Populus davidiana-rotundifolia complex. Bayesian clustering, analysis of molecular variance (AMOVA), Principle Coordinate Analysis (PCoA), ecological niche modeling (ENM), and gene flow (migrants per generation), were employed to detect and test genetic clustering, morphological and habitat differentiation, and gene flow between/among putative species. The nSSR data and ENM suggested that there are two separately evolving meta-population lineages that correspond to P. davidiana (pd) and P. rotundifolia (pr). Furthermore, several lines of evidence supported a subdivision of P. davidiana into Northeastern (NEC) and Central-North (CNC) groups, yet they are still functioning as one species. CpDNA data revealed that five haplotype clades formed a pattern of , but most haplotypes are species-specific. Meanwhile, PCA based on morphology suggested a closer relationship between the CNC group (P. davidiana) and P. rontundifolia. Discrepancy of nSSR and ENM vs. cpDNA and morphology could have reflected a complex lineage divergence and convergence history. P. davidiana and P. rotundifolia can be regarded as a recently diverged species pair that experienced parapatric speciation due to ecological differentiation in the face of gene flow. Our findings highlight the importance of integrative surveys at population level, as we have undertaken, is an important approach to detect the boundary of a group of species that have experienced complex evolutionary history.Species delimitation in tree species is notoriously challenging due to shared polymorphisms among species. An integrative survey that considers multiple operational criteria is a possible solution, and we aimed to test it in a species complex of aspens in China. Genetic [four chloroplast DNA (cpDNA) fragments and 14 nuclear microsatellite loci (nSSR)] and morphological variations were collected for 76 populations and 53 populations, respectively, covering the major geographic distribution of the  Species is a fundamental unit of biology, but there has been much debate about how to define species  is widely distributed in the Northern Hemisphere , amplified fragment-length polymorphisms (AFLPs), chloroplast DNA fragments, and mitochondrial DNA fragments  regions and 14 nuclear microsatellite loci (nSSR) for 375 individuals from 76 populations, and conducted morphometric analyses of leaf traits for representative populations across the distribution range of P. davidiana and P. rotundifolia. Subsequently, Bayesian clustering of nSSR genotypes were adopted to differentiate separate evolutionary lineages, Principal Component Analysis (PCA) was used to examine morphological variation across lineages, a maximum likelihood model (MIGRATE) was employed to assess gene flow between/among lineages, and ecological niche modeling was conducted to quantify niche differentiation between/among lineages. We aimed to address the following questions: (1) How many species are there in the P. davidiana-rotundifolia complex using an integrative survey, e.g., genetic variation, morphological variation and ecological divergence? (2) What lineage separation history has the species complex experienced?In the current study, we sought to identify independent evolutionary lineages within the Populus davidiana-rotundifolia complex were sampled throughout its geographical distribution in China. We sampled a total of 375 trees from 76 populations. From 3 to 5 trees at least 100 m apart in each population, leaf samples were taken and dried immediately in silica gel for DNA extraction. No population was encountered that appeared to contain both P. davidiana and P. rotundifolia. Latitude, longitude, and altitude for each sampled population were recorded using an Etrex GIS monitor  program  method  fragments: http://www.fieldgenetics.com). Two SSR loci that showed high null allele frequencies  steps. The optimal value of K was determined using the method of Pritchard et al. , effective number of alleles (Ae), observed heterozygosity (Ho), expected heterozygosity (He), Shannon's information index (I) , using STRUCTURE, we conducted a series analyses. Genetic diversity indices were estimated in GenAlEx version 6.5  as implemented in ARLEQUIN version 3.0 . Haplotypes and variable sites were identified in DnaSP v5  and nucleotide diversity (πs) at the species and the population level respectively using DNASP v5. Furthermore, using 1,000 permutations within PERMUT (available at: http://www.pierroton.inra.fr/genetics/labo/Software/PermutCpSSR) we estimated the population differentiation coefficients GST and NST, the total genetic diversity (HT), and average genetic diversity within population (HS). A significantly larger NST than GST implies the presence of significant phylogeographic structure . Historical gene flow among different evolutionary lineages and range sectors of the P. davidiana-rotundifolia complex was assessed using the software package MIGRATE version 3.2.6  and mutation-scaled migration rates (M = m/μ), under an assumption that θ is variable between/among populations and M is symmetric between any pair of populations. We adopted the mutation rate of 10−3 per gamete per generation in Populus  and mutation-scaled migration (M = m/μ) with ranges  and , respectively. The initial 100,000 steps were discarded as burn-in, and followed by two long chains of 10,000,000 steps, with sampling every 100 steps, under a constant mutation model. After checking for data convergence from two parallel runs, we estimated for each parameter the mode, mean, median, and 95% highest posterior density (HPD) values. The MIGRATE analyses were evaluated based on effective sampling size as well as the posterior distribution of each parameter.Based on nSSR variation, cpDNA variation, and previous phylogeographic hypotheses  to predict their potential distribution at present, during the Middle Holocene  and the last glacial maximum . To model the ecological niches of each evolutionary lineage, the maximum entropy machine-learning algorithm was implemented in MAXENT v3.3.3 software package  were downloaded from WorldClim database  can quantify the ability of the model to discriminate between sites with or without the presence of the species in question  in ENMTOOLS version 1.3  to 1 . We then performed an identity test with 100 replicates to estimate the similarity of the ENMs of the identified evolutionary lineages. The observed niche overlaps were compared with the null distribution, and tested for significance; histograms were drawn using R 2.13 (http://www.r-project.org/).To measure niche differences between evolutionary lineages or range sectors, we calculated Schoener's P. rotundifolia; 30 populations, 134 specimens for P. davidiana; Figure http://www.flywings.org.uk/MorphoJ_page.htm; Klingenberg, Finally, to test whether or not the differentiation of morphological traits corroborates with genetic divergence, we examined a set of morphological traits of leaves and analyzed their pattern of variation. We took images from 252 representative herbarium specimens, gathered from 53 of the sampled populations during fieldwork  was 34.408, effective allele number (Ae) per locus was 1.990, observed heterozygosity (Ho) was 0.390, and expected heterozygosity (He) was 0.376 . The log-likelihood value reached a plateau after K = 2, although it increased gradually as K raised from 2 to 8; meanwhile, the delta K had a single peak value at K = 2 . These results suggested that the hierarchical population structure is credible, as geographic isolation may have contributed to differentiation between lineages but not within them.Mantel tests revealed a significant correlation between geographical distance and genetic differentiation across the At the same time, AMOVA analyses revealed that 15.58% of genetic variation was attributed to genetic differentiation between the two groups , 17.47% was due to genetic differentiation among populations within groups, and 66.95% was ascribed to genetic differentiation between individuals within populations , of which four H13–H16 were present in P. davidiana's CNC range sector, whereas H17 was only in the westernmost edge of the NEC range sector. Notably, H13 also occurred disjunctly in the southern part of P. rotundifolia's range  and the diversity within populations (HS) based on cpDNA were higher in P. davidiana than P. rotundifolia (Table Hd) was 0.7523 for P. rotundifolia, 0.8884 for P. davidiana, and 0.8950 across all populations. Nucleotide diversity (πs) was 0.00112 for P. rotundifolia, 0.00207 for P. davidiana, and 0.00189 across all populations.The total genetic diversity  than vice versa  and to SWC (2Nem: 4.30) than from CNC to NEC (2Nem = 2.07) or SWC to NEC (2Nem = 2.90). Gene flow from SWC to CNC (2Nem = 5.14) was stronger than vice versa  Historical gene flow as estimated by MIGRATE among P. rotundifolia and the two range sectors (CNC and NEC) of P. davidiana based on nSSR data(A) Historical gene flow as estimated by MIGRATE between the two putative species of the .P. rotundifolia, 0.989 ± 0.004, 0.988 ± 0.007, 0.985 ± 0.006; P. davidiana, 0.954 ± 0.019, 0.948 ± 0.022, 0.957 ± 0.018. This indicates that all models were better than random expectation. According to variable jackknife analyses, the environmental variables that contributed most to potential models were Altitude, Isothermality (bio 3) and Mean Temperature of the Driest Quarters (bio 9) for P. rotundifolia, and Mean Temperature of the Driest Quarters (bio 9), Precipitation of Wettest Month (bio 13) and Precipitation of Warmest Quarter (bio 18) for P. davidiana   Figure . The AUC Figures .P. davidiana and P. rotundifolia  indicated that they occupy significantly different niches  and ecology; however, for other markers often used to distinguish taxa, i.e., morphology and cpDNA, separation is incomplete. Hence, P. davidiana and P. rotundifolia are functioning as two distinct species. Our findings support arguments that multiple criteria should be considered when delimitating closely related species in evolutionarily complex taxonomic groups , which co-occur in desert regions in Western China, and which underwent speciation during the Pleistocene . Otherwise, the haplotypes formed two pairs of groups, with each pair containing one group that was mainly in P. davidiana and another mainly in P. rotundifolia. Group III is almost exclusively P. rotundifolia, the exception being its presence in two nearby populations that are mainly P. davidiana according to nuclear data. However, it is sister to Group II, which occurs mainly in P. davidiana but also three populations of P. rotundifolia that are close to where the species overlap. A very similar pattern occurs in Groups IV and V: Group IV occurs in P. rotundifolia, plus one nearby population that is mainly P. davidiana according to nuclear data; Group IV is derived from Group V, all of whose haplotypes occur in P. davidiana, although haplotype H13 is also present in eight populations in the southeastern range of P. rotundifolia . The 21 detected cpDNA haplotypes were clustered into five groups Figure , with tha Figure . Taken aa Figure , with thOur data is also consistent with an alternative hypothesis, wherein the initially diverging lineages gave rise to the current NEC and SWC populations, which subsequently gave rise to the CNC populations through ongoing admixture and hybridization, with SWC populations contributing cpDNA haplotypes and NEC contributing most of the nuclear genomes (based on nSSR clusters). In this scenario, the sister relationship of two pairs of cpDNA haplotype groups could represent two independent rounds of hybridization and introgression between lineages shortly after initial divergence. A third possibility is that the initial split was between NEC and the common ancestor of CNC and SWC populations, following which CNC diverged from SWC, and then finally the CNC populations were homogenized by nuclear gene flow from NEC, which left their chloroplast genomes unaffected.P. rotundifolia. Hence it might have been caused by Quaternary climate oscillations.Regardless of exactly how speciation occurred, the interspecific sharing of H13 must reflect a gene flow event that occurred long after speciation, because it concerns one haplotype, rather than a clade or its common ancestor. Even so, the event must have occurred some time ago, because the haplotype is spread across eight populations of P. davidiana, yet multiple lines of evidence support a subdivision between NEC and CNC. First, as we have discussed above, NEC harbors predominantly haplotype Group I, and CNC has groups II and V , because NEC material is dominated by the earliest diverging haplotype group, I , indicating at most weak geographical structuring, hence minimal separation between NEC and CNC. Conversely, significant geographic structuring is revealed when both species are examined together , by the National Key Basic Research Program of China (grant 2014CB954100), Sichuan Provincial Department of Science and Technology (grant 2015JQ0018) and Sichuan University.