. Numbering code used in (DOC)Click here for additional data file.Table S2Morphological data: tarsus-length (mm), wing-length  and body mass (g). The sex of all birds was determined using a PCR-based assay. Please, see Methods section for further details.(DOC)Click here for additional data file.Table S3females = 29, Nmales = 52.Effect of geography and sex on morphological variation in the brown scrub-robin as revealed by a two-way ANOVA. N(DOC)Click here for additional data file.File S1Multivariate approach to test niche overlap – R script.(DOC)Click here for additional data file.


Trionyx triunguis populations from the Mediterranean and African continent based on mtDNA D-loop (776 bp) and nine microsatellite loci. A total of 102 polymorphic sites and 13 mtDNA haplotypes were described. Nucleotide diversity and haplotypes diversity were 0.047 and 0.974 respectively. Both mtDNA and nDNA supported the existence of two main management units as the Mediterranean and Africa. Based on the mtDNA results, the Mediterranean can be divided into two subunits; western Turkey and the eastern Mediterranean.We studied the genetic structure of  Trionyx triunguis mainly lives in fresh or brackish water lakes, rivers and estuaries but there are also records of this interesting animal from the marine habitats [Rafetus swinhoei or species of the genus Chitra and Pelochelys can reach lengths of greater than 1 meter [T. triunguis is very broad and in some parts of its range  it can still be found. Like its East Asian counterparts, T. triunguis is a species of conservation concern and its populations have been decimated by habitat destruction and harvesting [The Nile soft shell turtle habitats ,2. Softs 1 meter . Most of 1 meter , and are 1 meter . In thisrvesting –12.T. triunguis includes parts of the Mediterranean and Africa. In the Mediterranean, and perhaps throughout its range, the largest populations are found in Turkey [Trionyx triunguis mostly lives in estuaries although there are records of the species being found far out in the Mediterranean and Aegean Seas, where it is sometimes caught by fishermen, especially during winter months [The current distribution of n Turkey –16. Trior months ,17.T. triunguis within Trionychidae has been determined through the use of molecular and osteological data [et al. showed that T. triunguis is the sister group to a clade of giant trionychids (Pelochelys + Chitra), although this inferred relationship was only well-supported in Bayesian analyses, and the species was relatively distantly related to these other giant softshells [et al. and Gidis et al. reported genetic differences between the African and Mediterranean populations of the species, but additional data on the genetic diversity of this species are necessary to define units for conservation and management [The evolutionary relationship of cal data ,18. Usinftshells . Güçlü enagement ,20.Pelodiscus sinensis [T. triunguis in the Africa and the Mediterranean Basin, providing crucial insights into gene flow and conservation units.In the present study, we describe the genetic diversity among Nile soft-shelled turtle populations using nine microsatellite loci previously isolated from sinensis  and mtDNA total of 102  samples were obtained from 13 different localities in Turkey, Israel, Cote d’Ivoire, Congo and Gabon , Table 1Dogania subplana (AF366350) and Pelodiscus sinensis (AY962573) [2 (Fermantas Inc.), 2.5 mM dNTP, 0.5 mM each primer, 1.0 unit of Taq polymerase (Fermantas Inc.), and 1–2 μL (50 ng DNA) of template DNA. PCR conditions were used 95 °C denature for 1 min, 55 °C anneal for 1 min and 72 °C extension for 1 min for 35 cycles for mitochondrial D-loop. Amplicons were purified using the PCR Purification Kit (Invitrogen Inc.) and were analyzed on an AB3700 or 3730xl automatic sequencer using the amplification primers. Sequences were aligned using BioEdit 7.0.9 [We assessed the nucleotide variation of relatively fast-evolving fragments of mtDNA control region (D-loop) which is the most variable region of the mitochondrial genome in turtles ,23. Totansis AY9673 [25,26it 7.0.9 . Multiplit 7.0.9  using thT. triunguis populations using GenAlEx 6.3 [FST was determined through permutation tests against a null distribution generated from the data in GenAlEx. The number of migrants (Nm) between each population pair was calculated from genetic distances through the equation Nm = (1 − FST)/2FST [Five samples were collected from Anamur (An), 1 from Göksu Delta (Gk), 3 from Kazanlı (Kz) and two from the Seyhan River (Sy) and samples from these localities were grouped according to their geographic proximity as AnGkKzSy (11 samples) population. An analysis of molecular variance (AMOVA)  was perfAlEx 6.3 . StatistST)/2FST .p < 0.05) was taken as evidence of population expansion. ARLEQUIN was also used to calculate Tajima’s D and Fu’s Fs neutrality tests [D may be due to factors such as population expansion, bottlenecks, or heterogeneity of mutation rates [FS may also indicate population expansion [D and Fs values was tested by 1000 randomization replicates. Minimum-evolution (ME) analyses were performed using maximum-likelihood pairwise distance by MEGA 5.0 [We examined lineage history in several ways using the D-loop data. The distribution of pairwise differences among individuals, also known as the mismatch distribution, was calculated using ARLEQUIN 3.0 . The shaty tests ,36. A sion rates ,38. A sixpansion . The sigMEGA 5.0 .TM Software ver. 1.0.).Amplicons were obtained from nine of the 15 microsatellite loci  . One priHo) and expected heterozygosity (He) among populations for each locus were computed using GenAlEx 6.3 [For each locus, allelic frequencies in between populations and pairwise comparison of the population pairs were conducted using a Markov chain