Geum rivale and selfing Geum urbanum, sister species that hybridize in contemporary populations. We generated and used a draft genome of G. urbanum to develop dd‐RAD data scorable in both species. Coalescent analysis of RAD data from allopatric populations indicated that the species diverged 2–3 Mya, and that historical gene flow between them was extremely low (1 migrant every 25 generations). Comparison of genetic divergence between species in sympatry and allopatry, together with an analysis of allele frequencies in potential parental and hybrid populations, provided no evidence of contemporary introgression in sympatric populations. Cluster‐ and species‐specific marker analyses revealed that, apart from four early‐generation hybrids, individuals in sympatric populations fell into two genetically distinct groups that corresponded exactly to their morphological species classification with maximum individual admixture estimates of only 1–3%. However, we did observe joint segregation of four putatively introgressed SNPs across two scaffolds in the G. urbanum population that was associated with significant morphological variation, interpreted as tentative evidence for rare, recent interspecific gene flow. Overall, our results indicate that despite the presence of hybrids in contemporary populations, genetic exchange between G. rivale and G. urbanum has been extremely limited throughout their evolutionary history.Within the plant kingdom, many genera contain sister lineages with contrasting outcrossing and inbreeding mating systems that are known to hybridize. The evolutionary fate of these sister lineages is likely to be influenced by the extent to which they exchange genes. We measured gene flow between outcrossing  https://doi.org/10.1111/mec.14444 Allopatric G. urbanum populations occur in the south‐east of the UK, where rainfall is low (<650 mm/year), and in the south of its continental European range  to the outbreeder . We (i) test whether allele frequencies of a potential hybrid population disproportionately reflect allele frequencies of one likely parental population  score sympatric samples using species‐diagnostic SNPs to quantify introgression for individuals. Finally (v), we relate estimates of an individuals’ hybridity to its morphology to test whether a plant's morphology reflects its genotypic hybrid status and to test whether morphology varies with focal RAD loci.To test for recent gene flow between the 22.12.1.1G. rivale individuals were sampled from each of three high elevation tall herb communities located on three distinct Scottish mountain ranges above the altitudinal limits of G. urbanum (Table G. urbanum is absent (Table http://linnaeus.nrm.se/flora).Four um Table . One indG. urbanum plants were sampled from each of 10 populations in south‐east England . 0.5 g of young leaf tissue was removed from sampled plants and stored dry in silica gel prior to DNA extraction.Two nd Table , a regio2.1.2G. rivale and G. urbanum beyond hybrid swarms. Hence, we sampled sites (a) with both species present and (b) where each species lived individually. However, unlike “allopatric” populations (see above), the sites with only one Geum species had habitat characteristics that could allow introgression by interpopulation gene flow or could have allowed both species to co‐occur in the recent past. Hence, we broadly define “sympatric” populations as localities where (in the absence of selection) recent introgression should be possible. The Botanical Society of Britain and Ireland (BSBI) database was searched for the location of sites recorded as containing G. rivale alone (15), G. urbanum alone (14) or both taxa (10) in eight contiguous 10 km × 10 km squares within vice county 81 in the British Isles (Berwickshire) . Plant collections were made by taking cuttings of plants (a minimum of 5 m apart) from these sites in August 2013. At single taxon sites, two individuals were randomly sampled. At mixed sites, cuttings were collected from four randomly selected Geum plants located across the full range of ecological conditions present at the site. Initial field identification was to genus only. Ninety‐six of the 98 cuttings were rooted successfully in 20‐cm‐diameter pots containing commercial potting compost. Newly emerging leaves (0.1 g/plant) were frozen (−80°C) prior to DNA extraction. Plants were overwintered outside in a randomized array in Edinburgh to induce flowering.We aimed to test whether recent introgression affects le alone 5, G. urb2.22.2.1G. urbanum and G. rivale, respectively). Therefore, to ensure adequate depth of coverage, we adopted the ddRAD technique that allows modulation of the number of RAD tags generated per genome  or an Illumina miseq using 150 base PE reads (v2 chemistry).DNA quality was evaluated using the E‐Gel Precast Agarose Electrophoresis System (ThermoFisher Scientific), and samples quantified using the Qubit dsDNA BR Assay Kit (ThermoFisher Scientific). Sample preparation, library construction and PCR amplification for double‐digest RAD sequencing were modified from Peterson et al. . Superpo2.2.5miseq and hiseq Illumina technologies for ddRAD analyses, we used fastx trimmer (http://hannonlab.cshl.edu/fastx_toolkit/) to trim miseq reads to 125 bp . We de‐multiplexed and filtered reads for quality using process_radtags  on read mapping for one allopatric individual from each species .Mapping ddRAD sequences from the two M values, we used gsnap ; however, the percentage of read pairs that mapped jumped to 82.84% for M = 10. Similarly, the percentage of G. rivale PE reads that mapped in proper pairs increased gradually from 39.21% to 58.16% over M = 2 to M = 9, with a large increase to 79.93% for M = 10. Therefore, the structure of the Geum genome  appears to present different environments for mapping ddRAD data at a threshold genetic diversity of c. 8.55% . To avoid mapping to paralogous regions while maximizing the number of G. rivale reads correctly mapped to the heterospecific draft genome, we specified M = 6 throughout.For a range of 2.2.7stacks’ ref_map pipeline .We used 2.2.8Geum because reads from paralogous genome regions could map to identical sites. To identify genome regions that potentially attract paralogous reads, we used stacks’ populations module to calculate observed heterozygosity and FIS at each identified SNP. Thereafter, custom scripts were used to collate data and restrict further analysis to scaffolds free of SNPs that exhibited either excess heterozygosity (>0.5) or negative FIS. The heterozygosity and FIS calculations for this purpose only considered UK allopatric Geum samples that comprised four individuals from each of three Scottish “allopatric” G. rivale populations , and one randomly chosen individual from each of the 10 Southern England G. urbanum populations  plus one each from France and Portugal  and measure differentiation among populations (FST) for each species using data from the three allopatric G. rivale populations in Scotland  and the 10 allopatric G. urbanum populations in England . Estimates of FIS and FST only considered radtags present in both species and in all individuals of a given analysis.We applied the 2.3.3We used alternately fixed SNPs in allopatric populations of the two taxa  heterozygous sites exclusive to G. urbanum, (iii) heterozygous sites shared by both species and (iv) fixed differences between species. We counted these site types within 117 bp radtags (block), and treated each radtag as an independent block. For randomly mating populations, the polymorphisms at each block represent an independent outcome of the coalescent process, which is a function of the species’ history. As stacks ignores RAD tags that are monomorphic, we conditioned the likelihood on only observing variable blocks by normalizing the probabilities of blockwise mutational configurations by 1 − p_IBS, where p_IBS is the probability of identity in state for a block.We used an analytic likelihood framework to assess the support for alternative models of divergence between d Barton  and is bd Barton . Brieflyd Barton ”, above G. rivale and G. urbanum. Therefore, we compared three models:  species diverged at time T with no introgression,  a history of divergence with gene flow from G. urbanum to G. rivale and  divergence with gene flow from G. rivale to G. urbanum (four parameters). All models assume instantaneous species divergence and a constant Ne within taxa. We also constrained the effective population size of the ancestor (Nanc) to equal that of G. rivale  expected to result from the transition to inbreeding .We primarily wish to test for historical introgression between G. urbanum individual  curves for these parameters .Maximum‐likelihood estimates under each model were obtained in see Data . To esti2.42.4.1G. rivale or G. urbanum populations; f3 compares allele frequencies in two potential source populations  and a potential hybrid population (c). A significantly negative value of f3 indicates introgression , because we expected these populations  to most contribute to introgression in Berwickshire. As we are interested in detecting long‐term introgression, we omitted four obvious early‐generation hybrids in Berwickshire, described in the Section G. rivale and G. urbanum populations as potentially admixed . All allele frequency estimates involved at least 10 individuals per population.We calculated f3 as the mean of (c−a) (c−b) over all SNPs ) + ((1 − PX) × PY)]/n between the two species, separately for allopatric and sympatric samples: P represents the frequency of a focal allele in the sample of species X and Y, and n is the sequence length  for unrooted samples; that is, in the absence of gene flow, we expect equal dxy for sympatric and allopatric comparisons. The analysis excluded four individuals in the Berwickshire sample identified as obvious early‐generation hybrids or backcrosses , which we assume to be independent with respect to linkage, and determined the mean dxy and its SE across scaffolds. All analysed radtags were present in at least 12 individuals for each species/population type combination.To test whether potential gene exchange resulted in reduced differentiation between the species in sympatry, we calculated genetic differentiation dxy estimates, we similarly calculated dxy between the two most diverged allopatric UK G. rivale populations  corresponding to the two Geum species, with the possibility of genetic admixture of individuals. The SNP data were derived from a stacks analysis of all 96 successfully genotyped individuals in the sympatric Berwickshire sample together with data from the British and European allopatric populations of the two species. We analysed two differently filtered data sets: (i) SNPs were callable in any fraction of individuals, which retained more SNPs but included missing values for some individuals, and (ii) all SNPs were required to be callable in all individuals. Both analyses used SNPs that had been filtered for paralogs, and considered only a single SNP per scaffold to minimize the effect of linkage between SNPs.As a first approach to analysing introgression between the two 2.5.2G. rivale in origin . SNPs with either uncallable genotypes  or third alleles  were excluded. We tabulated the frequency of SNPs that were heterozygous for the species‐diagnostic alleles in each individual.In our second approach to analysing recent introgression, we used custom scripts and the species‐specific SNPs identified in Section  (above) stacks accounts for coverage when calling genotypes .In general, our analyses did not specify minimum coverage because 2.62.6.1G. rivale and G. urbanum, were measured: angle at which flowers are held  (FA), petal length (mm), petal width (mm), petal shape , sepal length (mm), stamen number, stipule length (mm) and stipule width (mm)  was conducted on the total sympatric sample using data on all eight morphological characters. Distinct groupings were recognized on scatterplots of the first two principal component scores and related to parental species and hybrid classes defined genetically by species‐specific SNPs. PCA based on the same characters was also used to summarize morphological variation separately within dxy in two ways. For the five radtags found on these scaffolds, we calculated dxy (as above) between the seven Berwickshire G. urbanum samples that putatively possessed these sequences due to introgression vs. either (i) Berwickshire G. rivale samples or (ii) the remaining Berwickshire G. urbanum samples. Both dxy calculations excluded four obvious early‐generation hybrids . The distribution of per cent variant bases per scaffold is shown in Figure Core genic regions were well assembled and appeared to be present in approximately three (haploid) copies as expected for an ancient hexaploid. We searched our assembled genome for 248 ultra‐conserved eukaryotic genes (CEGs), listed by Parra, Bradnam, and Korf , using t3.1.27 reads remained in the miseq data, and between 6.0 × 107 and 7.3 × 107 reads remained that derived from the five hiseq libraries. Following alignments, assembling radtags with stacks and applying corrections with rxstacks, our data set included 230,356 radtags for G. rivale and G. urbanum, collectively. However, coverage was highly stochastic. For example, only c. 2%  of radtags were represented in more than one half of our samples.Following quality filtering, 2.7 × 103.1.3stacks’ populations module was used to analyse the raw SNP data from the allopatric populations of the inbreeding taxon G. urbanum, FIS estimates were low and sometimes negative, which is unexpected for a highly inbreeding species. This suggests that paralogous reads have mapped to identical locations and thereby increased individual heterozygosity (see Table FIS) and, unless specifically noted, we henceforth only present results from paralogy‐filtered SNPs.When the 3.23.2.1G. rivale but invariant in G. urbanum. This type of polymorphic site occurred approximately four times more frequently than the reverse case , included one (of the two) sample(s) that was heterozygous at 92% of the 71 polymorphic SNPs analysed, suggesting that this sample was derived from a recent outcrossing event between two different inbred lines. In the second population, Selwyn Wood, only one SNP was recorded as polymorphic, suggesting that this population may have been founded by few (possibly a single) highly selfing individuals.For the allopatric populations sampled in Britain, we obtained G. rivale in Britain exhibited less population differentiation than G. urbanum populations , as expected from their contrasting mating systems.Allopatric populations of 3.2.3G. urbanum–G. rivale sample pairs. All three pairs had approximately 1,400 SNPs distributed among c. 660 blocks . Model comparisons for all three sample pairs reject a model of strict divergence and suggest that introgression has occurred between the Geum species , the model of gene flow from G. rivale to G. urbanum  fits the data significantly better than the model of strict divergence , whereas the model IMu➔r does not fit significantly better than div2 (Table r➔u and IMu➔r) also fit the data significantly better than the div2 model for the third pair (involving sample Ben Lui 4), but IMr➔u and IMu➔r have effectively equal support  yield similar estimates of Ne and divergence time , and all three models suggest that the species diverged approximately 2–3 Mya (Table u➔r and IMr➔u both suggest a low but significant long‐term rate of effective gene flow (M ≈ 0.04), of approximately one migrant every 25 generations .The three models  when calculated for Berwickshire G. rivale samples, but did not differ significantly from zero . f3 was positive (0.00174) for the Berwickshire G. urbanum samples and again did not differ significantly from zero .f3 tests provided no evidence for long‐term introgression from UK allopatric 3.3.2G. rivale and G. urbanum is similar for UK allopatric and sympatric samples (mean dxy (± SE) equals 0.0115 ± .0005 and 0.0112 ± .0005, respectively). By comparison, dxy between the two most diverged allopatric UK G. rivale populations equalled 0.00286 ± .00008. We note that, due to the fact that stacks ignores monomorphic radtags, these estimates should be viewed as relative measures (and upper limits) of dxy, and not as absolute estimates  or a fraction of  individuals analysed in the combined allopatric and sympatric populations yielded highly consistent results. Figure faststructure results for analyses based on 188 SNPs. All except one of the 36 individuals in the allopatric populations, and all except four of the 96 individuals in the sympatric populations, show <1% admixture. In the sympatric population, Q values  of the four individuals with substantial admixture are 0.496, 0.517, 0.663 and 0.935. These Q values provide no evidence for significant, advanced introgression. The substantially admixed individuals present are likely recently formed F1 or F2 hybrids, and different generations of backcrosses to G. rivale.3.4.2G. urbanum samples that had not been used to generate species‐diagnostic SNPs; 215 of 220 diagnostic SNPs were homozygous for the G. urbanum allele in all individuals. Five SNPs were homozygous for the putative G. rivale allele in one or more of the 10 G. urbanum individuals, indicating that these SNPs were not fixed between species. They were removed from subsequent analysis of 96 individuals of Geum from the Berwickshire population. The mean number of SNPs successfully scored per individual was 207.3  are common to both analyses. Early‐generation hybrids were significantly associated with “mixed” rather than “pure” sites within Berwickshire . We highlight, however, the lack of evidence for introgression even in the 10 sites that had both species present at the time of sampling: the vast majority of these 40 samples  have genotypes in which more than 97% of the species‐specific SNPs derive from only one of the two  of 96 han = 9, n = 12 respectively), they were also homozygous for alleles specific to the same taxon. Two further individuals contain alleles derived predominantly from G. rivale, but additionally possess a substantial fraction of G. urbanum alleles  are present in homozygous form. All other G. urbanum‐specific alleles  present on the same scaffolds were also homozygous. If these hybrids had been simple backcrosses, all G. urbanum alleles would have been present as heterozygotes.Neither of the two individuals possessing a hybrid index close to 0.5 are heterozygous at all SNPs, the situation anticipated if they were simple F1 hybrids . Furthermore, 76% are polymorphic in G. rivale only, 20% are polymorphic in G. urbanum only, and 4% are polymorphic in both species. These proportions are not significantly different from those found in the total sample of SNPs scored  of species‐specific alleles from one taxon, 33 of the 47 individuals assigned to s Figure . These cG. rivale and G. urbanum populations. However, two species‐diagnostic SNPs at radtags 20,454 and 24,791, respectively, and originally classified as specific to G. rivale, showed complete association in genotypic state within sympatric G. urbanum. The alleles were present at a mean frequency of 14.4%, distributed across six sites. In six individuals, both alleles were present in homozygous form, while a seventh individual was heterozygous at both SNPs. As the SNPs involved are located on different scaffolds, these results may indicate the presence of a section of genome spanning the two scaffolds, possibly derived from G. rivale, which is segregating in the G. urbanum population. When two additional species‐specific SNPs located on the scaffold, but up to 19.5 kb from radtag 24,791, were analysed, these were also found to possess alleles specific to G. rivale and to be in complete linkage disequilibrium with the original SNP scored. This provides further evidence for a large section of un‐recombined DNA, derived from G. rivale, segregating in the sympatric G. urbanum population.With one exception, the putatively introgressed alleles discussed above were randomly associated with each other within each of the sympatric dxy value calculated for the putatively introgressed G. rivale scaffolds also supported this hypothesis, as these dxy estimates mirrored the mean between‐species dxy estimates based on all radtags. The putatively introgressed scaffolds in G. urbanum showed little divergence from Berwickshire G. rivale samples (mean dxy = 0.00287); this divergence was very similar to that observed between allopatric G. rivale populations (see Section G. urbanum samples and the remaining G. urbanum Berwickshire samples (dxy = 0.01323); this dxy estimate was similar to that observed between species, when calculated for all radtags  and G. urbanum (N = 40) on the basis of species‐specific SNPs fall clearly into the two groups with positive and negative PCA1 values, respectively. The two individuals with intermediate PCA1 scores correspond to the early‐generation hybrids identified with clustering and species‐specific SNPs. The two genotypes identified as complex backcrosses to G. rivale, on the basis of species‐specific SNPs, group morphologically with individuals classified as G. rivale on the PCA plot.Morphological measurements of eight floral and vegetative characters were obtained from 87 of the 96 individuals from Berwickshire scored for species‐specific SNPs. Based on these data, PCA1 and PCA2 accounted for 82.4% and 6.7% of the variation, respectively, and distinguished two major groups, with two individuals falling between these on the first PCA axis . Greater flower angle is a feature characteristic of G. rivale and is most heavily weighted in PCA2.We