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Genetic differences between regions are usually studied for individual species. However, many species can reproduce with each other. We studied whether gene flow between two closely related sedge species influences regional differences. Our molecular genetic data support considerable gene flow between the species. Still, we detected clear genetic differences between species and regions, and more pronounced regional differences for the less common one. Thus, gene flow between the species appeared too weak to neutralize differences between the regional genetic structure of our study species. We encourage further regional differentiation studies in groups of cross-compatible species. Carex flava and C. viridula and their hybrid C. × subviridula in 37 populations in the north and centre of their distribution range in Europe  and Highland Switzerland) using 10 putative microsatellite loci. We ask whether regional differentiation was larger in the less common taxon C. viridula or whether, possibly due to hybridization, it was similar between taxa. Our results showed similar, low to moderate genetic diversity for the three studied taxa. In total, we found 12 regional species-specific alleles. Analysis of molecular variance (AMOVA), STRUCTURE and multidimensional scaling analysis showed regional structure in genetic variation, where intraspecific differentiation between regions was lower for C. flava (AMOVA: 6.84 %) than for C. viridula (20.77 %) or C. × subviridula (18.27 %) populations. Hybrids differed from the parental taxa in the two regions where they occurred, i.e. in Estonia and Lowland Switzerland. We conclude that C. flava and C. viridula clearly differ from each other genetically, that there is pronounced regional differentiation and that, despite hybridization, this regional differentiation is more pronounced in the less common taxon, C. viridula. We encourage future studies on hybridizing taxa to work with plant populations from more than one region.Regional genetic differentiation within species is often addressed in evolutionary ecology and conservation biology. Here, we address regional differentiation in two closely related hybridizing taxa, the perennial sedges  Plants and other organisms differ in their levels of genetic diversity and genetic differentiation . The extWhile genetic consequences of small population size, i.e. increased inbreeding and genetic drift, are expected to contribute to higher population differentiation and lower genetic diversity within populations . LovelesIt is generally assumed that species are reproductively isolated without gene flow between them, but in reality hybridization is a widespread phenomenon . Gene flHybrid individuals are expected to be more heterozygous than their parental taxa due to genetic admixing . Carex flava and C. viridula var. viridula sensu stricto (s.s.) (henceforth C. viridula) are wind-pollinated, self-compatible, caespitose perennials of the C. flava aggregate . Although there are no impediments to outcrossing, a large amount of seeds is produced by selfing , five sites in Lowland Switzerland  and 12 sites in Estonia  were found at two sites in Estonia and two in Lowland Switzerland, while none were found in Highland Switzerland. As hybrids, we classified partly sterile individuals that were morphologically intermediate between the parents, but often were more robust and pale . Magnesium source 1.2 mM MgSO4 was used, except for S177, where 1.6 mM MgCl2 was used. PCR amplifications were performed in 10 µL volumes containing 1–2 µL of genomic DNA, 1.2 µL GoTaq Flexi buffer (1×), 0.6 µL of each dNTP, 0.5 µL of untagged primer, 0.5 µL of fluorescent tag, 0.5 µL of the tagged primer, 0.05 µL of bovine serum albumin (BSA), 0.05 µL GoTaq Flexi DNA polymerase and varying concentrations of MgCl2 or MgSO4. Total genomic DNA was isolated from silica-dried leaves using the CTAB method (Each primer was optimized for a range of temperatures (B method . The extNe), percentage of polymorphic loci (PL), expected heterozygosity  and observed heterozygosity (Ho) were estimated across all loci using GENALEX 6.5 (FIS) shows the probability to observe alleles of an individual (I) that are identical by descent (IBD) in a subpopulation (S). FIS, calculated as (He − Ho)/He, allowed to estimate the prevailing mating systems by region  and taxon. Variation in these measures of population diversity was tested with ANOVA, using taxon, region of origin and the interaction of taxon and region of origin as fixed effects, implemented in the software R v 3.1.2  without a priori knowledge of taxonomy or population, and (ii) to identify the hybrid individuals with admixture analysis. The clustering was conducted with the admixture model and the correlated-allele-frequencies option using a burn-in of 10000 steps and 100000 replications, the remaining parameters were set to the default values. Five independent runs were done for the set of K = {1:10}. We used Structure Harvester v 0.6.92 (K) by using firstly the ΔK method of K = 2 we detected the posterior probability , which describes the proportion of an individual genotype originating from each of K categories. We used K = 2 as we expected two taxa contributing to the gene pool of hybrids. We chose a threshold value of 0.9, which was found efficient to distinguish pure individuals (q > 0.9 or q < 0.1) from hybrids and backcrosses (0.1 < q < 0.9)  enabled us to determine the distribution of microsatellite variation among groups of populations, among populations within groups and among individuals within populations, using Arlequin. We grouped the populations according to the tested hypotheses per taxa and regions. We tested the significance of the variance components by calculating their probabilities based on 9999 permutations of individual samples.C. flava in Highland Switzerland see .The total number of alleles observed per locus in the overall sample of 380 individuals from 37 populations and 24 sites of three regions ranged from 4 to 11, with overall 64 alleles scored over the 10 loci. Private alleles were detected at seven loci, four of which were found in C. flava  than in the less common C. viridula   and among three regions , percentage of polymorphic loci  and expected heterozygosity  indicate that differences between the two taxa in their levels of genetic diversity depended on the region  . The pere region .P-values for multiple comparisons, 10 out of 370 tests (10 loci × 37 populations) showed significant deviations from Hardy–Weinberg expectations (Appendix 1). Large positive inbreeding coefficients (FIS) were generally correlated with significant deviations from Hardy–Weinberg equilibrium (HWE). No locus in any population had a negative inbreeding coefficient that differed significantly from HWE. Locus S180 showed deviations from HWE in three populations , locus Cko2-135 in three populations , locus S177 in two populations  and locus CL101 and locus Cko2-112 in one populations . Some pairs (loci × population) could not be tested because loci were not polymorphic.After correcting C. viridula populations was highest in Lowland Switzerland , followed by Estonia . In Highland Switzerland only few populations of C. viridula were found and their genetic diversity was much lower than in the other two study regions . We expected C. viridula with its smaller populations to be more prone to inbreeding. Accordingly, C. viridula showed a deficit of heterozygotes in Lowland Switzerland populations and in some populations of Estonia. However, other Estonian populations and the ones in Highland Switzerland showed an excess of heterozygotes , intermediate in Lowland Switzerland  and lowest in Estonia . Independent of the region, C. flava populations varied in their inbreeding coefficients between −0.79 and 0.65  and in Estonia . In both regions hybrid populations showed an excess of heterozygotes , i.e. as pure individuals, only two putative C. flava and two C. viridula individuals had q < 0.90, suggesting that they rather were mixed genotypes.The Δa K = 2; . We usedC. flava complex co-occurred. For the C. flava morphotypes at the mixed sites 136 individuals of 159 putative C. flava were indeed pure C. flava , 21 of 159 had mixed genotypes  and two individuals (1.2 %) were even C. viridula-like. For the C. viridula morphotypes at the mixed sites, 102 individuals of 110 putative C. viridula were pure C. viridula , three had mixed genotype  and five (4.5 %) were C. flava-like. For the C. × subviridula hybrid morphotypes a wide range of admixture proportions were found (q ranged from 0.103 to 0.897), suggesting the presence of a broad range of hybrid generations and backcrossing to both parental taxa.The situation was more complex at ‘mixed’ sites, where two or more taxa of the see . K = 3 yielded different results than K = 2, also revealing differences between regions, namely admixture between plants of C. flava and C. viridula in Highland and Lowland Switzerland  and in Melchsee-Frutt (F1), where we exclusively found C. flava).With the log-likelihood distribution method, the value where the rate of increase in likelihood reaches a plateau without increase in variance corresponds to three clusters tzerland . The thiOverall, the STRUCTURE results indicate clear differentiation between the taxa and in addition further variation between regions. This is in line with the results of genetic diversity, which showed significant differences between regions (for the percentage of polymorphic loci) and significant region-by-taxon interactions , intermediate in Lowland Switzerland (FST = 0.31) and lowest in Highland Switzerland  than from C. viridula (FST = 0.16), while in Lowland Switzerland the hybrids were more differentiated from C. viridula (FST = 0.24) than from C. flava , followed by Highland (28.27 %) and Lowland Switzerland (17.51 %), i.e. between-taxa differentiation was highest in Estonia and lowest in Lowland Switzerland, where three of the five studied sites were mixed . AMOVA r FST = 0.7, intermC. flava (6.84 %) than for C. viridula (20.77 %) or C. × subviridula (18.27 %) populations  than for C. viridula  or C. × subviridula , which were also very different from each other  was slightly higher in C. viridula, though not significantly higher from C. flava with He = 0.25 and He = 0.21, respectively. Carex viridula showed highest genetic diversity in Lowland Switzerland, whereas C. flava was most diverse in Highland Switzerland, with He = 0.32 and He = 0.26, respectively. Both taxa had lower diversity in Estonia. Greater allozyme diversity and a lower inbreeding coefficient for C. viridula than for C. flava was also detected in earlier studies using allozymes by C. viridula s.l., united with C. lepidocarpa, C. demissa and C. viridula s.s., however. Meanwhile, C. viridula and attributed it to bottlenecks at arrival from Europe and to predominant selfing. Higher diversity in highland populations, as in our study in C. flava, was found in some studies, e.g. for Cystopteris fragilis (Pteridophyta) using isozymes (Primula farinosa (Primulaceae) using RAPD analysis (Campanula thyrsoides (Campanulaceae) , C. macrocephala , C. rugulosa  and C. scoparia , the mean gene diversities of microsatellites were higher  than in our study  (Hs = 0.10) for C. extensa (sect. Spirostachyae) despite using a wide study area. Possibly our gene diversity values were smaller than the ones detected in other