method. Linkage disequilibrium was also tested using the Markov chain permutations and Fisher’s exact test. All of these analyses were carried out using GENEPOP 3.3 . AllelicAlEx 6.3 .n < 10) and the number of studied loci was low (n < 20), genetic structures of the populations were determined through FST [FST and pairwise exact tests of genetic differentiation with GenAlEx 6.3 [Nm) among and within groups were calculated from pairwise estimates of FST (Nm = ¼ (1/FST − 1)) [FST calculated in GenAlEx 6.3 [In cases when the sample size was small ) . AnalysiAlEx 6.3 .Evidence for recent population bottlenecks was assessed with Bottleneck 1.2 software . In popuAn assignment test assigns individuals to a population through the genotype formed depending on loci according to Bayesian probability approach  implemenA total of 776 bp were aligned for 52 individuals. Thirteen mitochondrial DNA haplotypes were found . A totalHd) was 0.974 for the overall dataset.Sequence divergences for the mtDNA-D loop were 0.13% between WT and EM, 5.42% between WT and AG, and 5.16% between EM and AG. Nucleotide diversity was 0.047  and haplFST values) (p < 0.001). The AMOVA analysis showed that genetic variation among groups constituted 81% (p < 0.01) of the whole variation, whereas the genetic variation among populations was at 19% (p < 0.01).The most significant genetic difference between groups was calculated between those in AG and WT, whereas the least significant difference was determined between WT and EM  . NeverthThe highest gene flow was between WT and EM while the gene flow between AG and WT was rather limited .D value was found to be positive in all populations although it was not significant (p > 0.05). This shows that populations were not affected by factors such as population growth, selection and bottlenecks in the past. Mismatch analysis rejected the null model of population expansion .Except for the AG, Tajima T. triunguis was basically divided into two main clades, namely the Mediterranean basin and the AG. The Mediterranean clade was divided into two subclades; EM, consisting of Israel, Kazanlı, Anamur and Göksu, and WT consisting of Dalyan and Dalaman. The sequence of Amer and Kumazava from Egypt clustered with the Mediterranean clade [Based on Nei’s unbiased genetic distance for all haplotype pairs, an clade  . Linkage disequilibrium was not determined between two loci of any single population (p > 0.05). Nevertheless, allelic diversity in microsatellite loci occurred non-independently from each other. Significant differences were determined in terms of allele frequencies (p < 0.001) between populations.We analyzed polymorphism across nine loci in 102 individuals from three populations identifying a total of 15 alleles. The number of alleles per locus ranged from two (PT-04) to four (PT-01) although six microsatellites were fixed for one allele. Observed heterozygosity did not show a statistical difference from expected heterozygosity under the Hardy Weinberg equation. Observed heterozygosity range was 0.12 and 0.05, whereas expected heterozygosity was between 0.20 and 0.06. Mean values of FST estimations, significant genetic differences were determined between groups and populations (p < 0.01), 3% was among populations within groups (p > 0.05), and the remaining 51% was within the populations (p < 0.01). There was no measurable genetic differences between AnGkKzSy population and Dalyan and Dalaman populations , whereas a significant difference was determined between AnGkKzSy and Israel populations .According to ulations . Forty-sA UPGMA tree generated based on the genetic distance data between populations  displayeNm < 1). On the other hand, there is more gene flow between WT and EM . As a result of the Sing and Wilcoxon sign-rank tests performed for all groups and populations, no significant heterozygosity excess was determined in loci (p > 0.05). The values calculated under IAM, SMM and TPM models showed that the studied populations of T. triunguis did not suffer a bottleneck in the past. According to assignment test, all individuals in each group were classified correctly . In addition, AG has higher genetic diversity compared to WT and EM based on the number of observed haplotypes and alleles.In contrast to our results, Gidiş  turtles . FurtherT. triunguis populations shows that these populations are at Hardy-Weinberg equilibrium. According to both mtDNA and microsatellite locus analyses, there is significant genetic difference among the three groups of T. triunguis (p < 0.01); nevertheless, the AG was completely isolated from WT and EM. Both mtDNA and microsatellite analyses proved a significant genetic difference among 3 groups (p < 0.01) and AG seems to be isolated from WT and EM. Lower genetic diversity observed in WT and EM compared to those in AG mean that these populations have small population sizes and probably a decrease in diversity has occurred through founder effect. Genetic diversity in populations and, as a result, allelic richness, has a positive relationship with population size [Despite the fact that we analyzed nine loci, only three were useful for population level analysis. The number of these loci is limited for population genetic analysis. The most important feature in determining the genetic diversity of a species or a population is the amount of heterozygosity in that species or population . The cloion size . The AG,Nm = 0.3), there is a gene flow between these groups for microsatellite loci (Nm = 1.8). Such high gene flow observed for microsatellite loci is a function of the gene flow between AnGkKzSy and Dalyan and Dalaman populations . The limited gene flow in terms of mtDNA possibly resulted from nesting site fidelity of the females as in the case of marine turtles [According to mtDNA data, although gene flow