used PCA generated, above, to determine whether presence of putatively introgressed genetic material marked jointly by alleles at radtags 20,454 and 24,791 affected the morphology of 4G. rivale and G. urbanum have allowed us to measure historical and contemporary gene exchange in this outcrossing and selfing species pair. Coalescent modelling of RAD data from allopatric populations suggests that the sister species split 2–3 Mya. Although we find some support for models that include gene exchange between the species between the time they split and the isolation of the allopatric populations, the estimated long‐term rate of introgression is extremely low (1 genome per 25 generations); also, violation of model assumptions could yield a spurious signal of introgression (see below). Similarly, neither a comparison of genetic differentiation between allopatric and sympatric populations nor an f3 test provides evidence for significant recent introgression within an area of sympatry. This conclusion agrees with both clustering analyses and analysis of species‐specific markers, each suggesting little (if any) admixture in Berwickshire. Thus, our analyses suggest that, despite widespread contemporary hybridization, introgression between G. rivale and G. urbanum has not been important in their evolution. The only piece of evidence that contradicts this general conclusion is the detection of a >19.5 kb DNA sequence, apparently derived from G. rivale, segregating within the sympatric G. urbanum population. Although this introgressed genetic material significantly affects the morphology of G. urbanum plants, it is present at a frequency of 0.14 in Berwickshire G. urbanum population and involved a small fraction of the Geum genome (<1% of our species‐diagnostic SNPs).Our comparative genomic analyses of allopatric and sympatric populations of Geum species are considerably older (2–3 Myr) than estimates for other sister taxa that possess contrasting outcrossing and selfing mating systems. These range from 50 to 100 kya for Capsella grandiflora and C. rubella  is an order of magnitude lower than that from other inbreeding/outbreeding pairs: M. nasutus into M. guttatus , we potentially conflate changes in Ne with introgression, which, again, could lead to a spurious signal of introgression. Given these issues and the extremely low estimates of introgression, we do not interpret support of IM models over the div2 model as strong evidence for ancient long‐term gene flow between the Geum species .Our coalescent analyses necessarily rely on simplifying assumptions. First, the blockwise site‐frequency spectrum approach assumes a constant um Chen, ; by assudxy) in sympatry compared with allopatry, nor any signal of introgression in the f3 analysis  and the hybrid index based on species‐diagnostic SNPs identified the same four individuals as early hybrids and backcrosses; they yielded highly correlated (r = .99) estimates of genomic composition, and both approaches matched morphological classifications. Leaving aside the four early‐generation hybrids identified, our analyses suggest that 35% of the Berwickshire samples scored showed no detectable introgression. Within the remaining samples, the upper limit for the proportion of introgressed genome is between 1%  and 3% (species‐specific markers). At the least, the 3% estimate is likely to be inflated because the assumption that all our species diagnostic SNPs are alternately fixed between the species is unlikely to be true. These estimates apply to a broad “sympatric” region, including 10 sites where the Geum species co‐occurred and 29 sites where they were found individually at the time of sampling.We also found little evidence for recent introgression. Our first tests of introgression in contemporary populations involved comparisons of allopatric vs. sympatric populations: we did not find reduced species differentiation : DNA sequences:G. urbanum genome available the European Nucleotide Archive (ENA): http://www.ebi.ac.uk/ena/data/view/PRJEB23267.Read used to produce draft http://www.ebi.ac.uk/ena/data/view/PRJEB23012.ddRAD reads also available from ENA: https://doi.org/10.5061/dryad.k67fv.Morphological data available on Dryad: Scripts available on Github, at crispinjordan/Geum‐introgression.None declared.C.Y.J., K.G. and R.A.E. conceived the study; C.Y.J., F.T., M.T. and R.A.E. gathered data; C.Y.J., K.L., F.T. and R.A.E. analysed data; C.Y.J., K.L. and R.A.E. wrote the manuscript with input and revisions from all co‐authors. Click here for additional data file. Click here for additional data file.


This study of nuclear microsatellite loci demonstrates genetic relationships between twenty four Scots pine populations from Europe and Asia Minor. The analysed populations were assigned to several groups that corresponded to the geographical regions of their occurrence. Significant differentiation over short geographical distances was observed between isolated populations within the Iberian and Anatolian Peninsulas (Asia Minor), which contrasted with the absence of genetic differentiation between distant populations, for instance in central and northern Europe. Despite the signs of isolation found in peripheral stands, a similar level of genetic variation and no evidence of a recent bottleneck was found across the populations. FST = 0.035); however, the population structure was not homogenous, which was clearly evident from the allelic frequency spectra and Bayesian assignment analysis. Significant differentiation over short geographical distances was observed between isolated populations within the Iberian and Anatolian Peninsulas (Asia Minor), which contrasted with the absence of genetic differentiation observed between distant populations e.g., between central and northern Europe. The analysed populations were assigned to several groups that corresponded to the geographical regions of their occurrence. These results will be useful in genetics studies in Scots pine that aim to link nucleotide and phenotypic variation across the species distribution range and for development of sustainable breeding and management programs.Gene flow tends to have a homogenising effect on a species’ background genetic variation over large geographical areas. However, it is usually unknown to what extent the genetic structure of populations is influenced by gene exchange between core and peripheral populations that may represent stands of different evolutionary and demographic history. In this study, we looked at the patterns of population differentiation in Scots pine—a highly outcrossing and wind pollinated conifer species that forms large ecosystems of great ecological and economic importance in Europe and Asia. A set of 13 polymorphic nuclear microsatellite loci was analysed to infer the genetic relationships among 24 populations  from Europe and Asia Minor. The study included specimens from the primary continuous range and from isolated, marginal stands that are considered to be autochthonous populations representative of the species’ putative refugial areas. Despite their presumably different histories, a similar level of genetic variation and no evidence of a population bottleneck was found across the populations. Differentiation among populations was relatively low (average  Demographic and evolutionary processes interplay to shape genetic variation that is crucial to maintaining species’ adaptive responses to changing environments. Genetic variation among plant populations in the Northern Hemisphere has been shaped by range shifts and recolonization following the last glacial maximum (25–18 000 years ago) . The assForest trees are known to form large, wind pollinated populations and maintain a high level of genetic and phenotypic variation in comparison to other plant species . High diPinus sylvestris), which is one of the most ecologically and economically important forest-forming tree species in Eurasia. Some former phylogeographic studies of this pine based on isozyme polymorphisms, organelle DNA and palynological records have shown that the most abundant populations of Scots pine survived the cold periods of the Pleistocene within southern Eurasia in the Iberian, Apennine and Balkan Peninsulas and in the Anatolian and Caucasus Mountains (Picea abies)  . AlthougnSSR) markers. Nuclear markers in pines are distributed by seeds and pollen and could potentially be dispersed at large geographical distances. Therefore, the identification of populations with divergent genetic backgrounds could suggest the existence of distinct populations that do not share a recent history. To our knowledge, this species has not yet been investigated on such a large geographical scale using this type of neutral marker. In this study, we used a set of 13 nSSR loci to examine genetic diversity and test for the existence of populations of different origin. We aimed to check if there is any difference between core and peripheral populations considering the presumably homogenising effect of long distance gene flow. Information regarding the genetic relationships among populations is particularly important to advance studies of the genetic architecture of observed variation in phenotypic traits that have been shaped by selection and local adaptation within the Scots pine distribution.The aim of this study was to assess the genetic variation and structure of Scots pine populations from central and north Europe in relation to its marginal populations from the European and south-west Asiatic refugial areas based on the analysis of nuclear simple sequence repeat ; Spag7.14, PtTX2146, PtTX3107 and Spac11.4 (multiplex 2) and PtTX3025 and PtTX4011 (multiplex 3). The PCR for each sample was conducted in a total volume of 10 μL containing 5 μL of Qiagen Multiplex Master Mix (2×), 0.2 μL of primer mix (20 μM), 1 µL of Q-Solution (5×), 0.8 μL RNase-free water and 3 μL of DNA 84 template (approximately 10–20 ng). The following PCR amplification conditions were used: multiplex 1, initial denaturation at 95 °C for 15 min, 38 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 90 s, extension at 72 °C for 90 s and final extension at 72 °C for 10 min; multiplex 2, initial denaturation at 95 °C for 15 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 90 s, extension at 72 °C for 90 s and final extension at 72 °C for 10 min; multiplex 3, initial denaturation at 95 °C for 15 min, 10 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 40 s with temperature decreasing by 1 degree per cycle, extension at 72 °C for 90 s, 36 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 40 s, extension at 72 °C for 90 s and final extension at 72 °C for 10 min. The fluorescently labelled PCR products, along with a size standard (GeneScan 500 LIZ), were separated on a capillary sequencer ABI 3130 . The allele’s size was determined using GeneMapper