studies, because they generally addressed species with larger and less isolated populations.In previous studies on sedges  = 0.24) . On the Carex  was not higher in hybrid populations than in the parental taxa (He = 0.24 vs. 0.21 and 0.25). C. aquatilis × recta (sect. Phacocystis) similar gene diversity than for its parental taxa (He = 0.348 vs. 0.308 and 0.460) and for C. paleacea × recta higher diversity (He = 0.603 vs. 0.185 and 0.460). In contrast, high genetic diversity, using RAPD analysis, was found in Fallopia × bohemica (Polygonaceae) in Germany and Switzerland . The admixture proportions detected by microsatellites for hybrid individuals were very variable, indicating that these comprised FK = 2) and with some regional differentiation  taxa preserved despite intraspecific gene flow. Helianthus taxa (Asteraceae) remained largely reproductively isolated and morphologically and ecologically distinct despite high levels of interspecific gene flow. Senecio taxa (Asteraceae) and explained them with selection against hybrids and locally maladapted hybrid individuals.The results of our STRUCTURE analysis suggested that we dealt with two species and their hybrids  than for the more widespread C. flava or hybrid . In addition, groupwise FST values between regions were higher for C. viridula, especially between Estonia and both altitudes in Switzerland  and explained it with long-distance dispersal. Potential for long-distance pollen and seed dispersal was suggested to contribute to low geographic differentiation of circumpolar C. bigelowii (sect. Phacocystis)  . Differeocystis) , we suggFST = 0.32). This difference could originate from genetic drift in the hybrid populations  and the European Union through the European Regional Development Fund (Centre of Excellence EcolChange) supported this study.L.S., T.O. and M.F. conceived the study and its design. L.S. conducted field and lab work, analysed data and led the writing of the manuscript. T.O. advised lab work. Both T.O. and M.F. contributed to data analysis, data interpretation and writing of the manuscript. This manuscript forms part of the PhD thesis of the first author (L.S.). All authors have made a substantial contribution to the paper.None declared.The following additional information is available in the online version of this article—Table S1. Microsatellite loci used to study genetic diversity and species relationships in Carex flava , C. viridula var. viridula and their hybrid (C. × subviridula). Presented are primer sequences, 5′ tag, repeat motif, size range, annealing temperature Ta (°C), number of alleles detected for each locus. The 5′ tags M13R (AGGAAACAGCTATGACCAT) and CAGT (ACAGTCGGGCGTCATCA) were used for incorporation of the fluorescent tag. S082, S180, S245, S175, S119, S177 are from Table S2. Presence and frequency of private alleles in seven  of the 10 studied microsatellite loci in Carex flava , C. viridula var. viridula and their hybrid (C. × subviridula). The three other analysed loci  did not show private alleles.Figure S1. Estimating the optimal number of clusters with admixture analyses of plants of Carex flava , C. viridula var. viridula and their hybrid (C. × subviridula) from Estonia, Highland and Lowland Switzerland. (A) The ΔK method indicated two genetic groups. (B) The distribution of log-likelihoods indicated three genetic groups.Supplementry InformationClick here for additional data file.


Haemorhous purpureus). Mitochondrial DNA data show the presence of two distinct groups corresponding to the two subspecies, H. p. purpureus and H. p. californicus. The two subspecies likely survived in separate refugia during the last glacial maximum, one on the Pacific Coast and one east of the Rocky Mountains, and now remain distinct lineages with little evidence of gene flow between them. Southwestern British Columbia is a notable exception, as subspecies mixing between central British Columbia and Vancouver Island populations suggests a possible contact zone in this region. Z‐linked data support two mitochondrial groups; however, Coastal Oregon and central British Columbia sites show evidence of mixing. Contemporary population structure based on microsatellite data identified at least six genetic clusters: three H. p. purpureus clusters, two H. p. californicus clusters, and one mixed cluster, which likely resulted from high site fidelity and isolation by distance, combined with sexual selection on morphological characters reinforcing subspecies differences.Using a combination of mitochondrial and z‐linked sequences, microsatellite data, and spatio‐geographic modeling, we examined historical and contemporary factors influencing the population genetic structure of the purple finch ( Using a more representative sampling regime covering more of the purple finch's distribution, and several molecular makers, the aim of this study was to examine the evolutionary history and the effects of dispersal barriers on contemporary genetic patterns.The purple finch , Coastal Oregon (CoOR), Labrador (LAB), Michigan (MI), North Dakota (ND), Newfoundland (NL), Nova Scotia (NS), Southern California (SCA), Vancouver Island (VanIs), and Washington (WA) , 0.2 mmol/L dNTP, 2.5 mmol/L MgCl2, 0.4 μmol/L each primer, 1 U GoTaq® Flexi polymerase, and genomic DNA.A 707‐bp fragment of the ATP region of the mitochondrial genome, hereafter referred to as ATP6, containing both ATP 6 (271 bp) and ATP 8 (445 bp) was amplified using primers L8950 (CCAACCACAGCTTCATACCA) and H9694 (GCTAGTGGGCGGATGAGTAG) for 243 samples. The thermal cycling profile was one cycle of 120 s at 94°C, 45 s at 54°C, 60 s at 72°C; 37 cycles of 30 s at 94°C, 45 s at 54°C, and 60 s at 72°C; and a final cycle of 5 min at 72°C. The 25 μl PCR contained: Green GoTaq2, and crimson buffer (New England Biolabs).A 515‐bp fragment of the AldB region of the Z chromosome was amplified in 178 samples for a total of 306 alleles, using primers AldB6‐F and AldB8‐R , 1 μmol/L each primer, 0.5 U of taq polymerase, and 0.05 μmol/L fluorescently labeled M13 tag.Three 2.52.5.1AldB sequences from all known male birds were run through PHASE v. 2.0 , LAB, CoOR, and VanIs sites were removed from all pairwise FST analysis. p‐values were corrected using the modified false discovery rate (FDR)  were removed from the analyses. A total of 260 individuals from all 10 populations were successfully genotyped at the seven loci. Populations and loci were checked for deviations from Hardy–Weinberg equilibrium and linkage disequilibrium with GENEPOP . Individuals missing more than three of the seven loci  to map putative barriers according to Monmonier's algorithm  and LIG .Spatio‐geographic modeling was used to identify potential Pleistocene refugia. Occurrence information for the purple finch  was obtained from the Global Biodiversity Information Facility. After removing points with no UTMs, potential outliers (outside the expected range), duplicated data, samples from migratory periods, or any data from potentially unreliable sources, 5,710 points remained. Five of the 19 climate layers with ≥0.9 threshold in ENMTools  to 0.899 (ND), and nucleotide diversity ranging from 0.0004 (SCA) to 0.0056 (NS) Table . The aveH. p. californicus and H. p. purpureus formed two distinct clusters with no haplotype sharing , ΦST was 0.092, while for the five H. p. purpureus populations, ΦST values ranged from 0.000 to 0.194. Average ΦST between subspecies was 0.9193. All pairwise comparisons between subspecies were significant and CBC was significantly different from all other H. p. purpureus populations and the two H. p. californicus populations were significantly different from each other.Pairwise Φes Table a. BetweeThe significant differentiation between subspecies was supported by AMOVAs. The majority of the variance for the mitochondrial locus was explained by genetic differentiation between subspecies variation (90%), while 9% was explained by differences between individuals, and just 1% of the variation between populations.H. p. californicus cluster and two predominantly H. p. purpureus clusters , while coordinates 2 (8.9%) and 3 (3.2%) explained the rest. The first two coordinates explained 96% of the variation and clustered geographically proximal populations together   than between subspecies (0.438–0.805). All pairwise FST values between subspecies and between the two H. p. californicus populations were significant after corrections for multiple tests. The only H. p. purpureus comparison that was significant was NL–CBC.05 Table . The FSH. p. purpureus population to the H. p. californicus cluster and several H. p. californicus to a widespread cluster , while Microchecker suggested possible null alleles for Titgata 39 with NL and ND samples, for Titgata 02 among NL and CBC samples, and Ase18 among CBC samples. Analyses were run with and without CETC 215; however, the results were not different so CETC 215 was kept in the analysis.FST comparisons ranged from 0.028 to 0.174, with all 19 comparisons significant , while 8% explained variation between populations, and there was a negligible contribution of variation from comparisons between regions (subspecies) at 0.5%.Mean observed heterozygosities ranged from 0.352 (MI) to 0.587 (NS), and mean allelic richness ranged from 4.494 (SCA) to 6.583 (MI) Table . PopulatH. p. purpureus population clusters closer to H. p. californicus.Coordinates 1 and 2 explained a total of 70% of the variation , while coordinate 3 explained an additional 16%  and z‐linked data . However, it was not significant  for microsatellite data.Isolation by distance was significant using mtDNA  was 0.944 (±0.005), where 0.5 is random and values closer to one denote a greater ability to distinguish between suitable and unsuitable habitat , low among population variation of 1% in mtDNA, and similar haplotype diversity values among populations . The mtDNA haplotypes show separation of H. p. purpureus populations from H. p. californicus  and Swainson's thrushes (Catharus ustulatus)  Ruegg, . The conWootton, .H. p. californicus) individuals clustered with H. p. purpureus birds from CBC , three individuals from the well‐sampled CBC population shared a haplotype with the VanIs population at the z‐linked locus, and another CBC bird grouped with two WA individuals . The presence of H. p. purpureus nuclear DNA within the H. p. californicus range and H. p. californicus nuclear DNA in the H. p. purpureus range, along with the clustering of CBC and VanIs populations, suggests limited gene flow between the two subspecies at some western sites. This could occur through hybridization at a contact zone with male‐biased dispersal as the pattern is only seen in nuclear loci. Similar patterns of male gene flow are found in the spectacled eider (Somateria fischeri) , boreal chickadees (Poecile hudsonicus), and Sitka and white spruce (Picea sitchensis and P. glauca) , of banded birds return to breed in the same area. Similar patterns of low population structure of highly migratory species are found in northern pike (Esox lucius)  , in which males have darker heads, backs, rumps, and anterior lower parts; females are a darker olive. Both sexes lack mottling in the upper back and nape found in other nearby populations   south of James Bay, and in jack pine (Pinus banksiana) south of Hudson Bay  from other populations. Although no obvious physical barrier is present, other studies found population structure in both animal and plant species in eastern Canada. Rueness et al.  found reOur study has shown how the evolution of the purple finch has been influenced by a number of evolutionary processes and both historical and contemporary distributions. Prolonged isolation of the two subspecies was maintained during the last glacial maximum and contemporary physical barriers, and behavioral barriers including site fidelity, migration, and assortative mating, have maintained patterns of low gene flow between the two subspecies. Some secondary contact was found in a small portion of their range, although it was limited. The purple finch's evolutionary history demonstrates how population differences shape behavior and response to barriers to dispersal can influence evolution and, ultimately, speciation.We have no conflict of interest to declare.