between WT and EM is seems to be rather limited  gene flow estimates based on both mtDNA and microsatellite loci is negligibly low between AG and other populations, (ii) there are haplotypes and alleles unique to this population, and (iii) there are fixed differences between groups showing that the AG occupied the Mediterranean Basin in the past of their evolutionary history. This differentiation determined for  regions .et al. indicated that there could be gene flow between the Nile River and Middle African populations because of the connection between the Nile and Congo rivers [Gidiş o rivers . But, acet al. suggested that there are no significant differences between the Mediterranean and African populations and therefore they need to consider combining them into a single conservation unit [T. triunguis. Furthermore, based on mtDNA the Mediterranean basin also can be divided in two subMUs (WT and EM). The AG has the highest genetic diversity among all and constitutes the foundation for the evolutionary future of T. triunguis.Classification of a population as Evolutionarily Significant Units (ESU) or Management Units (MU) can be possible by determination of the genetic structure of that population ,56. Locaion unit . HoweverTrionyx triunguis has been declining due to habitat degradation and fragmentation. For instance, until the early 1990s, T. triunguis had a robust population in terms of number of individuals in Nahal Alexander, a population of Israel from the EM. However, after the sea infiltrated the land due to storms, habitat changes occurred and the population began to decrease rapidly [In recent years the populations of  rapidly . Such haTrionyx triunguis and provided inventory genetic data for future population restoration for the recovery of this declining species.We defined two main MUs for


Glis glis), a small arboreal mammal that is distributed throughout Central Europe, where forests are mostly fragmented at different spatial scales. The aim of this study was to investigate the effect of habitat fragmentation on genetic population structures using the example of edible dormouse populations inhabiting forest fragments in south western Germany. We genotyped 380 adult individuals captured between 2001 and 2009 in four different forest fragments and one large continuous forest using 14 species-specific microsatellites. We hypothesised, that populations in small forest patches have a lower genetic diversity and are more isolated compared to populations living in continuous forests. In accordance with our expectations we found that dormice inhabiting forest fragments were isolated from each other. Furthermore, their genetic population structure was more unstable over the study period than in the large continuous forest. Even though we could not detect lower genetic variability within individuals inhabiting forest fragments, strong genetic isolation and an overall high risk to mate with close relatives might be precursors to a reduced genetic variability and the onset of inbreeding depression. Results of this study highlight that connectivity among habitat fragments can already be strongly hampered before genetic erosion within small and isolated populations becomes evident.Habitat loss and fragmentation represent the most serious extinction threats for many species and have been demonstrated to be especially detrimental for mammals. Particularly, highly specialized species with low dispersal abilities will encounter a high risk of extinction in fragmented landscapes. Here we studied the edible dormouse ( Pseudocheirus peregrinus) and Australian squirrel gliders (Petaurus norfolcensis), were shown to display lower levels of allelic richness and observed heterozygosity in small isolated populations compared to populations inhabiting larger habitat patches Habitat loss, degradation and fragmentation represent the most serious extinction threats for mammals, affecting 40% of the species assessed Glis glis; rodentia) are extreme habitat specialists, occurring preferentially in deciduous mixed forests dominated by European beech (Fagus sylvatica) and oaks (Quercus spec.) Edible dormice  In accordance with patterns described by the island rule for small mammals, edible dormice inhabiting forest fragments were shown to be significantly larger and heavier than individuals in the continuous forest and occurred also in higher numbers Edible dormice provide an excellent model to study effects of forest fragmentation and potential isolation effects on the genetic and demographic structure in animal populations. They are extreme habitat specialist, have low dispersal abilities and were shown to be particularly sensitive to habitat fragmentation. We here characterised the genetic structure and variability of dormice inhabiting a large continuous forest and several comparatively small forest fragments. Results of our study highlight that connectivity among habitat fragments can already be strongly hampered before genetic erosion within small and isolated populations becomes evident.G. glis) is the largest European dormouse with a body mass between 80 and 120 g. In Central Europe, this nocturnal rodent occurs preferentially in deciduous mixed forests dominated by the European beech (F. sylvatica) F. sylvatica) and oaks (Quercus spec.). Reproductive activity seems to be energetically costly, not only for females but also for male edible dormice Regulation of Wildlife Conservation in the Federal Republic of Germany). However, in the state of Baden-Württemberg, they are not listed as endangered The arboreal edible dormouse (F. sylvatica) dominated mixed deciduous forests in south western Germany (cont and HLcont) located 0.7 km apart from each other  with an extension of about 300 m. At the eastern border of this monoculture runs a railway