software .The study comprised 24 Scots pine populations , 8 of whividuals . GenomicR22) for a minimum sample size of 22 individuals and inbreeding coefficient values (Fis). The deviation of genotypic frequencies from Hardy–Weinberg equilibrium (HWE) were also identified utilizing the inbreeding coefficients  for each population using the Bayesian method implemented in INEST 2.0 software (Fis = 0).Genotypic disequilibrium between pairs of loci was tested at the single population level and across all populations with a Fisher’s exact test using ARLEQUIN 3.11 . The allnts Fis;  with a csoftware . The evaFST and RST) was conducted using ARLEQUIN 3.11. Moreover, due to the presence of null alleles, the global, pairwise and within-geographic regions FST were calculated using FreeNA software. FreeNA applies the ENA  correction method to effectively correct for the positive bias induced by the presence of null alleles in the FST estimation. Bootstrap 95 % confidence intervals (CI) were calculated for the global FSTENA values using 2000 replicates across the loci. The statistical significance of the FST values was verified with ARLEQUIN 3.11.To estimate the proportion of the total genetic variation due to differentiation among populations, an Analysis of Molecular Variance (AMOVA) based on two distance methods  was significant, the permutation test proposed by To evaluate the ability of the stepwise mutation model (SMM) to differentiate among populations and geographical regions, which in turn indicates whether phylogeographical structures exist, the computed FST/(1−FST)) distances. The statistical significance of the correlation was calculated for all populations and sets of populations located along latitudinal and longitudinal transects using 9999 permutations with GenAlEx 6.The genetic population structure (in the case of microsatellite markers) can arise due to isolation by distance (IBD), range expansions, diffusion of genes through space in migratory events and/or allelic surfing (STENA matrix and GenAlEx 6 software. Phylogenetic relationships between the populations were investigated using POPTREEW (DST)  was applied to visualize the patterns of the genetic structure of the populations using a pairwise FPOPTREEW . Nei's sEW (DST)  was chosK) without considering sampling origins and assuming that each cluster is in optimal Hardy–Weinberg (H–W) and linkage equilibrium (LE). The correlated allele frequencies and admixture model used allowed for mixed recent ancestry of individuals and assigned the proportion of the genome of each individual to the inferred clusters. Moreover, because all the microsatellite loci used in this study were affected by null alleles (see Results section), the recessive alleles option was chosen. Twenty independent runs were performed for each K, from K = 1 to 24, with burn-in lengths of 500 000 and 100 000 iterations. The probability distributions of the data (LnP[D]) and the ΔK values . The number of alleles per locus ranged from 3 (psyl25) to 40 (Spag7.14), with an average of 12.3 [see Supporting Information—Table S2]. The estimated frequency of null alleles for most of the loci was moderate  with the exceptions occurring at three loci: psyl18 (8.7 %), Spag7.14 (8.3 %) and PtTX3107 (16.2 %) [see Supporting Information—Table S2].The 13 nuclear microsatellite loci investigated were polymorphic, providing a total of 160 size variants. There was no significant linkage disequilibrium between pairs of loci across all populations (AR22) were comparable in all investigated populations and ranged from 5.1 in the T1 population to 6.2 in the PL1 population. The mean effective number of alleles was Ae = 3.4. The lowest number of effective alleles was observed in the Tokat–Yıldızeli population from Turkey , whereas the highest number of effective alleles was found in the population from southwestern Poland . Twenty private alleles were also detected among some of the studied populations, and their frequency ranged from Pa = 0.0 % to Pa = 23.1%. Similar levels of gene diversity were found in all populations, which ranged from 0.48 to 0.53. However, pine populations from the peripheral stands appear to show greater diversity (with an average of 0.52) in comparison with populations from central and northern Europe (with an average of 0.48). The level of the overall observed heterozygosity (Ho) per population  was similar for all populations and slightly lower than the level of expected heterozygosity (He) . The inbreeding coefficients ranged from 0.017 in the F2 population from Finland to 0.282 in the T5 population from Turkey, with an overall mean of 0.120. However, the inbreeding coefficients may be highly overvalued due to the presence of null alleles. Because of this and based on our previous results that showed that null alleles were present at all investigated loci [see Supporting Information—Table S2], we used the IIM approach (see M&M) to partition out their influence on FisNull  value. Recalculated values of inbreeding coefficients, taking into account the frequency of null alleles (FisNull), were much lower than those obtained previously and ranged from 0.014 to 0.081 with an average of 0.045. The values of FisNull and Fis were significantly different from zero in all populations. However, it seems that deficiency of heterozygotes is mostly due to the presence of null alleles. Moreover, it should be noted that the highest values of both Fis and FisNull were observed in peripheral pine populations from Turkey and Spain.The basic statistics for genetic variation within the populations are summarized in FST) and the sum of the squared size differences (RST) showed that differentiation between Scots pine populations was low but significant . The majority of the variance was found within populations  [see Supporting Information—Table S3]. The greatest difference (0.11) was between the T4 population from Çatacık in Turkey and the H2 population from the Sierra de Neila in Spain, and the lowest (FST < 0.01) was between the PL2 population located in southern Poland and the PL4 population located in northern Poland.The analysis of molecular variance (AMOVA) based on the number of different alleles . This suggests an absence of phylogeographic structure and that gene flow is high compared with the mutation rate. However, the results of the Mantel test correlation between genetic distance and the logarithm of geographic distance among all P. sylvestris populations indicated that the genetic diversity is structured in geographic space . The strongest correlation was found along a longitudinal transect among populations from Anatolia and the Iberian Peninsula . The spatial genetic structure among all populations was rather weak, whereas only 15 % (R2 = 0.152) of the genetic divergence was explained by geographical distance. The strong structure  found among south peripheral populations is most likely due to the presence of geographical barriers along Mediterranean basin transects which intensify the effects of the process of isolation by distance (IBD).The permutation test, by which global STENA matrix is illustrated in the PCoA plot  provide the most probably representation of the overall genetic structure of the analysed Scots pine populations. The admixture proportions of each of the three gene pools (clusters) were estimated and differed among populations . The next largest groups included populations from the Balkans (Group 2), southern Spain (Group 3), north of the Iberian Peninsula (populations H4 and A) and the population from the Massif Central in France (Group 4). Two other groups were formed by the population from Scotland (Group 5) and the populations from central and northern Europe (Group 6). The AMOVA results and the pairwise FST values between these groups of populations are presented in see Supporting Information—Table S3).Based on these findings and the pairwise Despite the fact that some of the investigated populations were characterized by lower genetic diversity, no evidence for a recent genetic bottleneck or reductions in effective population size under the two-phased mutation model (TPM), infinite allele mutation model (IAM) or stepwise mutation model (SMM) was detected. Moreover, the mode-shift test, based on the frequency distribution of alleles, demonstrated a typical L-shaped mode for all populations, which is consistent with a non-bottleneck model.nSSR markers. All the 13 microsatellite loci studied were polymorphic with a mean of 12.3 alleles per locus. Considerably lower numbers of alleles were found at the psyl loci (an average of 6.1) compared with the Spag and PtTX loci . The most variable loci were Spag7.14, Spac11.4 and PtTX2146. Therefore, these loci appear to be the most informative for population genetics studies of P. sylvestris that require a high resolution of markers for fine-scale analysis. Consistent with earlier studies on the genetic variation of conifer species, we found that the analysed loci contained some level of null alleles  was found within the Scots pine natural range in Italy  and Spain (Group 3). The differentiation between the Iberian and Anatolian populations was also the greatest based on the morphological and anatomical traits of needles from Scots pines in the Mediterranean Basin , north of the Iberian Peninsula and the Massif Central (Group 4), Scotland (Group 5) and Poland and Finland (Group 6). In some previous mitochondrial DNA studies, unique haplotypes not observed in other parts of Europe were detected in Spanish and Turkish populations .Although only a small percentage of the total variation was due to differentiation among populations (approximately 4%), we found clear signals of population substructuring at The position of the Scottish populations is not straightforward. Scottish populations showed some similarity to groups from northern Spain and the Massif Central and populations from central and northern Europe. Previous pollen, allozyme and monoterpene studies suggested a west/east population subdivision within Scotland, reflecting different origins of the populations contributing to the postglacial colonization (Betula pendula) and Norway spruce (Picea abies) which at present co-occur with Scots pine across large parts of its natural habitat, we can conclude that Europe most likely was re-occupied by at least three main waves of recolonization. The source populations could have their origins in the regions of Alps, Balkans and east refugia with origin at intermediate latitude in Moscow region .This work was financially supported by W.W. and B.W. designed and conceptualized the study. B.W. and M.L. collected and analysed the data. B.W. wrote the manuscript; W.W. assisted in drafting the manuscript; B.W. and W.W. critically reviewed and revised the manuscript for content; all authors read and approved the final manuscript.None declared.


