The genetic structure of human populations is the outcome of the combined action of different processes such as demographic dynamics and natural selection. Several efforts toward the characterization of population genetic architectures and the identification of adaptation signatures were recently made. In this study, we provide a genome-wide depiction of the Italian population structure and the analysis of the major determinants of the current existing genetic variation.We defined and characterized 210 genomic loci associated with the first Principal Component calculated on the Italian genotypic data and correlated to the North–south genetic gradient. Using a gene-enrichment approach we identified the immune function as primarily involved in the Italian population differentiation and we described a locus on chromosome 13 showing combined evidence of North–south diversification in allele frequencies and signs of recent positive selection. In this region our bioinformatics analysis pinpointed an uncharacterized long intergenic non-coding (lincRNA), whose expression appeared specific for immune-related tissues suggesting its relevance for the immune function.Our study, combining population genetic analyses with biological insights provides a description of the Italian genetic structure that in future could contribute to the evaluation of complex diseases risk in the population context.The online version of this article (doi:10.1186/s12863-015-0293-x) contains supplementary material, which is available to authorized users. Understanding the genetic structure of human populations is crucial to reconstruct their history and to elucidate the genetic predisposition to diseases. In fact, the genetic structure of human populations was shaped by several demographic events and selective forces, which have contributed to the current diversification and to the differences in diseases prevalence and predisposition , 2. SomeIn recent years, the genetic structure of several human populations has been characterized both at worldwide and regional level using genome-wide markers. In Europe, the genetic variation pattern showed a southeast-northwest gradient with a strict correspondence between genetic and geographic distances –7. AlongLCT) which confers ability to digest lactose in adulthood. The lactase persistence shows a latitudinal cline with particularly high rates among Northern Europeans and it was demonstrated to be a target of natural selection [In addition to demographic processes, several evidence of positive selection differentially shaping the genome of human populations have been described , 12. In election . Moreoveelection . As an eelection .Although the genetic structure of different populations has been deeply characterized, the underlining biological processes are still poorly understood thus requiring further investigations, both at worldwide and regional level.In this paper, we exploited genome-wide genotypic data to recapitulate the genetic structure of the Italian population in its geographic context, refining the picture of the North–South gradient in genetic variation. A total of 210 genomic loci, sufficient to explain the latitudinal cline in genetic variation, were identified and characterized by different bioinformatics approaches.To investigate the genetic structure of the Italian population we assembled a genome-wide genotype dataset of 1736 Italian individuals, as detailed in the Methods section.smartpca tool of the EIGENSOFT package [r = 0.876, p = 8.805 × 10−7). The regional subdivision of the Italian population was also evaluated using the pairwise FST parameter as a measure of genetic distance. A significant correlation between the matrix of FST and the matrix of the kilometric distances between regional capitals was found . The second principal component  differentiated Sardinian individuals from the others, reflecting their known genetic diversity. The other PCs did not show any correlation with the Italian geography.After a quality-control procedure, the Italian genetic diversity was summarized by Principal Component Analysis (PCA) using the  package . To gainWe also evaluated the Italian genetic diversity in the surrounding geographic context through the analysis of available genotype data from populations of the European and Mediterranean area , 14 % were non-coding RNAs (n = 99) and the remaining 4 % were pseudogenes (n = 25). When we tested the enrichment in gene content of the 210 genomic intervals, a slight overrepresentation was observed (p = 0.0595) and it resulted statistically significant considering only the protein-coding genes (p = 0.0014). Moreover, the enrichment in genes causing Mendelian diseases resulted significant (p = 0.0223). Using the National Human Genome Research Institute (NHGRI) Genome Wide Association Study (GWAS) catalogue [p = 0.0126).Through a linear regression analysis, after applying a genome-wide atalogue , we founIL6, CHUK, CXCR4, CD79A, CNR2, FCGR2B and MAL) in common with the “Signal transduction” term and four genes  shared with the “Regulation of cellular metabolic process” clade pointing out an overall interconnection among these biological processes. Taking into account the “Cellular Component” ontology, “Membrane” resulted as the most significantly enriched term together with 7 other terms referring to the membrane portion of the cell  region was excluded from the analysis, since it harbors several genes with known immune functions. The overrepresentation of Gene Ontology (GO) terms was evaluated using MSigDB of the Gene Set Enrichment Analysis (GSEA) package (see Methods) . 8 GO teSignals of positive selection were identified using the integrated haplotype score (iHS) statistics , which wp = 0.017 and p = 0.004).Additional insights into the contribution of positive selection as a mechanism involved in the determination of the Italian North–South genetic gradient were obtained by comparison with literature data and testing the enrichment in gene lists for biological functions known to be target of positive selection. Taking into account recent publications based on genome-wide genotypic data and performed on different populations , 22, 23,LINC00381) with no functional information available. According to UCSC [TCONS_00022202) giving rise to two alternative spliced isoforms with high expression levels in white blood cells and in lymph nodes. Data from the ENCODE project [In particular, the locus on chromosome 13 at nucleotide position 74,690,999–75,337,499  or CD79A, a subunit of the B-cell antigen receptor. In agreement with this result, the Gene Ontology analysis for Cellular Component revealed an enrichment of genes acting at the plasma membrane level, thus modulating cell behavior in response to external stimuli. The enrichment of genes with a role in the immune system emerged even more clearly from the analysis of canonical pathways. The pathways identified by the IPA and MSignDB analyses highlighted different aspects of the immune response. The majority of them converged to the Nf-kB signaling as previously suggested [CHUK, NFKBIA) or regulators  were located in the Italian PC1-associated loci. Taken together, these data pointed out the immune response as the biological process mainly differentiated along the Italian peninsula, probably as a preferential target of natural selection.On the basis of the SNPs significantly contributing to the first Italian principal component, we identified 210 genomic loci that we considered as the main contributors to the North–South gradient. The evaluation of the loci by an interval-based enrichment approach revealed us that they were not randomly located in the genome but preferentially spanned genic regions and, in particular, regions containing protein coding genes. Moreover, the identified loci resulted enriched in disease-associated genes and risk-variants underlining the functional relevance of these regions. Within the most associated loci, genomic regions known for their contribution to the European genetic diversity were contained such as the  regions , 29. The SLC45A2 , HPS5 [3A2 [HPS5  and EXOCOC2-IRF4  were fouOC2-IRF4 . In addiOC2-IRF4 . The Genuggested , 34, 35 HBB, a gene known to harbor alleles conferring protection against malaria and to be a target of balancing selection [CR1) gene, suggested to be involved in malaria susceptibility [FCGR2B gene, demonstrated to harbor malaria protective alleles [The most likely explanation for the contribution of immunity to population differentiation is its function in host defense against pathogens , 37. In election , is amontibility , and the alleles , 43, werYersinia pestis [TLR1 involved in leprosy susceptibility [IFITM3 gene, whose expression was demonstrated to protect against influenza A infection [Vibrio cholera [Malaria was not the only pathology for which we recognized traces in the Italian population. The Toll-like receptor gene cluster, shown to modulate the response to a pestis  and its tibility , were alnfection . Further cholera , resulteTCONS_00022202 transcript appeared as the best candidate to exert its role in the immune system, because it is mainly expressed in lymph nodes and white blood cells. The transcriptional activity of the TCONS_00022202 