trail from north to south, encompassed by pasture (width 15 to 60 m). In the north the forest adjoins a pasture with 35 m width. In the south the nearest forest fragment is in about 500 m distance  between 2001 and 2009 . Upon fiEdible dormice show high site fidelity 2, 0.40 mM of each dNTP and 0.5 U Taq DNA Polymerase (Qiagen) and 1 µl template using a Gradient Cycler PTC-200 . A touchdown thermal cycling programme encompassing a 10°C span of annealing temperatures ranging between 60°C and 50°C was used for amplification. Following an initial denaturation step of 95°C for 3 min, cycling parameters were 20 cycles at 95°C for 30 s, 60°C annealing temperature (decreased 0.5°C per cycle) for 30 s and 72°C for 40 s and 15 cycles of 95°C for 30 s, 50°C for 30 s and 72°C for 40 s and a final extension step of 72°C for 5 min. All PCR-products were run on an ABI 310 sequencer  and their fragment length was determined with the programme Genescan v3.7 (Applied Biosystems 2001).DNA was extracted with a QIAamp Kit , following the manufacturer's instructions. For genotyping we used a total of 14 microsatellites. We used 11 microsatellites . We further tested whether the genetic population structures at the location Ulm can be explained by isolation by distance (MANTEL test). Beyond that effects of forest fragmentation on the population structure are of interest. The intention of our study was to show that populations living in forest fragments are strictly isolated having each their characteristic genetic population background whereas populations living in continuous forests share a common gene pool (STRUCTURE).Third, we graphically demonstrate our hitherto existing statistical results by using traditional cluster analyses, i.e. we apply a simple cluster procedure to group populations (UPGMA) with respect to the genetic distances between pairs of populations (NEI's distance). This graphically shows the temporal and spatial similarities within populations across years and differences among populations within different locations.Genepop version 4.1.3 The software programme Genepop was also used to test for deviations of observed genotype frequencies from Hardy-Weinberg proportion (HWE), the genotypic linkage disequilibrium between pairs of loci in the samples of each year, and finally the allelic and genotypic homogeneity among populations, and within and among populations sampled in different years. The homogeneity tests performed by Genepop are exact tests and in case of large sample sizes combined with Monte-Carlo-procedures. The basic level of significance α was set to 0.05 and, in the case of multiple testing we applied a sequential Bonferroni correction compensating for the risk of an inflating type 1 error.Derived from the degree of homozygosity we also obtained the effective number of alleles. Fstat version 2.8 cont: N = 11; HLcont: N = 9; Ulm – BGfrag: N = 10; BSfrag: N = 12; SFfrag: N = 14; JHfrag: N = 11). This measure takes into account sample size The allelic richness (AR) in populations was estimated using the programme The genotypic patterns of locus Glis483 indicated sex-linked inheritance as in males only homozygotes were observed, whereas in females also heterozygotes could be found. Consequently, in cases of testing Hardy-Weinberg proportion male genotypes at this locus were not considered. The appearance of stutter bands hindered a reliable fragment length analysis of the di-nucleotide microsatellite 11pilch and, therefore, we used a 4bp-allelic raster to minimize the misclassification of genotypes .cont and HLcont; Ulm: BGfrag, BSfrag, SFfrag and JHfrag), among yearly samples of populations, among individuals within populations, and within individuals.We considered different genetic sources that can explain the genetic variation of populations and which might be different in the populations of the continuous forest and the forest fragments, i.e. genetic differences between locations (Tübingen and Ulm), among populations of the two locations , the subprocedure MANTEL given in the programme Genepop was then used to test whether the genetic population structures in the location Ulm can be explained by an effect of isolation by distance.The procedure AMOVA was applied separately to the data sets from the two locations Tübingen and Ulm. Using estimates of the genetic differentiation between each pair of populations  cont and HLcont, loci Glis426 and 11pilch in BSfrag, locus Glis228 in SFfrag, and the loci Glis196 and Glis228 in JHfrag).Considering all loci, the number of alleles ranges from 2 to 8 in our sample over all years . The numHe≤0.420; 0.351≤Ho≤0.408; The mean expected and observed degrees of heterozygosity were similar across all forest fragments . As samples from population HLcont: 2001 versus 2008 or 2009, p<0.001; BGfrag and SFfrag: 2007 versus 2009, p « 0.001). The analyses of genotype variation between succeeding years yielded similar results . Three pairs of populations from different years showed significant differences after Bonferroni corrections .In all populations the allele frequencies were homogeneous in two successive years  but populations were genetically heterogeneous over longer time periods (HEcont and HLcont) were homogeneous in their allelic and genotypic structure across all loci in 2002 and 2003 (p≥0.175), and only in 2001 significant differences could be observed between the two study sites of the continuous forest (p « 0.001). In contrast, the fragmented and isolated populations around Ulm differed in their allelic and genotypic composition in all years between 2007 and 2009 (p<0.001).Differences in the genetic variation among sites within the two locations Tübingen and Ulm were found. Dormice inhabiting the two sites in the continuous woodland . 