Past events like fluctuations in population size and post-glacial colonization processes may influence the relative importance of genetic drift, migration and selection when determining the present day patterns of genetic variation. We disentangle how drift, selection and migration shape neutral and adaptive genetic variation in 12 moor frog populations along a 1700 km latitudinal gradient. We studied genetic differentiation and variation at a MHC exon II locus and a set of 18 microsatellites.Using outlier analyses, we identified the MHC II exon 2 (corresponding to the β-2 domain) locus and one microsatellite locus (RCO8640) to be subject to diversifying selection, while five microsatellite loci showed signals of stabilizing selection among populations. STRUCTURE and DAPC analyses on the neutral microsatellites assigned populations to a northern and a southern cluster, reflecting two different post-glacial colonization routes found in previous studies. Genetic variation overall was lower in the northern cluster. The signature of selection on MHC exon II was weaker in the northern cluster, possibly as a consequence of smaller and more fragmented populations.Our results show that historical demographic processes combined with selection and drift have led to a complex pattern of differentiation along the gradient where some loci are more divergent among populations than predicted from drift expectations due to diversifying selection, while other loci are more uniform among populations due to stabilizing selection. Importantly, both overall and MHC genetic variation are lower at northern latitudes. Due to lower evolutionary potential, the low genetic variation in northern populations may increase the risk of extinction when confronted with emerging pathogens and climate change.The online version of this article (doi:10.1186/s12862-017-1022-z) contains supplementary material, which is available to authorized users. The relative roles of selection and drift shaping genetic diversity among and within natural populations are a contentious issue in evolutionary biology , 2. GeneWhile the abundant center model has been influential for much of the theory, evidence for populations being more numerous or frequent at the centre of the distribution is, at best, limited . Here, hDisentangling whether current patterns of genetic variation along geographical gradients are brought about by central-marginal or colonization processes is not simple, because, due to the location of glacial refugia, the two patterns are geographically correlated . MoreoveIn population and conservation genetics, spatial population structure and gene flow are usually assessed with neutral markers, assuming that this variation also reflects adaptive potential  However,Batrachochytrium dendrobatidis (Bd), which has caused a large number of local and global amphibian extinctions , ted ones , we cannST and genetic diversity indexes (AR and HE) were substantially higher for the MHC locus compared to neutral microsatellite markers (even though correlated). These results suggest that the MHC II exon 2 locus is under diversifying selection and are in agreement with previous studies finding more differentiation in MHC than in neutral loci suggesting adaptation to local parasite faunas . There R. arvalis has a broad Euroasiatic distribution, from the North Sea coast to Siberia [Rana arvalis is located in the area of eastern Germany and western Poland  to northern Sweden (Luleå) in 2014 and 2015 . We colHaeIII restriction enzyme [HaeIII produces different, easily distinguishable banding patterns in R. arvalis and R. temporaria.Genomic DNA was extracted from 240 tadpoles  using a high salt extraction precipitation protocol (modified from ). Purityn enzyme . DigestiRana species, and tested another set of ten microsatellites without success  and ELR_2 (3′-GCATAGCAGACGGAGGAGTC-5′) were designed based on primers sequences developed for R. pretiosa and R. luteiventris [We amplified the complete second exon (272 bp) of the single MHC II gene (corresponding to the β −2 domain) in iventris , amplifywww.DnaBaser.com). The jMHC software [Sequencing data were extracted from the raw data and combined into single forward reads using FLASH , each of the eight amplicon pools were analyzed independently. In total, eight fastq files were generated and transformed into fasta (multifasta) files using Avalanche NextGen package (DNA Baser Sequence Assembler v4 (2013), Heracle BioSoft, software  was usedWe assigned the two most frequent variants within each amplicon as valid MHC alleles that occurred in at least 3% of the reads , 90. In Ai) per individual. This procedure is based on the break point in sequencing coverage between variants within each individual and avoids choosing a subjective threshold to separate true alleles from artefacts. In this procedure, variants are sorted top-down by coverage, followed by the calculation of the coverage break point (DOC statistic) around each variant. The variant with the highest DOC value is assumed to be the last true allele , therefele (see ).Quasipaa spinosa; GenBank: KM390904.1) natterjack toad , mouse  and turkey . According to the GT/AG rule [Odorrana tormota (JQ918829), the exon boundary was defined 16 bps downstream from intron-exon boundary determined in mouse. We removed the intron sequences for further analyses and ended up with a MHC II exon 2 fragment of 272 bps. Valid MHC exon 2 alleles were named following the nomenclature suggested by Klein [All valid allele sequences were imported and aligned in MEGA 6.0 . Allele FST was simulated with 100,000 iterations implementing a stepwise mutation model (SMM) in Lositan. In Bayescan, outliers were detected by implementing the multinomial Dirichlet model. Outliers identified by at least one of the two methods were deemed as being non-neutral ; and a southern cluster . We did the division after analyzing our data (see below) and in accordance with a bidirectional recolonization hypothesis of Scandinavia , 68. ForE), observed heterozygosity (HO) and allelic richness (AR), allele numbers rarified to the smallest sample size were calculated for each locality in FSTAT 2.9.3.2 [Input files were converted for different analyses program formats using the Excel add-in Microsatellite toolkit . The fre 2.9.3.2  Multi-lo 2.9.3.2 .ST between all the populations [ST/G’ST corrections [ST values when appropriate. We tested for deviation from Hardy-Weinberg equilibrium (HWE) in ARLEQUIN v. 3.5. [ST and log distance (km) for every population pair. Pair-wise FST was transformed based on Slatkin’s (1995) data adjustment (FST/(1-FST)). The Euclidean distance matrix was estimated using the R package Geosphere . To tesAdegenet .ST (as implemented in divMigrate-online) as the differentiation metric and set the filter threshold to 0.20; 0.30; 0.40, respectively. Confidence limits on gene flow estimates were determined by 1000 bootstrap replicates.We visualized the spatial structure of microsatellite data using discriminant analysis of principal components (DAPC) implemented in Adegenet . The optE, HO, and allelic frequencies) were calculated for each locality in Arlequin v 3.5 [To assess genetic diversity in the MHC II exon 2 standard diversity indices , as desST for MHC exon 2 and RCO8640, respectively, against the differentiation for neutral microsatellites, G’ST [To compare population differentiation among the differentially selected loci, as revealed by the outlier analyses (above), we plotted the standardized F’es, G’ST . We thenIn summary, populations were shown to be subjected to different selective regimes and combined with different historical demographic patterns affecting the strength of genetic drift, a complex pattern of differentiation have evolved along the gradient. Some loci are more divergent than expected by drift among populations due to diversifying selection while others are more uniform among populations due to stabilizing selection. Our data show a high number of MHC exon 2 alleles in comparison to other European amphibian species. Both overall and MHC genetic variation are lower at northern latitudes which suggest a high risk of extinction when confronted with emerging pathogens and climate change. These results emphasize the importance of latitudinal gradient studies in order to elucidate and understand the evolutionary processes shaping genetic variation among natural populations.Additional file 1: Table S1.Microsatellites used in the study. (PDF 40 kb)Additional file 2: Table S2.16 microsatellites outlier analyses results from Lositan and Bayescan. (PDF 31 kb)Additional file 3: Table S3.15 microsatellites and MHC II exon 2 outlier analyses results from Lositan and Bayescan. Analyses were performed independently considering: all the gradient, northern populations as a cluster and southern populations as another cluster. (PDF 31 kb)Additional file 4: Figure S1.ST “(FST/(1-FST))” as a function of the natural logarithm of distance (km) between locality pairs. MHC class II is represented by blue circles and MS are represented by black stars. (PDF 2099 kb)Isolation-by-distance a) for MHC class II and b) for microsatellite (MS) shown as Slatkin’s linearized pair-wise FAdditional file 5: Figure S2.Results of STRUCTURE analyses using the Admixture model. Average individual assignment probability (y-axis) of individuals for four values of K. Sampled populations are given below the plot and country, region or province of origin is given above. Delta K and Mean estimation Ln probability of the data are shown for different values of K. (PDF 326 kb)Additional file 6: Figure S3.Discriminant component analyses (DAPC) of 9 neutral microsatellites for all the individuals. All individuals from the north cluster together to the right of the figure. (PDF 1539 kb)Additional file 7: Table S4.Pairwise FSTs. a) MHC gene FST pairwise comparisons and b) neutral microsatellite FST pairwise comparisons, respectively. (PDF 49 kb)Additional file 8: Figure S4.Colour scheme for the allele distribution maps a) MHC gene and b) RCO8640 gene. (PDF 875 kb)Additional file 9: Table S5.Relative allele frequency for all the MHC alleles found along the latitudinal gradient. N = number of individual per population, AS = Alleles within sampling areas. (PDF 101 kb)Additional file 10: Table S6.Information about pond locations along the latitudinal gradient. (PDF 48 kb)Additional file 11: Table S7.PCR conditions for a total volume of 10 μl reaction for the neutral microsatellites finally used in the study. (PDF 69 kb)Additional file 12: Figure S5.a) Primers were modified for Illumina Miseq consisting of a 8 bps barcode assignment and a sequence of three N Barcodes represented in pink  and in orange . The NNN sequence is shown in blue. Possible primer pair combinations are presented in two different boxes . Every forward primer was combined with 9 different reverse primers for every pool. A total of four pools were constructed in the study: blue (A), purple (B), orange (C) and red (D). b) Outline of the library preparation. (PDF 173 kb)Additional file 13: Figure S6.Results of outlier analyses by Lositan software in MHC class II exon 2 and RCO8640 according to a) Infinite allele model approach (IAM) b) Stepwise mutation model approach (SMM). (PDF 140 kb)Additional file 14: Table S8.FST for the gradient excluding all population from Germany was 0.35. (PDF 17 kb)15 microsatellites and MHC II exon 2 outlier analyses results from Lositan and Bayescan. Analyses were performed independently considering: all the gradient and southern population, excluding all the German locations. Overall Additional file 15: Figure S7.Results of outlier analyses by Lositan software for 15 microsatellites neutral markers, MHC class II exon 2 and RCO8640 according to the Stepwise mutation model approach (SMM) and excluding all the German populations. (PDF 856 kb)