locus was further supported by recent data provided by the ENCODE project demonstrating that it is enriched in an enrichment in modifications typical of active chromatin and is highly transcribed in the NHEK cell line. The NHEK cell line derived from primary epidermal keratinocytes which represent an effective barrier to the entry of infectious agents and play an active role in the initiation of the immune response. These cells produce a variety of cytokines, growth factors, interleukins and antimicrobial peptides thus representing a cell model to investigate inflammation and immune response. Given that non-coding RNAs are emerging as important regulators of gene expression in the immune response [TCONS_00022202 transcript may represent a new immune-related molecule deserving further investigations.Recent studies highlighted the presence of adaptation signals in non-coding regions likely owing regulatory functions , 47. In response , 49, we TCONS_00022202 lincRNA was associated with the IFNα response in smallpox vaccine recipients, a phenotype that resembles the host response to the virus. Since the allele correlating with higher level of interferon expression was on the positively selected haplotype, we proposed that the observed signature of positive selection is the effect of adaptation to Variola virus. Moreover, the observation that 2 other SNPs (rs17070309 and rs12256830) associated with smallpox-induced cytokine response [Intriguingly, a polymorphism located about 22 kb upstream to the response  are locaIn conclusion, our study provides new insights into the Italian population structure by characterizing the main determinants of the current genetic diversity and results in the identification of immunity as the main biological process responsible for genetic differentiation in Italy positive selection target, likely triggered by infective agents. Interestingly, recent studies suggested an important role of loci involved in host defense against pathogens also in autoimmune disease susceptibility. For example, it was proposed that the genetic architecture of inflammatory bowel disease was shaped by pathogen-driven selection , 51. FurAll the reported genomic coordinates were based on the February 2009 assembly of the human genome (hg19/GRCh37). The statistical analyses, unless otherwise specified, were performed with R, version 2.15.3 .Before the quality control procedure a total of 1736 individuals was available for this study. In particular, 1648 individuals of self-reported Italian origin, recruited in North Italy had surname information accessible. Their genotype data were assembled from a study of cerebrovascular disease including 697 cases and 951 controls. Controls were recruited among blood donors and volunteer healthy people, 409 already analyzed in a study on obesity and 392 in the PROCARDIS study . All indThe geographic origin of individuals was defined through the analysis of their surnames. The birth place and the place of residence were not available for all the individuals and previous analyses demonstrated that they are not suitable to infer the individual’s geographic origin because of recent migrations . In Italr2 greater than 0.4, in windows of 200 SNPs (sliding window of 25 SNPs), was removed using the indep-pairwise command in PLINK. After the quality control procedure, a total of 1715 individuals and 172,111 SNPs was considered for the analysis. Finally, 1000 SNPs from the 8p23.1 genetic region, known to harbor a large inversion polymorphism [Managing of genotype data and quality control procedures were performed with PLINK 1.0.7 . For themorphism , 63, wersmartpca tool of the EIGENSOFT package version 3.0 using the default parameter and no outlier exclusion [Principal Component Analysis (PCA) was carried out using the xclusion . The corK = 2 to K = 7. The optimal number of K was estimated through the cross-validation procedure using the --cv = 10 option.The SNPs significantly associated with the first principal component were identified through a linear regression model in PLINK. PC1 was used as a response variable and the SNP as the explanatory one. The analysis of SNPs associated with Italian PC2 identified a small number of significant SNPs, likely because samples from Sardinia were too few. For this reason PC2 was not further examined. To infer the ancestry proportions in the European/Mediterranean dataset we applied the unsupervised clustering algorithm ADMIXTURE . The anarehh [fastPHASE [The presence of signal of selection was tested using the iHS statistics , calcularehh . This teastPHASE . For eacThe comparison with