13.05% of total genetic variation could be attributed to genetic differences between the two locations Tübingen and Ulm. The genetic differences among populations among years within one of the two locations contributed on average 6.41%. Finally, 4% of the total variation occurs by genetic differences among individuals within populations.We again removed the population of HLus years , the geoFCT = 0.015, p = 0.005; and Ulm: FCT = 0.182, p<0.001). However, differentiation within the fragmented populations was 10 times higher than in the continuous woodland. Differences between both locations became also apparent in the parameter that reflects the stability of populations across years. The structures within the continuous forest at both sites HEcont and HLcont remained stable across 9 and 3 years, respectively  whereas significant differences occurred in the populations of the forest fragments within 4 and 3 years, respectively . An effect of fragmentation might also be mirrored in the parameters measuring deviation from a random mating system: There was a significant higher genetic variation among individuals within populations living in the forest fragments  than in the populations from the continuous forest .The large differences in the genetic structure between dormouse populations inhabiting the continuous forest and the forest fragments were best seen through the comparison of the results from the AMOVAs done separately for both locations . The genFinally, the genetic differences among local populations in Ulm cannot be explained by an effect of isolation by distance .Structure separates individuals from the study sites near Ulm from those close to Tübingen  formed a cluster of genetically highly similar populations over a period of 9 years  were more distantly connected than the dormice inhabiting the two study sites in the continuous forest area (HEcont and HLcont) but again the yearly samples grouped tightly together for each population. The bootstrapping procedure clearly supported the grouping of the Ulm fragments into distinct populations whereas the patterns of the populations from Tübingen did not indicate strong isolation of the two subpopulations (HEcont and HLcont) over the total study period .Dormice inhabiting the continuous forest (HE 9 years . The difTetrao tetrix) Cricetus cricetus) Pseudocheirus peregrinus) and Australian squirrel gliders (Petaurus norfolcensis), which display lower levels of allelic richness and observed heterozygosity in small isolated populations than in populations inhabiting larger habitat patches The arboreal edible dormouse has low dispersal abilities e≤0.420 and Ho≤0.408, F. sylvatica) and oaks (Quercus spec.). It can therefore be assumed that during the glacial periods the geographical distribution of edible dormice with its low dispersal ability and strong habitat preference was restricted to the retreats of beeches and oaks in southern Europe. During the middle warm period , however, dormice extended their distribution range throughout Central Europe following the expansion of beech dominated forests Genetic variability in our study was very low in all dormouse populations (HSciurus vulgaris) found high levels of gene flow across a comparably strongly fragmented landscape S. vulgaris was shown to be not disrupted by fragmentation, the red squirrel was rarely found in small forest fragments and is therefore believed to be especially sensitive to forest fragmentation Within Europe genetic studies on arboreal mammals investigating the connectivity among fragmented forest patches are scarce. One study on the red squirrel  than those in the continuous forest , which can again support our assumption of a low number of immigrating individuals into the small forest fragments of the location Ulm.Genetic drift in small and isolated populations may lead to genetic differentiation of local populations which has been found for example in the common hamster in the Netherlands and Belgium Edible dormice inhabiting forests fragments represent genetically isolated populations. Our finding of no significant differences in genetic diversity between forest fragments and the large continuous populations can possibly be explained by comparatively high population densities within the small forest fragments. However, we consider degrees of heterozygosity, allele numbers, and allelic richness as diversity measures to be rather insensitive when genetic variability of a species is generally low as shown for the edible dormouse. Nevertheless genetic isolation might be a precursor to genetic erosion and inbreeding depression to occur in the near future, which may limit or even prevent adaptive responses to environmental changes.File S1Tables S1–S5.(RTF)Click here for additional data file.Figure S1frag sampled from 2006 to 2009; BSfrag and SFfrag sampled from 2007 to 2009; JHfrag sampled in 2009) in south western Germany to different source populations assuming different numbers of potential source populations .Assignment of dormouse individuals of four sites at the location Ulm (BG Different colours indicate the possibility of different source populations. Colours in each column show the likelihood (%) to which source population an individual can be assigned.(TIF)Click here for additional data file.Figure S2contAssignment of dormouse individuals of two sites at the location Tübingen (HEcont) in south western Germany to different source populations.and HL Samples from the population at site HEcont was taken from 2001 to 2009, and from HLcont from 2001 to 2003. For the assignment procedure we assumed different numbers of potential source populations . Different colours indicate the possibility of different source populations. Colours in each column show the likelihood (%) to which source population an individual can be assigned.(TIF)Click here for additional data file.