Lasiorhinus latifrons), whose conservation status has recently deteriorated, and whose phylogeographic history during the Pleistocene may be atypical compared to other species. Its contemporary range spans approximately 2000 km of diverse habitat on either side of the Spencer Gulf, which was a land bridge during periods of Pleistocene aridity that may have allowed for migration circumventing the arid Eyrean barrier. We sampled from animals in nearly all known sites within the species’ current distribution, mainly using non-invasive methods, and employed nuclear and mitochondrial DNA analyses to assess alternative scenarios for Pleistocene impacts on population structure. We found evidence for mildly differentiated populations at the range extremes on either side of Spencer Gulf, with secondary contact between locations neighbouring each side of the barrier. These extreme western and eastern regions, and four other regions in between, were genetically distinct in genotypic clustering analyses. Estimates indicate modest, but complex gene flow patterns among some of these regions, in some cases possibly restricted for several thousand years. Prior to this study there was little information to aid risk assessment and prioritization of conservation interventions facilitating gene flow among populations of this species. The contributions of this study to that issue are outlined.Major prehistoric forces, such as the climatic shifts of the Pleistocene, can remain visible in a species’ population genetics. Inference of refuges via genetic tools is useful for conservation management as it can identify populations whose preservation may help retain a species’ adaptive potential. Such investigation is needed for Australia’s southern hairy-nosed wombat ( The Pleistocene period was marked by oscillations in global climate aridity and, as a consequence, habitat fragmentation with animal species potentially surviving in refuges , 2. DescLasiorhinus latifrons), a large, herbivorous, fossorial marsupial from semi-arid regions of southern Australia  wombat , but unactivity . In addiactivity , 17. In activity . The mosactivity , 19. Thuactivity –22. Thesactivity .http://maps.iucnredlist.org/map.html?id=40555), but prior to European settlement there may have been a narrow habitat conduit around the northern end of Spencer Gulf  no impact of the disappearance of the land bridge, and so no east or west post-Pleistocene refuges; 2) east and west refuges with no or very little gene flow between them, followed by subsequent secondary contact; 3) east and west refuges with little or no admixture; and 4) post-Pleistocene populations surviving only on one side of Spencer Gulf with subsequent recolonization across the current range. We expect that these competing scenarios will be reflected in mitochondrial DNA (mtDNA) and microsatellite DNA data as follows:Scenario 1: ‘no refuges’ would be supported by low divergence between populations on either side of the Spencer Gulf land bridge. This could be due either to insufficient time post the Spencer Gulf barrier, or ineffectiveness of the Eyrean barrier to prevent movement and gene flow.Scenario 2: ‘multiple refuges with secondary contact’ would be supported by divergence between the descendants of putative refuge populations, with estimates of time of divergence consistent with impact of the re-flooding of the Gulf, and maximal genetic diversity where the genes from the different refuges are admixed, unless population size reduction and genetic drift cause loss of variation at the contact zones.Scenario 3: ‘multiple refuges without admixture’ also predicts divergence between the refuges, like Scenario 2, but does not predict higher diversity in between, given no admixture. As small numbers of wombats spread out into the colonised areas, many genotypes may have survived, but rare alleles may have been lost during successive drift events.Scenario 4: ‘recolonisation from one side of Spencer Gulf’ would be supported by a lack of strong genetic division, and by genetic diversity gradually reducing with increasing distance from the refugium, or homogeneity or isolation-by-distance if gene flow has been sufficient. In this case, genetic subdivision should be unrelated to putative phylogeographic barriers and times of high aridity when the gulf was dry.We investigated the effects of environmental influences since the late Pleistocene on SHN wombat population structure, and attempted to uncover the population genetic patterns that existed prior to land-use by European settlers and the relationships among extant populations. We applied microsatellite and mitochondrial DNA data, collected from sites across the entire current range of the SHN wombat, to estimate genetic differentiation and gene flow at different time scales. We provide genetically based recommendations for conservation management of the SHN wombat, building upon previous preliminary studies of the genetics of this species –34 and tP = 0.250; Kulpara: A = 6.00, B = 6.00, P = 1.000; Wauraltee: A = 8.00, B = 6.75; P = 0.500; Wilcoxon signed-ranks tests). Set B DNA was extracted via same-day 5% Chelex extractions performed in the field . The NSW National Parks and Wildlife Service approved the holding of samples from South Australia in NSW (Permit Numbers: A941 and B1025). All efforts were made to minimize impacts on animals, and so DNA was extracted mainly from hair samples collected on sticky tape from wombats as they entered or left their burrows , 36–38. he field  [41]. Seb. Cytochrome b is useful when looking for longer-term signatures of isolation owing to its relatively well-studied rate of evolution in mammals . Th. Th14]. Scenario 1 proposed that the Eyrean barrier did not prevent gene flow and hence there would be low divergence between populations on either side of the Gulf. The genetic analyses present considerable support for this scenario. First, nuclear variation indicated relatively close genetic similarity of many pairs of locations on opposite sides of the Eyrean barrier / Spencer Gulf, providing evidence for gene flow across the barrier. In particular, the Eyre Peninsula (west) cluster is weakly diverged from the Murraylands (east). The Gawler Ranges (west) also has some strong connections with eastern sites, including in the Murraylands  between the two refuges. There was also support for this scenario, as two potential refuges were indicated by mtDNA and nuclear DNA variation. While mtDNA divergence was slight, and the most common haplotype shared between east and west, the east had three haplotypes not found in the west and the west had one not found in the east, resulting in a significant AMOVA differentiation test. Nuclear variation was less clearly split, as expected given its higher effective population size but nonetheless there were some relatively large and significant differences between the most westerly and the most easterly sampled locations. The mtDNA cytochrome b divergence dates back to approximately 250 000 years ago, well before the Last Glacial Maximum (24 500 BCE), whereas the nuclear DNA divergences are much more recent, less than 100 000 years ago (the longest divergence time in Scenario 2 of highest diversity at the point of contact between two refuges was not fulfilled by the data. Average expected heterozygosity (HE) was perhaps even relatively low where eastern and western putative populations would be expected to meet , plausibly consistent with little impact of the Gulf as a post-Pleistocene barrier. Nevertheless, across the species’ broad range, some geographic groups might have been separated for some thousands of years.Overall the data give the greatest support to −2 instead of 10−3), divergence between the east and west ends of the species’ distribution seems to predate the 500 years that is conventionally accepted as the time when humans began to impact on natural evolution [Microsatellite and mtDNA data are consistent with SHN wombat population differentiation caused by periods of Pleistocene aridity, as has been established for many other taxa in the region , 27, 28.volution . Thus, avolution , 80, evevolution . But addvolution , 81. Thevolution . We do nSix distinct geographic population groupings were identified. The largest population of SHN wombats exists towards the western edge of its distribution, the FWC, on which basis this would be the most likely population to survive into the future. However, this population represents only one of six genotypic groups, and only the western (much less variable) mitochondrial grouping that putatively reflects only one of two mid-Pleistocene refuges. Much genetic information and evolutionary potential could be lost if genetic variation from the other five genotypic zones were not also conserved.The Yorke Peninsula populations have diverged from the others at least in part as a consequence of fragmentation and genetic drift. However, two sites, Tiparra and Wauraltee, preserve private alleles, and may harbour genetic variation of value to the species as a whole. Only seven animals were found with thorough sampling at the Tiparra site.In summary, we found evidence that the SHN wombat species is genetically subdivided into six genotypic clusters, some of which may have persisted for thousands of years. There is some evidence for two refuges, or possibly isolation by distance, on each side of the land bridge across the Spencer Gulf that existed during periods of Pleistocene aridity. This substructure should be considered in efforts to preserve the maximum potential for adaptation in the species, including conservation interventions involving facilitated gene flow . It alsoS1 FileL. latifrons populations.Microsatellite genotypes for 24  Set A: 12 populations, 8 loci. Set B: 16 populations 9 loci.(XLSX)Click here for additional data file.S2 FileL. latifrons populations.Mitochondrial DNA haplotypes for 7  Frequencies of 6 RFLP haplotypes.(ARP)Click here for additional data file.S1 TableLasiorhinus latifrons.Allele frequencies in samples from all collection sites analysed for (DOCX)Click here for additional data file.S2 TableSignificance of Wilcoxon paired-samples signed-ranks tests of genetic diversity measures tested between all pairs of 24 sampling sites of SHN wombats.(DOCX)Click here for additional data file.S3 Tablestructure, using microsatellite data (four loci) from 24 L. latifrons populations.Average probability of membership for individuals in six genetic clusters inferred by (DOCX)Click here for additional data file.S1 TextChelex Extractions of Hair Samples. Phenol/IAC Extractions of Hair, Blood, and Tissue Samples. Amplification of Microsatellite Loci. Genotyping Process.(DOCX)Click here for additional data file.