Macrobrachium rosenbergii, the giant freshwater prawn, is an important source of high quality protein and occurs naturally in rivers as well as commercial farms in South and South‐East Asia, including Bangladesh. This study investigated the genetic variation and population structure of M. rosenbergii sampled from four rivers in Bangladesh (sample size ranged from 19 to 20), assessing sequence variation, both in the mitochondrial cytochrome oxidase subunit 1 (CO1) gene and in 106 single nucleotide polymorphisms (SNPs) sampled randomly from the genome with double digest RAD sequencing (ddRADseq). The mitochondrial variation presented a shallow genealogy with high haplotype diversity (h = 0.95), reflecting an expansion in population size for the last ~82 kyr. Based on the CO1 variation the current effective population size (Ne) was 9.7 × 106 (CI: 1.33 × 106 – 35.84 × 106) individuals. A significant population differentiation was observed with the mitochondrial CO1 sequence variation and based on the ddRADseq variation, which could be traced to the divergence of the population in the Naf River in the South‐East border with Myanmar from the other populations. A differentiation in mtDNA haplotype frequencies was also observed between the Biskhali River and the Karnaphuli Rivers in eastern Bangladesh. This study demonstrated the use of high‐throughput genotyping based on the ddRADseq method to reveal population structure at a small geographical scale for an important freshwater prawn. The information from this study can be utilized for management and conservation of this species in Bangladesh. The species is found naturally in rivers and adjacent freshwater reservoirs  in South and South‐East Asia, from western Pakistan to western Java of Indonesia  enter into the freshwater system to grow until sexual maturity  and climate change effects   and South‐eastern Bangladesh (the Naf River), based on seven microsatellites, did not reveal significant differences among the sites, but the pairwise distances corresponded with the geographical distances , in comparison with other species. Another transcriptome study of this same species by Jung et al.  and other variants of reduced‐representation sequencing have proven to be an effective tool to delineate genetic structure for various species, with a larger power than traditional genetic markers, by analyzing variation in thousands of loci, for example, in the American lobster M. rosenbergii within Bangladesh by assessing genomic variation in samples from four of its main rivers: the Bishkhali in the West, the Meghna, and the Karnaphuli in the East and the Naf River at the boundary with Myanmar in South‐East by applying more genetic markers than in a previous study by Khan et al. , Meghna River , Karnaphuli River , and postlarvae (PL) from the Naf River   Figure . The qua2.22.2.1KX585687–KX585769): corresponding with 132–1,447 bps of M. rosenbergii mitochondrial genome  in a 10 μl reaction volume. The DNA template (1 μl) was sequenced with the Big Dye Terminator kit 3.1 (AB), precipitated with ethanol and run on a Genetic Analyser (3500xL Applied Biosystems). The sequences were edited using BioEdit Sequence Alignment Editor Hall,  and alig2.2.2M. rosenbergii, including haplotype diversity (h), nucleotide diversity (π) and the partition among sample sites with analysis of molecular variance (AMOVA) applying both the distance method (Φ) and the conventional F‐statistics from haplotype frequencies were calculated using ARLEQUIN v3.5  was calculated using the allele richness function in HIERFSTAT package in R .Genetic diversity of the combined CO1 fragments of  Goudet, . Evoluti Goudet, , impleme2.2.3M. rosenbergii were estimated by analyzing the mismatch distribution, using sum of square deviation (SSD) , where L is the length of the sequence. The demographic changes were further analyzed with the Bayesian Skyline Plot (BSP), based on the 83 CO1 sequences to estimate the past population dynamics from the time of sampling, assuming no population structure. The BSP analysis was implemented in BEAST v1.7.5  was estimated with the BSP analysis, using MCMC procedures by moving backward until the time of the most recent common ancestor was reached  following manufacturers protocol and eluted in sufficient volume of water for size selection. The fragmented DNA was size selected using a Pippin Prep (Sage Science) with 2% ethidium‐free gels and external size standard. Size selection used the narrow setting with a median fragment size of 400 bp (± 18 bp). The resulting fragments were amplified using PCR primers from Elshire et al.  final c. Librari2.3.2process_radtags command in STACKS v.1.09 . Variant detection and genotyping were performed using denovo pipelines: ustacks, cstacks, and stacks in STACKS v.1.09  with bootstrapped confidence intervals and testing the deviation from Hardy–Weinberg, using the HIERFSTAT , and followed by the assignment of individuals to different clusters defined with and without prior information, as implemented in the ADEGENET package , and nucleotide diversities ranged from 0.0022 to 0.0031 for the different samples , but not for the haplotype frequencies . The greatest differentiation occurred between the two most geographically distant locations, BR and NR  and between BR and KR based on haplotype frequencies  . Deviation from equilibrium was also observed with the Tajima's D and Fu's Fs, which were both negative and significant , suggesting expansion in population size or range from a bottleneck or alternatively due to recovery from a selective sweep. The BSP analysis showed that the population has a current effective population size (Ne) of 9.7 × 106 (CI: 1.33 × 106 – 35.84 × 106) individuals and has undergone a steady increase in Ne for the last 81.6 kyr (with a lower confidence limit close to 35 kyr)  of the Bangladesh M. rosenbergii, based on the median of the mismatch distributions (τ), was 100.35–186.37 kyr ago.The mismatch distribution for the samples Figure  followed3.23.2.1SD = 20.7) using the 7 depth base calling. The deviations of expected heterozygosity from observed heterozygosity (FIS) for the SNP dataset were overall negative, ranging from −0.22 to −0.66 within populations  and two with positive FIS values . By omitting these 19 loci the overall heterozygosity decreased from 0.15 to 0.10, and the FIS did not differ from 0  due to multiple testing using the FDR method  for SNPs among all sampling locations were significant , whereas the others were not (Table FST) was about two times larger when the loci which deviated from Hardy–Weinberg  were omitted, but the pattern remained the same. The differentiation between BR and KR, observed with the mtDNA, increased when omitting the loci which were not in HWE .The DAPC analysis with prior information of the sampling sites, based on the SNP dataset, reveal distinct differentiation among populations, with the highest overlap between KR and MR Figure b. Indiviot Table . The pai4Homarus americanus  and the Paira rivers (36 ind.), South‐West Bangladesh and the Naf River (36 ind.), South‐East Bangladesh, performed by Khan et al.  were not significant, they were almost similar to the values observed in our study (0.011–0.035). The detection of significant differentiation between the populations in the present study might have been supported by the larger number of the nuclear markers. The result from the DAPC ordination method supported the differentiation of the Naf River population but indicated that there might also be barriers to admixture between the rivers. When considering the combination of base pairs at the different sites within the genome, the most individuals sampled in one river were more similar to the individuals from the same river than to the individuals in other river.Analyzes of genome wide SNPs revealed a clear split between the population in the Naf River, at the boundary of Bangladesh and Myanmar, and the populations of the three other main rivers of Bangladesh which appear to be more connected, with an indication of gene flow and admixture among the rivers. Despite the divergence of the population in Naf River from other locations, two individuals (of 20) from the Naf River showed the closest genetic similarity with the individuals from the Bishkhali and the Meghna Rivers. This could possibly be explained by recent anthropogenic introductions, either due to transportation for aquaculture or accidentally through transfer of ballast water. The population sampled in Meghna River showed closest similarity with the Karnaphuli and the Bishkhali rivers, indicating connectivity and ongoing gene flow among these rivers either due to natural or human‐mediated mixing. Prawn can survive and grow in salinity up to 15 ppt (Chand et al., n et al. , based oM. rosenbergii sampled from Bangladesh: first, in the Naf River in between Bangladesh and Myanmar, and the second, in the Meghna, Bishkhali, and the Karnaphuli rivers. The haplotypic variation revealed high genetic variability within populations. Mitochondrial CO1 sequence variation revealed high overall haplotype diversity in Bangladesh M. rosenbergii, and revealed some extent of population differentiation, indicating similar pattern of population structure to the nuclear data, although the support was weaker. Based on our findings of these genetically distinct populations, we suggest that they should be considered as separate management units for sustainable management, harvest, and conservation. Fishing efforts can be controlled based on the genetic patterns to avoid over or underexploitation of different populations. As Bangladesh M. rosenbergii has high genetic variation, prawn hatcheries could be more sensible when they use ovigerous females from the same area in order to reduce threats to the local population diversity due to accidental escape from aquaculture. Further information about the biology of the species, such as variation in time of reproduction, behavior and habitat, is warranted to evaluate whether the observed population structure can be explained by different sources rather than the geographical origins.To conclude, SNP variation revealed at least two distinct populations of None declared. Click here for additional data file. Click here for additional data file.