literature data was performed selecting articles reporting genome-wide analyses of positive natural selection carried out on reference populations belonging to the HapMap project or to the Human Genome Diversity Project, published up to 2013.The genomic intervals corresponding to each PC1-associated SNP were defined on the basis of the LD feature of the genome through the Gene Relationships Across Implicated Loci (GRAIL) tool . The toohttp://www.ncbi.nlm.nih.gov/gene/) limiting the query to RefSeq records. The list of genes involved in Mendelian diseases was defined filtering the Online Mendelian Inheritance in Man (http://omim.org/) catalogue to exclude unconfirmed diseases, traits not involved in disorders and inconsistent or tentative records. The list of SNPs associated with complex disorders was retrieved from the NHGRI GWAS catalogue  [http://www.broadinstitute.org/mpg/cms) [The interval-based enrichment tests were performed with INRICH v.1.0  using 1,rd 2014) . The manmpg/cms) .http://www.broadinstitute.org/gsea/msigdb/index.jsp) querying Gene Ontology as source annotation database and considering the categories with a corrected p-value less than 1 × 10−4. The canonical pathways were investigated using QIAGEN’s Ingenuity® Pathway Analysis  and the first 10 significant canonical pathways were reported.The gene-set enrichment analysis was performed using the MSigDB tool of the GSEA package (The functional categories reported in Fig. The list of the 210 loci associated with Italian population PC1 is available in Additional file


Taraxacum kok-saghyz (TKS), is a perennial species native to Central Asia that produces high quality, natural rubber. Despite its potential to help maintain a stable worldwide rubber supply, little is known about genetic variation in this species. To facilitate future germplasm improvement efforts, we developed simple-sequence repeat (SSR) markers from available expressed-sequence tag (EST) data and used them to investigate patterns of population genetic diversity in this nascent crop species. We identified numerous SSRs  in 1,248 unigenes from a larger set of 6,960 unigenes  and designed PCR primers targeting 767 of these loci. Screening of a subset of 192 of these primer pairs resulted in the identification of 48 pairs that appeared to produce single-locus polymorphisms. We then used the most reliable 17 of these primer pairs to genotype 176 individuals from 17 natural TKS populations. We observed an average of 4.8 alleles per locus with population-level expected heterozygosities ranging from 0.28 to 0.50. An average pairwise FST of 0.11 indicated moderate but statistically significant levels of genetic differentiation, though there was no clear geographic patterning to this differentiation. We also tested these 17 primer pairs in the widespread common dandelion, T. officinale, and a majority successfully produced apparently single-locus amplicons. This result demonstrates the potential utility of these markers for genetic analyses in other species in the genus.The Russian dandelion,  Hevea brasiliensis. Most H. brasiliensis cultivation occurs in equatorial regions of Southeast Asia, though there is some production in the Amazon basin [H. brasiliensis [Microcyculs ulei), which is spread by human activities as well as natural spore dispersal [Worldwide natural rubber production is largely reliant on the rubber tree, on basin ,2. Unforon basin . Though iliensis ,5, the diliensis . Moreoveispersal , will evTaraxacum kok-saghyz  is a perennial, diploid species that produces large quantities of rubber throughout the plant, especially in its roots. Initial attempts at developing TKS into a viable source of natural rubber were prompted by limited access to rubber plantations during World War II [d War II . When thd War II –14, we sHere, we report the development of simple-sequence repeat (SSR) markers derived from available TKS expressed-sequence tags (ESTs) and their use in population genetic analyses of wild-collected TKS accessions. These EST-SSRs represent the first published nuclear sequence-tagged site (STS) markers for this fascinating species, and their use provides novel insights into the distribution of genetic variation within and among natural populations of TKS.A total of 16,441 ESTs obtained from the root tissue of young (1 to 3 month) TKS seedlings were downloaded from GenBank (accession numbers DR398435 to DR403165 and GO660574 to GO672283). Sequences were then assembled into contigs using CAP3  with theT. officinale collected from Athens, GA to facilitate an investigation of the transferability of our markers to other