Simulium hirtipupa Lutz, 1910 in populations from the biomes Caatinga, Cerrado, and Atlantic Forest. In this study, we carried out phylogeographic and population genetic analyses using a fragment of the mitochondrial gene COI. The 19 populations studied were clustered into seven groups, most of which are associated with geography indicating certain genetic structure. The northern region of the state of Minas Gerais is most likely the center of origin of this species. The average intergroup genetic distance was 3.7%, indicating the presence of cryptic species. The species tree as well as the haplotype network recovered all groups forming two major groups: the first comprises groups Gr-Bahia , Gr-Pernambuco, and Gr-Mato Grosso do Sul. The second included groups comprising populations of the states of Goiás, Tocantins, Minas Gerais, Bahia, São Paulo, and Espírito Santo. The mismatch distribution for groups was consistent with the model of demographic expansion, except for the Gr-Central-East_1 group. The diversification in this group occurred about 1.19 Mya during the Pleistocene, influenced by paleoclimatic oscillations during the Quaternary glacial cycles.High morphological homogeneity and cryptic speciation may cause the diversity within Simuliidae to be underestimated. Recent molecular studies on population genetics and phylogeography have contributed to reveal which factors influenced the diversity within this group. This study aimed at examining the genetic diversity of  The family Simuliidae has a wide geographic distribution occurring in all continents except Antarctica. Over 25 species are of medical and veterinary importance since black flies are etiological agents of human onchocerciasis. As well, high biting activities of females can reduce animal productivity of birds and mammals. Moreover, their immature stages play an important role in nutrient turnover in streams. Due to the extensive cryptic speciation and high morphological homogeneity observed in the Simuliidae, much of the diversity within this group may be underestimated –6.Morphological and cytotaxonomic studies that have been historically used to assess the biodiversity of this group, and recently molecular markers have been incorporated into studies of systematics, phylogeny, population genetics, and phylogeography . NonetheDNA barcode studies using a small portion of the gene cytochrome c oxidase subunit I (COI) for species identification  have proIn simuliids, the DNA barcode approach has been used in studies focusing on phylogeny ; populatPhylogeographic studies that combine genetic and geographic data, have allowed inferences to be made on vicariance and dispersal processes that drive animal and plant species distributions . AquaticSimulium hirtipupa Lutz, 1910 is restricted to Brazil where it is present in four of the five major geographic regions, it is not present in the North region [Simulium criniferum [S. hirtipupa mentioned above may contribute to the understanding of which environmental and geological factors have most likely influenced the diversification and dispersal of this group in the Neotropical region.h region ,26. Receiniferum . In the Simulium comprises several species complexes [S. hirtipupa are unknown, although they are thought to be zoophilic [S. criniferum is the closest relative to S. hirtipupa and the females of the former are anthropophilic [S. hirtipupa might be both more complex than previously hypothesized and could also reflect interactions between cryptic differentiation and dispersal. Thus, in order to examine the genetic diversity and to elucidate the diversification process of this nominal species of Brazilian simuliid, we utilized a fragment of the mitochondrial gene COI and present here a large-scale phylogeographic and population genetic analyses of S. hirtipupa. Specifically, the objectives of this study are threefold: (i) examine the population structure and genetic diversity of S. hirtipupa across a large part of its distribution), (ii) infer the time of divergence and demographic history, and (iii) verify the occurrence of cryptic taxa.Cryptic differentiation is common to many Simuliidae species . Accordiomplexes ,6,14. Cromplexes ,6,28–30.oophilic . The recpophilic . These rBlack fly collections were made on public or owner-permitted private lands. A permit to collect zoological specimens was provided by SISBIO (permanent permit number 10873–1 to NH). No collections involved endangered or protected species.S. hirtipupa . The COI gene was amplified from all DNA samples using the Folmer et al. [TM visualized on 1% agarose gels. Samples were then purified using ExoI and SAP (Fermentas@) at 37°C (30 min) and 80°C (15 min). DNA sequences were generated using an ABI 3730 DNA Analyzer at the Centro de Estudos do Genoma Humano at the Universidade de São Paulo (CEGH-USP).PCRs were performed using the Swift™ Maxi Thermal Cycler  and TC-512 Techne Thermal Cycler , 25 μl reaction volumes and 1–3 μl of DNA template. Each reaction had a final concentration of 0.5X buffer, 3mM MgClr et al.  forward 2P model, with Gamma shape 0.603, was used to calculate pairwise distances using MEGA 6 and Arlequin 3.5.All DNA sequence trace files were edited using BioEdit v. 5.0.6 . The besBayesian Analysis of Population Structure) [The Bayesian population mixture analysis was implemented in BAPS v. 4.13 5.3.1 (ructure)  to identD [Fs [Basic statistics of mtDNA diversity, including nucleotide and haplotype diversity, analysis of molecular variance (AMOVA), pairwise Fst estimates, as well as Tajima’s D  and Fu’sD [Fs  neutraliD [Fs  and compD [Fs .Demographic history was inferred based on estimated and observed mismatch distributions and calculated Sum of Squared Deviations (SSD) and Harpending’s raggedness index (rg) also in Arlequin 3.5 . The corhttp://www.fluxustechnology.com/sharenet.htm) to infer the relationships among haplotypes and their geographical distribution.A haplotype network was constructed using median joining (MJ) method Network 4.6.0.0 .The Bayesian species tree and the time of divergence among populations were estimated using BEAST 1.8.2 6  . In thisA continuous Bayesian phylogeography approach under a lognormal relaxed random walk (RRW model) diffusion model across continuous space was applied using BEAST v1.8.2 to infer the historical dispersal routes. The maximum clade credibility tree was used to generate a visual representation of the historical movements in Google Earth v7.1.5 (Google Inc.) using the Continuous Tree module in SPREAD v1.0.662 .Simulium hirtipupa from 19 populations in Brazil. Of the 121 base substitutions identified, a total of 98 were transitions and 23 were transversions. The sequenced region contained 153 polymorphic sites defining 148 haplotypes, which ranged from 5 to 22 per population . For instance, the genetic distance between the groups Gr-Pernambuco and Gr-Goiás (4.51%) was higher than that between the groups Gr-Pernambuco and Gr-Mato Grosso do Sul (2.95%).The Mantel test results indicated a weak correlation between genetic and geographic distance  Gr-Goiás, Gr-Minas Gerais, Gr-Central-East_1, and Gr-Central-East_2, and II) Gr-Mato Grosso do Sul, Gr-Pernambuco, and Gr-Bahia. Most groups had support values higher than 0.95, except for the group Gr-Central-East_2, whose support value was 0.68. However, the group Gr-Central-East_2 clustered together with the group Gr-Central-East_1 with high support value, both of which comprise haplotypes from shared populations . On the The haplotype network  exhibiteThe hierarchical analysis revealed significant genetic variation between groups and within populations indicating high level of genetic structuring and recent restricted gene flow between the groups except between groups Gr-Central-East_1 and Gr-Central-East_2, which comprise the same populations.FCT = 0.83, p = 0.00) followed by variation within populations of 13.66% (FST = 0.86). Only 3.22% of the variation was attributed to variation among populations within groups (FSC = 0.19) (Fst (P = 0.05) values. They showed significant genetic differentiation among most populations (88%) with pairwise values ranging between -0.05 and 0.93  . These rand 0.93 . ExceptiD value for all individuals was positive and non significant, as well as for most populations and groups. The exceptions, where the Tajima’s D values were negative and significant for populations, were 12MG, 28TO, and 17PE and for groups Gr-Goiás, Gr-Mato Grosso do Sul, and Gr-Pernambuco  comprising populations from the states of Pernambuco, Mato Grosso do Sul, and Bahia, and a second group (II) clustering populations from the states of Goiás, Tocantins, Minas Gerais, Espírito Santo, São Paulo, and also Bahia. Our data also indicate that genetic differentiation in S. hirtipupa occurred about 1.19 Mya during the Pleistocene when the two major groups might have been formed, the first around 1.16 Mya and the second around 1.14 Mya. The diversification of simuliids has occurred more recently such as in Simulium angulistylum  [Simulium tani  [Simulium siamense  [Simulium aureohirtum  [Our results revealed two major groups within ars ago) , Simuliuars ago) , SimuliuS. hirtipupa, since 65% of the distance values between populations were higher than 3.5%, which is the same value found among most groups. Previous studies have indicated the presence of cryptic species within Simuliidae with genetic distances of 4.5–6.5% [Genetic distance values revealed in this study indicate the presence of cryptic species within 4.5–6.5% . Moreove4.5–6.5% ,47. In cS. hirtipupa. The genetic distance and the presence of exclusive haplotypes in group Gr-Minas Gerais provide robust support for considering this lineage as a cryptic species. Since most groups comprise specimens from different populations, further studies are necessary in order to point out which other populations may