North Africa is characterized by its diverse cultural and linguistic groups and its genetic heterogeneity. Genomic data has shown an amalgam of components mixed since pre-Holocean times. Though no differences have been found in uniparental and classical markers between Berbers and Arabs, the two main ethnic groups in the region, the scanty genomic data available have highlighted the singularity of Berbers. We characterize the genetic heterogeneity of North African groups, focusing on the putative differences of Berbers and Arabs, and estimate migration dates. We analyze genome-wide autosomal data in five Berber and six Arab groups, and compare them to Middle Easterns, sub-Saharans, and Europeans. Haplotype-based methods show a lack of correlation between geographical and genetic populations, and a high degree of genetic heterogeneity, without strong differences between Berbers and Arabs. Berbers enclose genetically diverse groups, from isolated endogamous groups with high autochthonous component frequencies, large homozygosity runs and low effective population sizes, to admixed groups with high frequencies of sub-Saharan and Middle Eastern components. Admixture time estimates show a complex pattern of recent historical migrations, with a peak around the 7th century C.E. coincident with the Arabization of the region; sub-Saharan migrations since the 1st century B.C. in agreement with Roman slave trade; and a strong migration in the 17th century C.E., coincident with a huge impact of the trans-Atlantic and trans-Saharan trade of sub-Saharan slaves in the Modern Era. The genetic complexity found should be taken into account when selecting reference groups in population genetics and biomedical studies. North African human populations are the result of an amalgam of migrations due to their strategic location at a crossroads of three continents: limited to the south by the Sahara desert, which has acted as a permeable barrier with the rest of the African continent; the Mediterranean basin in the coast, which has allowed the transit of maritime civilizations from Europe; and the connection to the Middle East by the Arabian Peninsula and the Sinai, which has permitted constant migrations by ground. The human presence in North Africa dates back 130–190 Kya  and diffHistorical records affirm that North Africa was populated by different groups supposed to be the ancestors of the current Berber peoples (Amazigh), by the arrival of Phoenicians in the second millennium B.C., and the posterior conquest of the area by the Romans. The Roman control persisted until the 5th century C.E., although non-Romanized Berber tribes persisted all over the region. The Arab expansion started in the Arabian Peninsula in the 7th century C.E. through Egypt and expanded until reaching the westernmost part of North Africa . The rule of the Arab dynasties ended with a decline in the 16th century, when the Ottoman Empire took control of the region until the colonization during the 18th and 19th centuries by European countries . The comIn addition to the migration complexity found in North Africa, a cultural diversity is characterized by the presence of two main branches of languages, both included in the Afro-Asiatic family : the AraThere is scanty genome-wide data in North African groups that can help to unravel the complexity of human migrations in the area. The pioneer study of ∼900 K SNPs by To explore the genetic complexity of North African groups and correlate it with the cultural diversity present in the area, we have genotyped ∼900 K SNPs in four additional Berber groups from Morocco, Algeria, and Tunisia. We combine this data with some published and some new reference panels, and apply haplotype-based approaches to characterize the ancestry components of North African groups and estimate admixture dates of the migrations that have shaped the genetic landscape of the region. Our results highlight the idea of a heterogeneous genetic landscape in North Africa due to recent demographic events, which should be taken into account when performing biomedical studies.k = 3) (k = 4), this “European component” is divided into one more linked to European populations, and another predominant in Middle East (blue). North African individuals, regardless of their Berber or Arab language affiliation, show a mixture of these components. These four components are correlated with geography , with the North African  and sub-Saharan  components significantly increased in the West, whereas the European  and Middle Eastern  components increased in the East     online ak = 3)  online. the East  online. the East  online. the East , which s× 10−16)  online. × 10−16)  online.In the PCA, the Berber-speaking individuals cluster with the rest of North African non-Berber samples without making differentiable clusters. The two groups of Berbers from Morocco (Tiznit and Errachidia) lie intermingled with non-Berber Moroccan samples; and Tunisian Berbers from Sened lie within their geographically non-Berber closer relatives, the Libyans, and the Algerians. The Algerian Berbers from Timimoun show a higher diversity, making a gradient towards sub-Saharan African samples and exhibit a higher frequency of the sub-Saharan ancestral component in the ADMIXTURE analysis. Finally, Tunisian Berbers from Chenini, which were the only Berber-speaking group included in a previous North African genome-wide analysis , form a In order to analyze in depth the complex structure and heterogeneous genetic patterns of North African populations, we explored patterns of haplotype sharing using ChromoPainter and fineSTRUCTURE . Both thAs shown in the description of the genetic clusters above, the geographical North African populations contain a mixture of individuals with different genetic histories. Representing each North African individual’s ChromoPainter inferred haplotype sharing patterns as a mixture of that from four genetically different groups  helps to illustrate this substructure . Under tSince Berber-speaking groups have been suggested to be descendant of isolated, fragmented and endogamous populations , an analWe used GLOBETROTTER to identify and date admixture events, using the same four surrogate groups: Yoruba (YRI), as representative of sub-Saharan Africa; Basques, as European proxies; Syrians, as Middle Easterns; and Tunisian Berbers from Chenini, as North African donors. Results suggest a history of gene flow that can be dated to as long ago as the first century B.C.  online. sampled populations are studied from a genetic point of view, genetic groups, or clusters, can be established based on genetic similarities rather than their geographical origin. Some individuals show high correspondence between their geographical origin and their genetic affinities, as shown for example in European populations  groups based on cultural practices, such as language. Although historically and sociologically this consideration is assumed, no genetic differences have been reported between Arabs and Berbers when analyzing individual genetic markers , some of which have likely diverged from one another relatively recently, are difficult to differentiate. We have addressed this issue by the use of haplotype-based methods that can have more precision to detect signals of historical and recent admixture events . Our datMoreover, we infer a lower proportion of sub-Saharan ancestry older than previously described in all admixture events dated from the first century B.C., which could be attributed to more ancient slave trade during the Roman or Islamic periods, such as the servile Haratin population of Nilo-Saharan origin in Berber groups such as the Sanhadja and Zenata . CautionThe recent genetic heterogeneity of North African groups described in the present analysis highlights the pivotal need of taking into account genetic substructure in future GWAS applications and other analyses of complex traits when North African samples are considered. The inclusion of cases and controls without accounting for substructure or the simple distinction of Arab and Berber groups  in Spain (2013/5429/I), as well as the approval from the local committees of the Charles Nicolle Hospital in Tunis, Tunisia; the CRASC  in Oran, Algeria; and the Comité d’Éthique du CHU  Mohamed VI in Marrakech, Morocco. DNA was extracted from blood samples using standard protocols, all samples were genotyped with Affymetrix 6.0 array and genotype calling was performed using the Affymetrix genotyping console 4.1.3.840 (data available in https://figshare.com/articles/North_African_Berber_dataset/3501761). The genotype calling was performed with the present data set and data from HapMap  were performed using the SmartPCA program from the EIGENSTRAT stratification correction method implemented in EIGENSOFT 4.2 package . Fst valtruct1.1 .Runs of homozygosity (ROH) analyses were performed in order to test for inbreeding among North African populations (excluding Mozabites from HGDP to increase the number of SNPs), using SNPs at all allele frequencies and allowing for linkage disequilibrium between them, leaving a total of 200,538 SNPs. Runs were identified in PLINK using a window of 5,000 kb and sliding it across the genome allowing for one heterozygous and one missing call per window. The minimum length of each ROH was set to 500 kb with 25 SNPs and a maximum gap of 100 kb between two consecutive SNPs.7, the thinning interval to 2000 and the burn-in value to 500 . The penalty for a change in recombination rate was set to 20. The number of individuals on each population was normalized to 13. The effective population size Ne was then estimated using the slope of a simple linear regression of population-based (ρ) on pedigree-based, sex averaged, deCODE recombination rates (r) (To estimate the effective population size (Ne) of each population we followed an approach similar to ates (r)  without We used SHAPEIT  to phaseWhen running ChromoPainter, we treated separately each sampled individual in our dataset as our recipient, painting this recipient using all other sampled individuals as donors. We first inferred the global mutation probability and the switch rate for chromosomes 1, 7, 14, and 20 in 10 iterations of the EM (expectation maximization) algorithm, the obtained values were: 0.00037 and 340.06914, respectively. We then fixed these parameters to infer the final ChromoPainter coancestry matrix that measures the amount of haplotype sharing among individuals summed across all chromosomes.FineSTRUCTURE  was usedWe ran GLOBETROTTER separately on each of our North African clusters A-M to test for instantaneous episodes of admixture. For this analysis we ran again ChromoPainter v2 in a separate analysis using the same switch rate and global mutation parameters cited above, but individuals were a priori classified into groups based on fineSTRUCTURE clustering results. Syrians (representing the Middle East), Basques (representing Europe), YRI (representing sub-Sahran Africa), and North African cluster from Tunisia Chenini were considered as both donors and recipients, whereas the rest of North African groups A–M were considered as recipients.In order to check the accuracy of the reference source populations chosen for sub-Saharan Africa and Europe we ran again the ChromoPainter analyses with other available populations of the area keeping all the parameters as previously. We included GBR, TSI, and IBS populations from 1000 Genomes Phase 3  to use an individuals were sampled with replacement, where n is the size of the North African cluster. The proportions of haplotype sharing for these resampled individuals were averaged to produce the average proportion of haplotype sharing for this resampled North African cluster, which was then used in NNLS to obtain the ancestry proportions for this resampled cluster. After estimating the X chromosome and autosomal ancestry proportions for the resampled cluster  the differences between the X chromosome and autosomes were recorded. Therefore, for each specific North African recipient cluster and each donor group, we had 10,000 bootstrap values that provided the bootstrap distribution of the observed X chromosome vs. autosomal ancestry difference. To estimate significance, we counted how many times this difference was greater  than 0  out of 10,000 bootstraps, and this proportion was multiplied by 2 to get a two sided P-value. With 13 recipient and four donor groups, a Bonferroni-adjusted threshold of 0.05/52 = 0.00096 was used for significance. We analyzed chromosome X vs. only chromosome 2, to have a comparison where both sides had roughly equal number of SNPs ; it gave quite similar results to the X chromosome vs. whole autosome analysis ; the phasing output by default treats each male individual as double homozygous, which is convenient when running the same analysis as autosomes. We then ran ChromoPainter on each individual sample for the X using the same parameters, donors and recipient clusters as for the autosomes (see above). The average ancestry proportions that each North African recipient group shares with the four surrogate groups were inferred by performing a NNLS on the X chromosome haplotype sharing results analogous to the autosomes (see above). Finally, to test for sex-bias gene flow, we subtracted the X and autosomal contributions from each surrogate for each North African recipient cluster. To evaluate the robustness of this difference, we performed bootstrap analysis for each North African recipient cluster separately. About 10,000 independent bootstrap draws were performed. For each bootstrap draw, analysis  online, parately  online. Molecular Biology and Evolution online.Supplementary DataClick here for additional data file.