Macrolophus pygmaeus (Rambur) (Hemiptera: Miridae) is widely distributed throughout the Palaearctic region. The aim was to explain the current geographic distribution of the species by investigating its genetic population structure. Samples of M. pygmaeus were collected in 15 localities through its range of distribution. A sample from a commercial producer was also analyzed. A total of 367 M. pygmaeus were genotyped for nine microsatellite loci. Isolation by distance was tested by Mantel's test. The molecular structure of M. pygmaeus populations was inferred by UPGMA, AMOVA, Principal component and Bayesian analyses. The average number of alleles per locus per population was 5.5 (range: 3.1–7.8). Istanbul (Turkey) and Nimes (France) had the lowest (0.291) and the highest (0.626) expected heterozygosity (He), respectively. There was an increase in He from the Canary Islands to Nimes, and a progressive decrease thereafter. A significant negative correlation was found between allelic richness and He, and the distance of each population to the easternmost locality (Canary Islands). Significant linkage disequilibrium was observed in the populations from Turkey. FST (0.004–0.334) indicated a high population differentiation, with isolation by distance supported by a high correlation. Bayesian analyses, PCA, and UPGMA pointed to three main clusters: (1) Greece and Turkey, (2) Italy and France, and (3) southern Iberia and the Canary Islands. The recent evolutionary history of M. pygmaeus is inferred from the data as follows: (1) the reduction in the geographic distribution of the species to the Iberian, Italian, and Balkan peninsulas, and possibly southern France, during glaciations and re-colonization of northern Europe from its southern refuges; (2) the maintenance of high diversity in Iberia and Italy (and possibly southern France) during contraction periods, and bottlenecks in the Balkans; (3) introgression of the Italian–French lineage in northern Spain, naturally or through trade. Macrolophus pygmaeus (Rambur) (Hemiptera: Miridae) is a zoophytophagous mirid reported on several plant species; many of them belonging to the Solanaceae, Asteracea, and Lamiaceae families , and Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) (Myzus persicae (Sulzer) (Hemiptera: Aphidae) (Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)  , aphids Aphidae) , and Lepchiidae) . In Eurois mirid . In areaMacrolophus pygmaeus has been the object of attention of many works from an agricultural and ecological point of view. However, the study of the population structure of the species was never addressed, because no suitable markers were available until the recent development of microsatellite markers by Sanchez, La-Spina, and Perera  (Thaumetopoea pityocampa Den. & Schiff. (Phlebotomus papatasi (Scopoli) (Bactrocera oleae (Gmelin) (Cicada spp. (Hemiptera: Cicadidae)(M. pygmaeus populations throughout much of its geographic distribution was explored using a set of nine microsatellites markers. The aim of this study was to investigate the population structure of M. pygmaeus and to understand the factors that may have contributed to shaping it.achaise) , Thaumet Schiff. , PhlebotScopoli) , Bactroc(Gmelin) , Cicada icadidae), among micadidae), but thiM. pygmaeus were used in this study, averaging 23 individuals per populations (range: 9–47) (Solanum lycopersicum L. (Solanaceae). The sample from England was collected in a small domestic tomato greenhouse with wide openings, which allow the free movements of insects between the exterior and interior of the greenhouse and where no M. pygmaeus releases were effectuated, thus it was considered as a native population.A total of 367 e: 9–47) . Adults e: 9–47)  and molee: 9–47) . Adults e: 9–47) . A totale: 9–47)  and Fig.M. pygmaeus preserved in ethanol were vacuum-dried for 20 min and ground individually in 1.5-mL microcentrifuge tubes in 350 μL of homogenization buffer (Buffer CTL) followed by addition of 25 μL Proteinase K (20 mg/mL) to the homogenates. The samples were mixed briefly and incubated overnight at 37°C. A 24:1 mixture of chloroform: isoamyl alcohol was added to the homogenate, vortexed to mix, and centrifuged at 10,000 ×g for 2 min at room temperature. The supernatant of each sample was transferred to a new 1.5-μL microcentrifuge tube and mixed with one volume of Buffer CBL and 2 μL of RNAase solution followed by 10 min incubation at 70°C. At the end of the incubation, one volume of 100% ethanol was added to each sample, mixed, and transferred to HiBind® DNA Mini columns and centrifuged at 10,000 ×g for 1 min. DNA bound to columns was washed once with 500 μL of HB buffer and twice with 700 μL of DNA wash buffer followed by centrifugation step at 15,000 ×g for 2 min to remove residual wash solution. DNA was eluted by adding 100 μL of the Elution Buffer (preheated to 70°C) directly onto the HiBind® column matrix, and centrifugation at 10,000 ×g for 1 min after 2 min incubation at room temperature.DNA was extracted using the E.Z.N.A insect DNA Isolation Kit  following manufacturer's instructions. Briefly, 2, 0.01% of Tween and 0.5 U of DNA polymerase , in a total volume of 20 μL. Forward primers were 5' labeled with 6-FAM, VIC, NED, or PET dyes . PCR amplifications were carried out with an Eppendorf Mastercycler EPgradient  under the following conditions: 2 min at 94°C followed by 36 cycles of 15 sec at 94°C, 30 sec at 61°C, and 30 sec at 72°C, and then a 10 min incubation at 72°C. The PCR products were run as 1:50 dilution on an AB3730 DNA Analyzer (Applied Biosystems) with ROX-labeled size standard (Applied Biosystems). Fragments were detected with Peak Scanner™ software v1.0 (Applied Biosystems) and verified manually. Three