represent cryptic species. For instance, a chromosome analysis of populations from the states of Bahia and Goiás showed that they differed from each other in one fixed inversion in three chromosomes, therefore indicating that S. hirtipupa, is a complex with at least two cytoforms [Our results indicate the likely presence of cryptic species within ytoforms .Populations 22MG (Gr-Minas Gerais) and 3MGa showed a strong molecular divergence with average genetic distance of 4.2% , which iPopulation 22MG is relatively more recent than the other populations. According to the divergence time estimate , populatS. hirtipupa have had distinct demographic histories underlying the haplogroups observed in this study. The haplotype diversity was high and nucleotide diversity low for most groups. This suggests that S. hirtipupa has undergone a rapid population expansion from an ancestral population from its putative center of origin in the northern region of the state of Minas Gerais. This also suggests that the time elapsed has been sufficient for the recovery of haplotype variation via mutation, albeit not long enough for the accumulation of large sequence differences. These indices were low for groups Gr-Goiás and Gr-Central-East_1, indicating that they may have experienced either a prolonged/severe population bottleneck in recent times or selective sweeps [Simulium species [The genetic diversity indices indicated that the populations of e sweeps . The gen species ,19,40, hFs) in our study corroborate a recent dispersal of populations of S. hirtipupa as has been observed for other species [The population expansion is also observed in the mismatch distribution analysis, which shows concordance with the spatial expansion model, since its distribution was unimodal for all groups, except for the group Gr-Central-East_1 that was multimodal. The unimodal distribution showed few pairwise differences in the mismatch distribution analysis, except for group Gr-Bahia for which the pairwise differences observed ranged from 8 to 12 , indicat species , 50.The bimodality or multimodality observed in group Gr-Central-East_1 could indicate that the population has experienced either chronologically distinct expansions or demographic equilibrium ,51,52. BFst index showed that the populations in the Gr-Goiás group are not genetically similar thus indicating the lack of present gene flow.Within group Gr-Goiás, the most frequent haplotype is most likely the ancestral haplotype as it showed the highest number of connections with other haplotypes, which can indicate a recent expansion of those populations , which iS. hirtipupa in the northern region of the state of Minas Gerais, followed by dispersal to other regions within the state of Minas Gerais and to the states of Bahia, Pernambuco, and Mato Grosso do Sul around 1.25 Mya. The populations of these states formed distinct groups  that still show low genetic differentiation (average 2.97%) between them when compared to other groups, even though they correspond to a more ancestral dispersal. S. hirtipupa then dispersed along the eastern coast of Brazil and to the state of Goiás, in the central region of Brazil. From there, S. hirtipupa dispersed to the state of Tocantins around 874 Kya. Then in a more recent period, dispersal occurred in the opposite direction from the state of Goiás to the state of Minas Gerais and from the eastern Brazilian coast (populations from the states of Espírito Santo and São Paulo) to the states of Minas Gerais and Bahia, and another route to the state of Pernambuco, reaching its present distribution  dispersed along the São Francisco river basin from the river’s east bank circumventing its headwaters and going along its west margin and then reaching the costal areas of the Northeast region. This dispersal would have resulted in adaptation to local habitats and population differentiation before the river changed its course [Calomys expulsus  from the river’s west bank circumventing its headwaters and then reaching the east bank [In this region, there are reports of other groups that dispersed circumventing the São Francisco river. The species s course . The samast bank .There are two dispersal routes towards the state of Pernambuco, north of the São Francisco river. The first dispersal route originates from the state of Goiás, and the second originates from the center of origin in the northern part of the state of Minas Gerais. One haplotype is shared between the populations from the states of Pernambuco and Goiás, which can be related either to an ancestral contact or to independent diversification. The high genetic diversity and geographic isolation of this group is concordant with the Carnaval-Moritz model of Pleistocene refugia for the Atlantic Forest .S. hirtipupa. Until the Mindel glaciation period (0.4 Mya) the São Francisco river flowed northwards towards the state of Piauí, after which its course changed in the direction of the eastern coast of the Northeast region. After a period of high humidity, it changed its course from endorheic to its present exhoreic pattern [The average genetic distance of the population from Pernambuco when compared to the other populations, excluding the populations from group Gr-Goiás, was lower (3.3%), even when compared to the group Gr-Mato Grosso do Sul (average 2.95%). The population from the state of Pernambuco and the populations from the state of Mato Grosso do Sul are located on opposite ends of the sampled area. Thus, the change in the river course most likely did not have an impact on the dispersal of  pattern ,58.Neither the São Francisco river nor the landscape of the state of Bahia acted as geographical barriers for the three populations in the group Gr-Bahia. Population 26BA from the Correntina river, a tributary on the west bank of the São Francisco river, clustered together with specimens from populations 1BA and 15BA, which are east of this river in the Eastern Atlantic Basin of Brazil. Dispersal most likely occurred from population 26BA to 1BA and from this to 15BA. Gene flow was observed in both directions between populations 1BA and 15BA , howeverS. hirtipupa. The populations of the Atlantic Forest included in this study showed no concordance with a latitudinal division of this biome, except for group Gr-Pernambuco in the northernmost part. These populations clustered into two groups, Gr-Central-East_1 and Gr-Central-East_2, which also encompassed populations from the biomes Cerrado and Caatinga with high genetic similarity (1.37%), indicating the occurrence of gene flow or shared recent history [Contrary to that which has been reported for other groups of animals, the rivers from the Brazilian east coast have not acted as physical barriers for  history . In the Melipona quadrifasciata  concluded that, even though its time of divergence was more recent, the river basins on the east coast of the Atlantic Forest also did not act as barriers to gene flow for those populations [Gymnodactylus darwinii  revealed that the rivers Doce, São Francisco, and Paraguaçu (Bahia de Todos os Santos) acted as barriers to gene flow for lizard populations within that complex [Partamona rustica  whose origin was probably on the west bank [Similarly, a study on ulations . On the  complex . Also, m complex . The Sãoest bank . Moreoveest bank . Contrasest bank , which aest bank .et al. (2014) [S. guianense are also congruent with the freshwater ecoregions [The populations analyzed in this study partially occupy the terrestrial ecoregions called North and Southeast by Vasconcelos . (2014) . The pop. (2014) . The onloregions .S. hirtipupa, is the geographic divide between the São Francisco and the Tocantins basins. This watershed divide is a sedimentary plateau that comprises two subunits: the Chapadão Ocidental do São Francisco and Patamares do Chapadão in the state of Goiás [Pyrrhura pfrimeri (Aves: Psittacidae) [Gracilinanus agilis (Mouse Opossum: Didelphidae) [A probable barrier between groups Gr-Goiás and Gr-Bahia is the Serra Geral de Goiás. The high genetic distance between these groups and the dispersal analysis indicated no gene flow between these populations. The Serra Geral de Goiás, dating back to the Cretaceous , and theof Goiás , which aof Goiás ,70. Thislphidae) .Some studies point out that elevation may act as a barrier to gene flow ,72–74. EAnother limitation to dispersal is the flight range of simuliids. Studies point out that the maximum flight range was 400 km and winds can help in their aerial dispersal . Our resClimate oscillations in the Pleistocene as well as vegetation expansion and retraction during that period  seem to Simulium hirtipupa.Our results revealed that, despite the genetic divergence from the Early Pleistocene, not enough time has elapsed for haplotype differentiation, especially on the extremes of the sampled area. Despite this, the data already indicate the presence of cryptic species and that the genetic diversity of some of the groups in the central area are partially associated with geography. Additional studies including populations from unsampled areas as well as nuclear markers will help further elucidate the evolution of S1 Fig2P model.Intrapopulation  and interpopulation  genetic distances (%) based on sequencing of a fragment of the mitochondrial COI gene. Analyses were conducted using the K(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 FigThe mismatch distribution for the groups was consistent with the model of demographic expansion, except for the Gr-Central-East_1 group. The lines represent the observed (black line) and expected (grey line) frequency of pairwise differences under the sudden population expansion model.(PDF)Click here for additional data file.S1 TableUnderlined numbers indicate shared haplotypes and in bold haplotypes repeated in the same population.(PDF)Click here for additional data file.