species within the genus.Seeds derived from 17 wild-collected TKS populations in Kazakhstan were obtained from the USDA-ARS Western Regional Plant Introduction Station. Seeds from each population were placed on moist filter paper and stored in darkness at 4C for 48 hours before being allowed to germinate at room temperature. Following germination, seedlings were transplanted into pots in the University of Georgia greenhouses . Once established, 600 mg of fresh tissue was collected from each individual for DNA extraction using the Qiagen  DNeasy Plant Mini Kit . Following extraction, DNA was quantified using a NanoDrop  and diluted to 2.5 ng/μl in preparation for genotyping. We also collected tissue and extracted DNA (as above) from four individuals of T. officinale individuals. Reactions were performed in a total volume of 15 μL containing 2.5 ng of template DNA, 30 mM Tricine pH 8.4-KOH, 50 mM KCl, 2 mM MgCl2, 125 μM of each dNTP, 0.1 μM reverse primer, 0.02 μM forward primer, 0.1 μM fluorescently labeled M13 primer , and 1 unit of Taq polymerase. The PCR conditions were as follows: 3 min at 95C; 10 cycles of 30 s at 94C, 30 s at 65C and 45 s at 72C, annealing temperature decreasing to 55C by 1C per cycle, followed by 30 cycles of 30 s at 94C, 30 s at 55C, 45 s at 72C, followed by 20 min at 72C. PCR products were visualized on 1% agarose gels to identify primers that successfully produced amplicons. If the above cycling conditions were unsuccessful at producing a single-banded amplicon in TKS, the annealing temperature was increased or decreased, as appropriate, to achieve more or less stringent reaction conditions. For primer pairs that produced a single-banded amplicon, the reactions were diluted 1:15 and visualized on an ABI 3730xl  with GGF 500 ROX size standard , which includes standards ranging in size from 88 to 435 bp. Data files were imported into GeneMarker  for genotype calling. The trace files were manually inspected to ensure accurate genotyping and 48 primer pairs that produced readily-interpretable, single-locus polymorphisms in the aforementioned test panel of eight TKS individuals were then used to genotype the full panel of TKS individuals following the same general methods. As more individuals were genotyped, we reduced our final dataset from the original 48 primer pairs to a subset of 17 loci that consistently provided easily interpretable, single-locus polymorphisms. The remaining 31 loci were excluded due to missing data or more complex  banding patterns that emerged when the larger set of samples was considered.All genotyping was done using a modification of the three-primer PCR protocol described by Schuelke , as adapST and GST were calculated for each locus. An analysis of molecular variation (AMOVA) was also performed in GenAlEx using 999 permutations to determine the amount of genetic variation that is significantly attributable to populations. We performed a principal coordinates analysis (PCoA) by using a standardized genetic distance matrix as calculated in GenAlEx. A Mantel test was performed to test the correspondence between a pairwise geographic distance matrix (and its log transformation) and a pairwise Nei’s genetic distance matrix among all 17 populations.The multi-locus genotype data were analyzed using GenAlEx (version 6.501) . For eacAssembly of the 16,441 TKS ESTs resulted in a unigene set of 6,960 contigs and singletons. Of these, a total of 1,248 (17.9%) were found to contain at least one SSR, on par with the values from other plant species . Of the T. officinale to assess cross-taxon transferability. Twelve of these seventeen primer pairs successfully produced single-banded amplicons in T. officinale  in southeast Kazakhstan near the border with western China . These 1ficinale , demonstP > 0.05). Similar to our results, Gupta et al. [Helianthus suggest that SSRs found in CDS vs. UTRs exhibit similar levels of polymorphism [The mean number of alleles per locus was 4.8 ± 0.6  across the full TKS dataset . Furthermore, we found only one population-level FIS value that was significantly different from zero . This particular population (35169) had a significantly positive F value overall, though it had negative FIS values (higher observed heterozygosity relative to expected heterozygosity) for six of the sixteen loci with FIS values (one locus was monomorphic in this population). Two loci in population 35169 had exceptionally elevated FIS estimates compared to the average across loci, which may indicate the presence of population specific null alleles, or may be the result of selection. As a whole, these results suggest