RPW), Rhynchophorus ferrugineus (Olivier), was initially reported in China in the 1990s and is now considered one of the most successful invasive pests of palm plants in the country. A total of 14 microsatellite loci and one mitochondrial cytochrome oxidase subunit Ι (cox I) gene fragment were used to investigate the genetic characteristics and structure of R. ferrugineus in southern China. High levels of genetic differentiation among populations and significant correlations between genetic and geographical distances indicated an important role of geographical distance in the distribution of the RPW in southern China. High gene flow between Fujian and Taiwan province populations illustrated the increased effects of frequent anthropogenic activities on gene flow between them. Genetic similarity  indicated that RPW individuals from Taiwan and Fujian invaded from a different source than those from Hainan. To some extent, the genetic structure of the RPW in southern China correlated well with the geographic origins of this pest. We propose that geographical distance, anthropogenic activities, and the biological attributes of this pest are responsible for the distribution pattern of the RPW in southern China. The phylogenetic analysis suggests that the most likely native sources of the RPW in southern China are India, the Philippines, and Vietnam.The red palm weevil ( It was not until the 1990s that scientists began to pay attention to this pest. In southern China, ornamental palm plants are widely used as roadside shade trees, especially in some tourist cities. The first detection of the RPW in Guangdong Province (Zhongshan City) was reported in 1997  and reverse primers (5′‐TCCTAATAAACCAATTGC‐3′)  were usn et al. .Following PCR product detection by 2% agarose gel electrophoresis and sequencing , the preliminary sequence analyses were assessed. The allele sizes of microsatellite loci were manually checked and scored based on trace data for further analysis, and mitochondrial PCR products were directly sequenced from both strands with both primers.2.32.3.1A), mean observed heterozygosity (Ho), and expected heterozygosity (He), were estimated using Excel Microsatellite Tools . To minimize the bias of genetic diversity statistics affected by null alleles, genotypes were corrected based on the allele frequencies by the EM algorithm using FreeNA  and the semi‐logarithmic geographical distance matrix (km) among all populations were determined by GENEPOP. The mean allelic richness (AR) was estimated using the FSTAT program  repetitions. We used the admixture ancestry model /4FST  analysis was performed using the BEAST v 2.1.3 program  network analysis was performed in NETWORK 4.6.1.1 , which relates to the inbreeding level in each population, produced positive values in all of the populations except the GXNN population under the corrected data. Nonsignificant heterozygote deficiency was observed in the GXNN population (p > .05) (data not shown).The mean numbers of alleles and AR at a microsatellite locus were 3.74 and 3.32, respectively. The results showed that He and Ho ranged from 0.273 to 0.650 and from 0.250 to 0.621, respectively. Null alleles were observed at five microsatellite loci with a frequency ranging from 0.022 to 0.283. Null alleles existed in the P1F8 locus in Fujian and Hainan populations; both P1E2 and P2F11 showed null alleles in the GXNN, GDSZ, HNWC1, and HNWC2 populations, and the markers P1C8 and P3A8 showed null alleles in the GXNN and GDSZ populations (data not shown). After correcting the data set for null alleles using the EM algorithm, most of the expected and observed heterozygosity values were slightly higher than the raw data, ranging from 0.273 to 0.660 and 0.250 to 0.677, respectively, with the lowest expected and observed heterozygosity values in the TWTZ population and the highest in the GXNN population . Thirty haplotypes with global values of haplotype diversity and nucleotide diversity and an average number of nucleotide differences were found in the eight populations   based on microsatellite data suggested a high level of genetic differentiation among populations. Considering the existence of null alleles, we estimated the corrected pairwise FST value using the ENA algorithm (FSTENA) across all microsatellite loci and populations, and the results showed that the mean value was 0.265   network analysis of s Figure . To cleas Figure .cox I sequences with 29 sequences obtained from GenBank  and 0.450 for mitochondrial genes (p = .001) when the four previous Fujian populations   were observed between three population pairs  and between the population pairs , indicating small genetic differentiation between these population pairs. The microsatellite data showed low levels of gene flow between the south‐eastern group  and the southern group . Mitochondrial sequences showed a low average estimate of gene flow (Nm = 0.11) throughout the populations.The gene flow values suggested the presence of variable levels of gene flow between populations Table . The higp = .00003) and TPM (p = .00131) models and in the HNWC1 population according to the IAM model (p = .03925). Additionally, the test was nonsignificant for all populations under the SMM model  and Fu's Fs  tests, mismatch distribution analysis showed a multimodal mismatch graph when all populations were considered as a single group . The insular location of Taiwan may contribute to the low level of genetic variation. A narrow ocean barrier coupled with a small population size may dramatically influence the genetic diversity. Wu et al. . Founder effects produced by genetic drift occurring in a small number of individuals probably contributed to the low genetic diversity in the TWTZ population.By genotyping different populations from the major RPW outbreak centers in China using microsatellite markers, we determined that genetic variation was generally low within the southern China populations. These results were consistent with those previously found for the RPW in Fujian Province . We therefore deduced that individuals in the GXNN population had experienced a bottleneck effect and did not meet the criteria for HWE.Our results showed that four of the seven new populations significantly deviated from HWE and that three populations  displayed significant heterozygote deficiency and positive High haplotype diversity (Hd > 0.5) was present in most populations, especially in individuals from the two southernmost provinces, Hainan (HNWC1 and HNWC2) and Guangdong (GDSZ), indicating that these populations have experienced more propagule pressure. Multiple introduction or repeated introduction events will enhance diversity among invasive populations. The local climate seems to play a role in the expansion process of invasive species. Located in the tropical zone, with relatively high temperatures and rainfall amounts and a wide diversity of potential hosts, Hainan is considered an optimal habitat for the RPW. The high level of ecosystem invasibility of Hainan Island promotes colonization by invasive populations. The RPW has infested many coconut palms in Hainan and has had a serious economic impact on the island. Shenzhen City has the most ports in China and is also an important border city in Guangdong Province; the  trade of palms from native or infected areas to new areas across this province increased the possibility of infection by invasive species. Based on the palm transportation records in China, frequent human‐aided dispersal events occurred in these two provinces  to China played a major role in the distribution of haplotypes in southern China. From palm plants and offshoot transportation, we can deduce that the RPW in China originated from different populations and followed various paths. The origin sources of this pest in southern China are likely from India, the Philippines, and Vietnam. To some extent, the construction of city gardens, which promoted palm plant transportation in China, widely influenced the distribution of the RPW. On the other hand, the successful invasion of the RPW in southern China is probably due to its biological attributes together with the existence of numerous suitable habitats and climates across southern China (Qin et al., None declared.All the authors in this manuscript contributed to the work. Y.M.H. and G.H.W. conceived and designed the research; X.Z., J.Z., Z.M.C., and G.H.W. collected RPW samples and generated and analyzed the data; and J.L.L. and G.H.W. conducted the experiments and wrote the manuscript. All authors read and approved the manuscript. Click here for additional data file. Click here for additional data file.


Prunus mira Koehne, an important economic fruit crop with high breeding and medicinal values, and an ancestral species of many cultivated peach species, has recently been declared an endangered species. However, basic information about genetic diversity, population structure, and morphological variation is still limited for this species. In this study, we sampled 420 P. mira individuals from 21 wild populations in the Tibet plateau to conduct a comprehensive analysis of genetic and morphological characteristics. The results of molecular analyses based on simple sequence repeat (SSR) markers indicated moderate genetic diversity and inbreeding  within P. mira populations. STRUCTURE, GENELAND, and phylogenetic analyses assigned the 21 populations to three genetic clusters that were moderately correlated with geographic altitudes, and this may have resulted from significantly different climatic and environmental factors at different altitudinal ranges. Significant isolation-by-distance was detected across the entire distribution of P. mira populations, but geographic altitude might have more significant effects on genetic structure than geographic distance in partial small-scale areas. Furthermore, clear genetic structure, high genetic differentiation, and restricted gene flow were detected between pairwise populations from different geographic groups, indicating that geographic barriers and genetic drift have significant effects on P. mira populations. Analyses of molecular variance based on the SSR markers indicated high variation (83.7% and 81.7%), whereas morphological analyses revealed low variation (1.30%–36.17%) within the populations. Large and heavy fruits were better adapted than light fruits and nutlets to poor climate and environmental conditions at high altitudes. Based on the results of molecular and morphological analyses, we classified the area into three conservation units and proposed several conservation strategies for wild P. mira populations in the Tibet plateau. Prunus mira Koehne (2n = 2x = 16) is a perennial woody plant that belongs to the genus Prunus of the family Rosaceae [P. davidiana, P. kansuensis, and P. ferganensis [P. mira is also considered a valuable “living fossil” of peach species because of its long life cycle (over 1000 years). Its fruits are rich in nutrients , fatty acids , and active medicinal ingredients (arbutin). Therefore, in addition to supplying fruits, P. mira is used in Chinese traditional medicine to treat irregular menses, fractures, and congestion [P. mira has high tolerance to environmental stress  and high yields, it has great potential with regard to peach breeding [ganensis . P. mirangestion . Considebreeding .P. mira is widely distributed along the Yarlung Zangbo Grand Canyon and its tributary basins (Parlung Zangbo Basin and Nyang River Basin) in the Tibet plateau [P. mira densely inhabits [P. mira wild resources from different areas develop different phenological phases [P. mira is March 15–25 in Shannan and March 9–20 in Linzhi. Recent field investigations revealed that morphological characteristics such as fruit size, nutlet size, and nutlet surfaces, were considerably different between P. mira wild resources in Shannan and those in Linzhi. Previous studies [P. mira resources, as well as many excellent breeding resources in the Shannan and Linzhi areas. Unfortunately, because of deforestation, over-harvest, and road building, the natural distribution and population size of P. mira have been remarkably reduced [P. mira [P. mira genetic and morphological variations is still lacking. The knowledge of genetic and morphological variations can reveal the evolutionary history and existing situations associated with a plant species [P. mira is crucial for the protection of this endangered species. For decades, numerous molecular markers such as single nucleotide polymorphisms (SNP), random amplified polymorphisms DNA (RAPDs), simple sequence repeats (SSRs), and inter-simple sequence repeats (ISSRs), have been developed to analyze the genetic diversity and population structure of plant species [Prunus species [P. mira. Morphological classification