The data presented here may aid in identifying drugs that are more appropriate and/or require pharmacogenetic testing in these populations. Our findings emphasize on the importance of distinct, ethnicity-specific clinical guidelines, especially for the African populations, to avoid ADRs and ensure effective drug treatment.Interethnic variability in drug response arises from genetic differences associated with drug metabolism, action and transport. These genetic variations can affect drug efficacy as well as cause adverse drug reactions (ADRs). We retrieved drug-response related single nucleotide polymorphism (SNP) associated data from databases and analyzed to elucidate population specific distribution of 159 drug-response related SNPs in twenty six populations belonging to five super-populations . Significant interpopulation differences exist in the minor (variant) allele frequencies (MAFs), linkage disequilibrium (LD) and haplotype distributions among these populations. 65 of the drug-response related alleles, which are considered as minor (variant) in global population, are present as the major alleles (frequency ≥0.5) in at least one or more populations. Populations that belong to the same super-population have similar distribution pattern for majority of the variant alleles. These drug response related variant allele frequencies and their pairwise LD measure (r Pharmacogenomics studies interindividual variability in drug response, which is mainly caused by particular genetic variants associated with drug absorption, distribution, metabolism and elimination (ADME) , 2. DiffExcept for a small fraction of the total genetic variants, the majority  are commonly shared across populations . But thiDue to interpopulation genetic variations, drugs as well as markers used for pharmacogenotyping in one population may not be appropriate for another population. For example, HLA-b*58:01 allele is associated with allopurinol-induced severe cutaneous adverse reactions and rs9263726 can be used as a surrogate biomarker for the Japanese, but not the Australian and the Han Chinese populations , 20. PopMany aspects of the population history are reflected in genetic information . SNPs anSince allelic distribution and linkage disequilibrium (LD) of SNPs vary among populations, frequencies of different SNP alleles associated with drug response and patterns of LD should be analyzed separately for different populations. Here, we present the variant allele distribution, pairwise LD and haplotypes frequencies of 159 drug-response associated SNPs in five super-populations  and twenty six individual populations belonging to these super-populations.https://www.ncbi.nlm.nih.gov/snp) at the National Center for Biotechnology Information (NCBI) was searched using the keyword ‘drug-response’. After filtering out the duplicates and insertion/deletion (indel) polymorphisms, 159 SNPs were selected for further analysis. Drugs related to these SNPs along with their applications were searched in ClinVar archive at NCBI (https://www.ncbi.nlm.nih.gov/clinvar) [https://www.pharmgkb.org/) [The dbSNP database (clinvar)  and the kb.org/) .https://ldlink.nci.nih.gov/) [2≥ 0.8) were selected and compiled in a non-redundant list.We used the LDhap module at LDlink (ih.gov/)  to retriih.gov/)  for fiveih.gov/) . The Refhttps://www.metaboanalyst.ca/MetaboAnalyst/ModuleView.xhtml) [2 and D'). Euclidean distance based Ward’s algorithm was applied in hierarchical clustering to generate population dendrogram. All graphs were generated using the GraphPad Prism® (Version 6) software.The statistical tools available at Metaboanalyst (w.xhtml)  were useWe compiled the allele frequencies of 159 drug-response related SNP loci in a total of 32 populations  . DefininSNPs can be arbitrarily divided into many classes based on their allele frequencies , 43. In Private alleles, which are only present in a particular population among a broader collection of populations, are very useful in population genetics and human evolutionary genetics . We foun2 and D' are the two most widely used measures of LD. r2 is more robust and correlates better among different population samples [2 ≥ 0.8 in at least one of the five super-populations .r samples . We founulations . 4 of thSNP loci . 7 SNP ppulation . East AsWe found 10 haplotypes  having ≥ 2 variant alleles as well as with frequencies ≥ 0.2 in at least one of the five super-populations . All the2 and D' estimates of pairwise LD among these SNPs for multivariate clustering through principal component analysis (PCA), partial least square- discriminant analysis (PLS-DA), and hierarchical clustering  cannot cluster the component populations as distinctively as in PCA and hierarchical clustering  of the 159 SNPs, and both ring Figs  and 4. Wulations . The 1stulations . As showilar way . In bothing Figs  and 4. Aulations . It is tAmong the 159 drug-response related SNPs, we found 13 SNPs that have MAFs ≥ 0.2 in all super- and sub-populations. These SNPs are responsible for variable responses to bisphosphonates rs2297480), carbamazepine, phenytoin and antiepileptics (rs3812718), fentanyl, morphine and opioids (rs2952768), cisplatin  oxazepam and lorazepam (rs1902023), salbutamol and salmeterol (rs1042713), antineoplastic agents such as cyclophosphamide, fluorouracil and paclitaxel (rs1042522), platinum and platinum compounds (rs3212986), nicotine rs4680), debrisoquine (rs1135840), ethambutol, isoniazid, pyrazinamide and rifampin rs1041983), and sildenafil (rs5443)- drugs that are prescribed for conditions like epilepsy, postmenopausal osteoporosis, pain relief, osteosarcoma, urinary bladder cancer, ovarian cancer, medulloblastoma, brain cancer, breast cancer, neutropenia, stomach cancer, non-small-cell lung carcinoma, colorectal cancer, esophageal cancer, pancreatic cancer, uterine cervical cancer, anxiety, insomnia, asthma, tuberculosis, etc , 37, 41.983, and 80, debri0, carbamOne of these (rs2228001) variant-drug combinations has reached level 1B of clinical annotation . Level 118 SNPs have MAFs ≥ 0.2 in only one super-population . 11 of tThe minor allele (C) at the rs4917639 locus- (located in CYP2C9 gene) is present with ≥0.2 frequency only in the African super-population. Individuals with the CA or CC genotype may require decreased dose of warfarin compared to those with the AA genotype and there is level 2A clinical annotation for this variant-drug combination . An ethnrs885004 and rs8099917 have MAFs ≥ 0.2 only in Admixed American super-population. These may cause variable response to anthracyclines and related substances (rs885004) and peginterferon alfa-2a, peginterferon alfa-2b, and ribavirin, telaprevir, boceprevir (rs8099917). There is level 1B clinical evidence for rs8099917 associated variable efficacy of peginterferon alfa-2a, peginterferon alfa-2b, ribavirin, telaprevir and boceprevir in chronic Hepatitis C treatment . So, patTwo SNPs (rs2231142 and rs11045879) have MAFs ≥ 0.2 only in the East Asian super-population . These mrs17708472, rs2884737 and rs1876828 have MAFs ≥ 0.2 only in the European super-population. These SNPs may be responsible for variable response to warfarin  and budesonide, corticosteroids, fluticasone propionate, fluticasone/salmeterol and triamcinolone (rs1876828). Genotype at rs17708472 and rs2884737 may influence warfarin dose requirement [2 = 1) among them  have MAF ≥ 0.8 in all East Asian sub-populations. These share absolute LD  have been extensively studied in the context of pharmacogenomics because of their important roles in drug metabolism [CYP genes in all the super-populations are listed in CYP2D6*2 allele)- paroxetine, nortriptyline, codeine, doxepin, trimipramine, clomipramine, atomoxetine and amitriptyline and rs776746- tacrolimus combinations [CYP2D6*2 allele)-tramadol combination. So, tramadol and codeine, both of which are used to treat pain, may have less than optimum response in the majority of individuals with African ancestry. It may have serious clinical implications as there are racial/ethnic disparities in pain epidemiology, access to quality pain care, pain assessments and treatments and pain-related outcomes [CYP2D6 biomarker panel may improve the accuracy of CYP2D6 metabolizer status prediction [CYP allele frequency variations among the African populations as reported in a previous study [Cytochrome P450 family genes  in the East Asian populations on chromosome 2. Although the frequency of this haplotype (0.1052) is < 0.2, all alleles except the first one are the minor (variant) alleles at the corresponding SNP loci (rs6166_ rs6165_ rs10497203_ rs7582141_ rs6432512_ rs264651_ rs264588_ rs264631) in the global population. All of these SNPs, except rs6166 and rs6165, are responsible for variable responses to radiotherapy for prostate cancer. However, currently none of these variant-drug combinations qualifies for level 1A or level 1B clinical annotation [_T_AT_C), phenylthiocarbamide tasting (chromosome 7: T_A_C), ethambutol, isoniazid, pyrazinamide, rifampin (chromosome 8: _C_G_GC and T_T_A_A), anthracyclines and related substances (chromosome 9: A_A), warfarin, proguanil, mephenytoin, amitriptyline, citalopram, clomipramine, clopidogrel (chromosome 10: A_A_C_G_G_T), warfarin, acenocoumarol, phenprocoumon, vitamin K-dependent clotting factors (chromosome 16: _A_G_A_T_GC) and peginterferon alfa-2a, peginterferon alfa-2b, and ribavirin, telaprevir, boceprevir (chromosome 19: G_T_T and G_T_G) [CYP2C gene region and all except one SNP in the haplotype on chromosome 16 are located in VKORC1 gene region. These are two very important pharmacogenes. Both of these haplotypes  are present in global population with frequency ≥ 0.2. Clinical annotations for rs12777823-warfarin and rs9923231-warfarin combinations have already been discussed. Among the other SNPs in the haplotype on chromosome 10, level 1A clinical annotation is available for rs4244285-clopidogrel, rs4244285-amitriptyline and rs1799853-warfarin combinations [Apart from the 10 haplotypes with at least two global minor alleles and frequencies ≥ 0.2 in at least one super-population , there inotation . SNPs ind G_T_G) , 37. It Presence of long stretches of genomic regions with high LD in a particular population means that a number of neighboring SNPs are in strong or absolute pairwise LD with the functional or causal variant within that population. So, SNPs that are in strong or absolute pairwise LD with the causal variant will give similarly strong association signal. In that case, trans-population analysis, which utilizes differences in LD patterns across different populations, can be used to narrow the list of possible causal variants . Hence, 2 ≥ 0.8) in African super-population, compared to 11 pairs in global population (2> 0.9) in all super-populations (2≥0.8) with one another. Majority of these SNP pairs (32) maintain r2 ≥ 0.9, which is highly distinctive of the East Asian population  of 159 drug response-related SNPs for multivariate analysis using PCA, PLS-DA and hierarchical clustering  or D′  [2 or D' both depend on the allele frequencies, r2 is a more stringent measure and depends more on allele frequencies [Linkage disequilibrium (LD) that exists among DNA variants in the current human genome is the result of historical evolutionary forces, particularly finite population size, mutation, recombination rate, and natural selection . LD betwuencies) , 100. Thquencies –103.2 values, Americans of African Ancestry (ASW) and African Caribbeans in Barbados (ACB) clustered with the African sub-populations. Based on the historical records, the African Americans and the African Caribbeans in Barbados are descended from slaves who were imported mostly from West Africa during the eighteenth century [2.In PCA, PLS-DA and dendrogram with MAF and r century . African century , 82. Amo century . Since,  century , they ar century , 105. As2. A dendrogram recapitulates the relationships among population groups. Individuals who cluster near each other in the tree could either share a recent common ancestry and/or experienced gene flow [2. So, human migration patterns and demographical history can be more accurately reconstructed with allele frequencies than pairwise LD measures. More dependency of r2 on allele frequencies in comparison to D' may explain why r2 is better than D' at reconstructing such patterns and history.Latin American populations- Colombia, Mexico, Peru, and Puerto Rico- have distinct patterns of continental genetic admixture . Puerto ene flow . Finnishene flow . MAF basAlthough most of the large inter-continental differences in allele frequency may not result from positive selection , there mCYP genes in African populations [Considering the high similarities of MAFs in sub-populations belonging to the same super-population, it may seem tempting to study only the super-populations to predict drug responses in all of its sub-populations. But such generalizations may not be appropriate. Only 26 sub-populations were included in this study and most of these samples cannot be considered representatives of their source populations. There can be marked differences in the allele frequencies of important pharmacogenes among the sub-populations belonging to the same super-population. Such differences were observed in the allele frequencies of ulations . There mulations . So, difulations . Individulations . Adminisulations . Deficieulations . Furtherulations .In our study, we looked at only the SNPs that may cause variable drug responses. But other factors e.g., diet, chemical exposures from the environment, disease state, etc may be sources of variability in drug response as well , 119. EpThere is a global concern to increase pharmacogenetic testing to ensure drug safety and enhance drug efficacy , 126. HoS1 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(DOC)Click here for additional data file.