positive and three negative controls were included on each 96-well PCR plate and some individuals from each population were scored twice.The nine microsatellites makers Mp13, Mp24, Mp26, Mp27, Mp29, Mp33, Mp34, Mp42, and Mp54 developed by Sanchez, La-Spina, and Perera were used in the genetic analyses  and expected (He) heterozigosity were calculated using the FSTAT 2.9.3.2 software  was analyzed by the Pearson's correlation test; the sample from the commercial producer was not included in the analyses. Deviation from Hardy–Weinberg equilibrium (HWE), fixation index (FIS) values, and Genotypic linkage disequilibrium (LD) was calculated for each population using FSTAT (Dc)  correction method   using FSTAT (Dc) ; Dc is c markers . Besides alleles . An UPGM alleles . The con alleles . The levsoftware ; probabisoftware . The fren method . Pairwise-Turkey , Table 1ST/(1−FST) and the geographic distances was calculated with the Mantel's test using the R software   .r = −0.661, t = −3.17, df = 12, P = 0.007), once Ten-S was excluded. The maximum number of private alleles was found at Val-S (8) and Cas-P (6), followed by Cay-T (4), Por-P (3), Kal-T (3); the remaining of the localities had less than 2 private alleles , two and four individuals shared two genotypes; and in the population from Istanbul (Turkey), two genotypes were found twice. The average number of alleles per locus per population was 5.5, ranging from 3.1 to 7.8 alleles per locus. The effective number of alleles ranged from 1.4 to 3.8 , and allelic richness from 2.0 to 3.6  . A signi alleles .Ho was 0.467, ranging from 0.237 in Ist-T to 0.567 in Arg-S. The average He was 0.539, ranging from 0.291 to 0.626 in Ist-T and Arg-S, respectively . Heterozygosity deficit, with significant departure from HWE, after the sequential Bonferroni correction, was observed at Mp24 in the Ben-S and Kal-G populations, Mp26 (Por-P), Mp34 , and Mp54 (Cay-T) (The average ectively . There w (Cay-T) . The aveST values estimated using the ENA and the conventional method were very close in most of the cases and, although FST values were generally a little higher than FST-ENA, there was no increase in the bias at high FST values  and southern Spain  , between the two populations from Portugal (0.002), and between populations in southern Spain (0.004) ; the highest FST values in the Iberian Peninsula were found between the Arg-S and the rest of the populations , and the geographic distances . In the AMOVA analysis, most of the genetic variations were due to differences between individuals within populations (84.18%)  . The low (0.004) . The aveulations . PairwisT values . The FSTulations . The pope others . Pairwisulations . Signifiulations . The hyp(84.18%) . HoweverIn the PCA, the first two axes explained 31.5 and 22.9% of the variance in the experimental data, respectively. Populations were clustered into groups based on geographic proximity, except for Col-E, which fell in between groups of the Iberian and the Italian–French populations . The axiad hoc statistic ΔK reaching its maximum at K = 3. According to these analyses, the population of M. pygmaeus could be split in three main clusters. M. pygmaeus individual. Moving from east to west, the following clusters may be observed in Macrolophus pygmaeus had high average coefficients of ancestry (0.757–0.880) in cluster 3, with a high percentage of the individuals (81–94) being assigned to this cluster  in Italy showed a shared ancestry with the Italian–French and Iberian populations, with some of the individuals from Sanda assigned to the Iberian cluster  (Hemiptera: Miridae)  (Stenotus rubrovittatus (Matsumura) (Hemiptera: Miridae)  (Lygus heperus Knight (Hemiptera: Miridae) had similar numbers  than M. pygmaeus  is also symptoms of a bottleneck. Cicada orni L. (Hemiptera: Cicadidae) in the Aegean islands compared with mainland Greek was possibly due to a reduced gene flow and bottlenecks during the process of colonization of the islands. When just a small numbers of individuals takes part in the colonization of new areas, the genetic variability of the founder population is expected to be lower than the original one   and Stenr locus) . Lygus hpygmaeus . Signifipygmaeus . The varpygmaeus . In the the loci , and in inal one .M. pygmaeus: one cluster including the populations from Greece and Turkey; a second cluster including the populations from Italy and France; and a third cluster with most of the populations from the Iberian Peninsula and the Canary Islands. The consistency of nodes for the three main clusters was supported by acceptable bootstrap values in the UPGMA analyses; the ad hoc statistic (ΔK), used to determine the true number of populations using the software STRUCTURE, reached its maximum value at K = 3. Structure is not considered as suitable to describe relationships among population, but it produces the appropriate number of evolutionary cluster under many circumstances  and western populations  . This high degree of population differentiation in the main peninsulas could be explained by the isolation of M. pygmaeus in these areas during long periods of time. Finding the highest number of private alleles in southern Iberia and Turkey is in agreement with the hypothesis of the old colonization of these two areas. Many species of animals and plants have evolved into different genetic lineages in the three Mediterranean peninsulas during periods of isolations in the glaciations /Melanargia lachesis (Hübner) (Satyrinae: Lepidoptera) sibling species complex represented three major genetic lineages, each originating from one of the three major refugial areas in southern Europe. ST values = 0.060) in the widely distributed and abundant butterfly Polyommatus