is a traditional and intuitive means of characterizing plant species, and it has been widely used to evaluate morphological variation and differentiation within and among populations [ plateau ,8. Shanninhabits ,7. Shanal phases ,10. For  studies ,12 revea reduced . Althoug[P. mira –8, they  species –15. Ther species –19. Amon species ,22. Therulations . The samP. mira populations that were collected from the Tibetan plateau. This study aimed to (1) comprehensively reveal the genetic diversity and population structure of P. mira; (2) estimate morphological variation among and within populations; and (3) propose effective conservation strategies for the studied populations.Therefore, in this study, we used 25 SSR markers and 11 morphological characteristics to assess the genetic diversity, population structure, and morphological variation of 21 wild P. mira populations along the Yarlung Zangbo Grand Canyon and its tributary basin in the Tibet plateau  contained 16 populations (P1–P16). Among these populations, P1, P2, P3, P4, P5, and P14 were distributed in low-altitude areas (2921–2979 m), and P6, P7, P8, P9, P10, P11, P12, P13, P15, and P16 were located in medium-altitude areas , which contained one of four fluorescent dyes—FAM, NED, VIC, and PET. Polymerase chain reaction (PCR) amplification was conducted in a 20 μL reaction volume that contained 20 ng DNA, 0.2 μL 2× Tab Master Mix, 2 μL 10× buffer, 0.5 μL dNTP (0.25 mol·L-1), 0.5 μL 10% DMSO, 14.8 μL ddH2O, and 0.5 μL of each primer (0.8 μmol·L-1). The PCR protocol was as follows: 95°C for 5 min; followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 5 min. PCR products were analyzed using an ABI 3500XL  DNA Analyzer. The amplicon fragments were sized using Gene-Marker , and the details are shown in The total genomic DNA of all dried leaves was extracted using the CTAB method , and DNA persica –30, one  cerasus , and twoP. avium  . Permutations of the most likely results among all runs for each K were performed in CLUMPP [GenAlEx version 6.501  was usedo et al.  was usedo et al.   in Arlequin version 3.5 [FST/(1—FST)) and geographic distance (km) in GenAlEx version 6.501.sion 3.5 . This ansion 3.5  in NTSYSsion 3.5 . Mantel A), effective allele number (Ae), private allele number (Np), Shannon’s information index (I), expected heterozygosity (He), observed heterozygosity (Ho), gene flow (Nm), and F-statistics indices . The Hardy-Weinberg equilibrium (HWE) was tested for each locus and population using Arlequin version 3.1 with 100,000,000 steps in the Markov chain and 100,000 dememorization steps. Polymorphism information content (PIC) was calculated using the following formula [Pi and Pj are the frequency of the amplified alleles, and n is the number of alleles at each SSR locus.GenAlEx version 6.501 was used to calculate the following genetic indices for loci, populations, and genetic clusters identified in the STRUCTURE analysis: allele number , and draw the boxplots of the 11 morphological characteristics. For clustering and statistical analyses, the three types of nutlet surfaces were classified as follows: 1 (smooth), 2 , and 3 (deep groove).In all, 20 fruits and 20 nutlets were randomly selected per individual to measure and calculate the following 11 morphological characteristics: fruit weight, fruit length, fruit width, fruit diameter, fruit shape index (fruit length/fruit width), nutlet weight, nutlet length, nutlet width, nutlet diameter, nutlet shape index (nutlet length/nutlet width), and nutlet surface. The weights of fruits and nutlets were tested using an electronic balance (accuracy of 0.001 g). The lengths, widths, and diameters of fruits and nutlets were measured using a digital Vernier caliper (accuracy of 0.001 mm). The mean values of all morphological characteristics were used to construct an unrooted UPGMA tree in NTSYS PC version 2.10 based on Nei’s genetic distance (1978). IBM SPSS Statistics 19 software was used to conduct Duncan’s multiple comparison analysis, calculate coefficients of variation  to 15 (BPPCT-025 and PMS67), with an average of 8.6 alleles per locus (Ae) was between 1.5 (UDP96-019) and 7.5 (BPPCT-025), with an average of 3.6. The mean expected heterozygosity (He) was 0.52, and it exceeded the observed heterozygosity (Ho = 0.46). F-statistics showed moderate FST (0.15) and FIS (0.13) values across the 25 loci, indicating moderate genetic differentiation across all sites. In addition, Shannon’s information index (I) ranged between 0.18 (Pchgms 4) and 2.07 (BPPCT-025), with an average of 1.19. The polymorphism information content (PIC) was between 0.82 (BPPCT-025) and 0.32 (Pchgms4), with an average of 0.62. Of the 25 SSR markers, six  were moderately informative (0.25 < PIC < 0.50), and 19 were highly informative (PIC > 0.50), indicating their high potential regarding the genetic diversity analysis of P. mira populations.Twenty-five SSR markers were selected to identify genotype 420 er locus . The effP. mira populations was tested using Bayesian clustering methods in STRUCTURE version 2.3.4. The classification of populations was highly correlated with their geographic altitudes. When K = 2, 16 low- and medium-altitude populations (P1–P16) from Linzhi group and five high-altitude populations (P17–P21) from Shannan group were assigned to two different clusters with high ancestry values  across the entire distribution of P. mira populations  and Cluster II  and pairwise gene flow values (Nm) among all populations and the three genetic clusters. Genetic differentiation between the 21 populations was significant and high as evidenced by a global FST value of 0.16 , whereas the genetic differentiation between Cluster III and the other clusters was significant and slightly high , which ranged from one to four alleles per population. Interestingly, P18 had the highest number of private alleles (Np = 4), although it contained the lowest genetic diversity. Genetic diversity was also estimated within each genetic cluster (The mean ulations . The hig cluster . ClusterFIS) ranged from 0.06 in P19 to 0.26 in P12 and P18, with an average of 0.17  significantly deviated from the HWE.The inbreeding coefficient  within populations ranged from 1.30% to 36.17%  were observed between pairwise populations from different geographic regions, thus indicating that the gene flow cannot combat genetic differentiation caused by genetic drift [P. mira populations.Our findings indicated that Cluster III was significantly and highly differentiated from the other two clusters. Five populations from Cluster III and 16 populations from Cluster I and Cluster II are distributed in Shannan and Linzhi, respectively. In the central region of the Tibet plateau, numerous mountains, rivers, and valleys shape geographic barriers between the two geographic groups, thereby leading to the observed high genetic differentiation. However, low gene flow values  in P. mira populations. The genetic diversity of P. mira in this study was higher than that previously reported for P. mira  [A = 2.7, Ae = 2.1, Ho = 0.32–0.34, He = 0.23–0.46) [P. mira. Moderate or even high levels of genetic diversity have been reported in some endangered species [P. mira, high adaption to various environments and wide distributions might have helped shape and preserve the genetic diversity of populations under poor living conditions [P. mira is relatively large, but more recent periods of excessive logging and fragmentation due to habitat destruction may have led to the initial loss of genetic diversity, despite the short period of fragmentation. Therefore, this factor might be also responsible for the moderate genetic diversity in P. mira.Molecular analyses revealed moderate genetic diversity ( = 0.35)  and othe23–0.46) ,50. Theo species ,52. Reganditions ,54. For nditions . The effP. mira. Deforestation and geographic isolation have led to the heavy fragmentation of P. mira habitats [P. mira could be attributed to habitat fragmentation. Previous studies [P. mira resources near rural areas in the Tibetan plateau. In this study, we found 7 populations near rural areas that deviated from HWE, thus indicating that recent human activities might have significant effects on these P. mira populations.Positive inbreeding coefficients were detected within each population and at each locus, indicating the significant excess of homozygotes in habitats ,48,55. Hhabitats –58. ThusP. mira populations, which is higher than that reported previously based on SSR (81.0%), ISSR (71.0%), and AFLP (74.48%) data [P. mira, an outcrossing system and long life cycle (over 1000 years) can improve gene flow and preserve shared ancestral genetic information among populations, thus significantly improving genetic variation within populations.Perennial woody plant species are expected to preserve high variation within populations because of their long life cycles –61. An A%), ISSR 1.0%, and AFLP 74.% data [4P. mira individuals are distributed over a narrow altitude range within each population, although they are widely distributed in the Tibet plateau. Combined with our molecular results, we concluded that the morphological differences may be caused by the relatively isolated distributions and various climates and environments associated with P. mira populations from different geographic altitudes. For instance, high-altitude populations contained larger and heavier fruits and nutlets than the medium- and low- altitude populations. These results were consistent with the findings of Pluess et al. [Both molecular and morphological data revealed three clusters that were correlated with geographic altitudes. Interestingly, our morphological analyses showed fewer differences within populations, and this was contrary to our molecular analysis results. The morphological variation among the three clusters was higher than that within clusters. s et al.  in that P. mira populations were moderate, some populations still exhibited high risk of genetic variation loss, and human activities and climates had significant effects on most P. mira populations. Therefore, there is also an urgent need to conduct P. mira resource conservation.Studying genetic diversity, population structure, and morphological variations could provide basic information about plant conservation . AlthougP. mira populations. Each cluster contained unique genetic and morphological variations and had different evolutionary histories. Therefore, the conservation efforts should be divided into three units that correspond to the three genetic and morphological clusters. Because the six populations in Cluster I were found in national nature reserves, more attention needs to be paid to the other clusters. Furthermore, in situ conservation should prioritize populations with high genetic diversity in Clusters II and III such as P11 and P17. Because human activities have had significant effects on many P. mira populations , we propose the strengthening of conservation awareness in local farmers and the hanging of placards to reduce deforestation and prevent continuing habitat deterioration. We also suggest ex situ conservation for individuals with unique traits and populations with high inbreeding to prevent the potential loss of genetic variation. Furthermore, improving the genetic diversity within populations requires the monitoring of crossing among populations.The analyses based on SSR markers and morphological characteristics revealed three completely consistent clusters among the 21 wild P. mira populations in the Tibet plateau. Moderate levels of genetic diversity were detected in P. mira populations. Significant homozygote excess was observed within each population, and this might be the result of sibling mating caused by habitat fragmentation. Furthermore, human activities might be responsible for the deviation of numerous P. mira populations from the HWE. Both molecular and morphological analyses assigned the 21 populations to three clusters, which were significantly correlated with the geographic altitudes. Because of the presence of geographic barriers and genetic drift, populations in Shannan were highly differentiated from populations in Linzhi. Lastly, we formed three conservation units and proposed several conservation strategies for these studied populations.This study comprehensively analyzed the genetic diversity, population structure, and morphological variation of 21 wild S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigP. mira individuals can be divided into three clusters based on visual inspection. The first cluster was smooth , the second cluster exhibited nutlet surfaces with shallow grooves , and the third cluster displayed nutlet surfaces with deep grooves .The nutlet surfaces of 420 (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.