coridon (Poda) (Lepidoptera: Lycaenidae) was best explained by the long disjunction in two ice-age refugia of the Adriato-Mediterranean  and Ponto-Mediterranean . In the same way, Maniola jurtina (L.)  shows two major genetic lineages: the Atlantico-Mediterranean and the Adriatic-Pontic-Mediterranean subpopulations , Central Mediterranean , and Eastern Mediterranean (Cyprus)   (Orthoptera: Acrididae) (Tomicus destruens Woll. (Coleoptera: Scolytinae)  indicate a moderate population differentiation in M. pygmaeus between southern Iberia and the Canary Islands; the lack of significance and high variance in FST values between some populations in southern Iberia and the Canary Islands could be due to the low number of individuals in the Canary Island sample. The connection of M. pygmaeus subpopulations between the Iberian Peninsula and the Canary Islands was probably facilitated by the reduction in the Strait of Gibraltar and by the stepping-stone corridor of islands between the Canaries, Madeira, and the Iberian Peninsula in the interglacial periods during the Pleistocene  between the populations of M. pygmaeus in northern Spain (Argentona) and the Liguria coast in Italy were low and not significantly different from zero, which indicates null or low population differentiation between these two geographic areas. These two populations were also included in the same cluster in the UPGMA analysis. In contrast, FST values (0.099) between northern Spain and southern France indicated a moderate differentiation in M. pygmaeus populations. These results are intriguing because a higher similarity would be expected between population in northern Spain and southern France due to their geographic proximity. If these were really what happened naturally, we would have to admit that the species is able to disperse through vast open seas, through a hypothetical bridge between northern Spain, Corsica or Sardinia, and the Liguria coast  and northern Spain  was lower than that between southern and northern populations in Spain . If these results were to be explained by physical barriers interfering with the movement of the species, we would have to admit that the mountains in central Spain are a stronger physical barrier for the dispersal of the species than the Alps and the Pyrenees put together. The high degree of similarity between population of M. pygmaeus in northern Spain and Italy could be explained by: (1) selective environmental factors ; (2) the ad-mixture of wild population and commercial strains native to the Italian peninsula in northern Spain. B. oleae in the Mediterranean basin. Besides, the possibility of human and environmental factors interfering with the distribution of populations in M. pygmaeus the high correlation between genetic differences among populations, and geographic distances also indicates a contribution of geographic distance to the genetic structure of M. pygmaeus populations. B. oleae.Bayesian analyses, PCA, and UPGMA analyses indicates the existence of three main population clusters in mstances . AMOVA aciations . Habel eciations  concludeulations . Three s(Cyprus) . Zygouri(Cyprus)  found thagaceae) . In otherididae)  and the lytinae) , 2009, sistocene . Habel eistocene  reportedia coast , which sM. pygmaeus as follows: (1) the reduction in the geographic distribution of the species to the Iberian, Italian, and Balkan peninsulas during the Pleistocene glaciations; the survival of the species in southern France is not excluded, and M. pygmaeus quite likely also survived in northern Africa. Abies alba Mill. (Pinaceae). (2) The maintenance of high populations and diversity of M. pygmaeus in Iberia, Italy, and possibly southern France, in the periods of contraction, with a reduction in population size and bottlenecks in the Balkans. (3) The introgression of the Italian–French lineage in northern Spain , and the likely expansion of the species from the Balkans to Asia Minor, given the similarities of subpopulations and the lower diversity in the latter area. Based on the genetic similarities of the British, the Iberian, and the Italian–French populations to northern European populations, it is possible that M. pygmaeus spread to northern Europe from its southern refuges in Iberia, Italy, and likely from the south of France.Based on the results of this study, we predict the recent history of M. pygmaeus populations. The use of microsatellite markers may have a very practical approach in biological control, especially in conservation programs, as it would allow the identification of the source of the individuals colonizing crops. Microsatellite marker may also help to understand the introgression of commercial strains into wild populations. This study gives for the first time insight into the population structure of a mirid species in the Palaearctic region, which may also help to explain the current geographic distribution and speciation of other plant bugs in the area. The findings of this study provide a better understanding of the evolution and ecology of M. pygmaeus and could be used to study other mirid species with a similar biology. Divergences in ecological traits  in M. pygmaeus subpopulations in the different geographic areas may also produce diverse outcomes in population dynamics and when using the species for pest control. More populations have to be analyzed to determine the population structure of M. pygmaeus in middle and northern Europe, and to understand the role of northern Africa as a refuge for the species during the glaciations and its connections with other geographic areas in the Middle East, Asia Minor, and the Balkans.This study proves that microsatellite could be used as powerful markers to determine the genetic structure of