that there are little or no systemic deviations from Hardy-Weinberg equilibrium within the other 16 populations, though it should be noted that average sampling per population was somewhat limited. It is thus possible that deeper sampling would reveal significant deviations that previously escaped detection. Within populations, the mean percent polymorphic loci was 94.5% ± 1.6% and contained one outlier value of 70.6%.Averaging across the 17 loci, the observed heterozygosity levels within populations ranged from 0.28 to 0.47 (0.37 ± 0.01), whereas unbiased expected heterozygosity levels ranged from 0.28 to 0.50  have been used to assess levels of diversity in asexual and sexual dandelion populations in central Europe [T. officinale [T. brevicorniculatum [T. brevicorniculatum [A number of previous studies have used molecular markers (including SSRs) to investigate genetic diversity in wild l Europe . Additioficinale . In TKS,iculatum . Specifiiculatum . While ticulatum .ST across all 17 loci, and considering all 17 populations, was 0.11 ± 0.007 (mean GST was 0.06 ± 0.007). Levels of pairwise FST in all population comparisons were also relatively low (P > 0.05). These results are generally consistent with the expectation for plant species with wind-based seed dispersal. Indeed, Nybom [ST level of 0.13 for such species in a meta-analysis of SSR-based studies compared to species with ingested seeds (FST = 0.21), attached seeds (FST = 0.33), and gravity-dispersed seeds (FST = 0.34)  having less genetic structure relative to both short-lived perennials (FST = 0.31) and annuals (FST = 0.40) [ST of species with different breeding systems, it has been shown that both selfing species (FST = 0.42) and those with mixed mating systems (FST = 0.26) exhibit higher levels of differentiation compared to outcrossing species (FST = 0.22) [ST = 0.101) when compared to species with seeds dispersed via animal ingestion (GST = 0.223) or gravity (GST = 0.189), but was not statistically different from species with animal-attached seeds (GST = 0.114) [In terms of population structure, the average Fvely low . In addid, Nybom  found antes from ). Additi = 0.40) . However = 0.22) . An earl= 0.114) .ST and PCoA analyses, an analysis of molecular variance (AMOVA) attributed only 7% (P = 0.001) of the genetic variance to population-level differentiation. This relatively low level of genetic structure could be a byproduct of relatively high levels of seed and/or pollen dispersal. Further insight awaits direct investigation of pollen vs. seed-mediated rates of gene flow . RelatiThis study has resulted in the development of a set of well-characterized, polymorphic genetic markers for use in TKS, along with additional, untested PCR primer pairs for the potential development of additional markers. The majority of these markers were found to produce apparently single-locus amplicons in a related species, indicating their potential utility for investigating patterns of genetic variation in other species within the genus. Moreover, our work has provided valuable insight into patterns of genetic diversity in wild populations sampled from throughout the native range of this prospective industrial crop species. The apparently low level of population genetic structure, which may be attributable to potentially high levels of seed and/or pollen dispersal, suggest that much of the neutral genetic diversity within Russian dandelion resides within populations, and can thus be captured by targeted sampling from a subset of the species range. The extent to which these populations vary in terms of adaptively important genetic variation remains to be seen.S1 FigThe x-axis indicates the number of repeat units.(TIFF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableST and above diagonal values represent the P value for the corresponding FST.Below diagonal values are pairwise F(XLSX)Click here for additional data file.S3 TableNumbers represent allele sizes in base pairs. Missing data is represented by ‘0’.(XLSX)Click here for additional data file.


As a multi-ethnic country, China has some indigenous population groups which vary in culture and social customs, perhaps as a result of geographic isolation and different traditions. However, upon close interactions and intermarriage, admixture of different gene pools among these ethnic groups may occur. In order to gain more insight on the genetic background of X-Chromosome from these ethnic groups, a set of X-markers (18 X-STRs and 16 X-Indels) was genotyped in 5 main ethnic groups of China . Twenty-three private alleles were detected in HAN, Uygur, Tibetan and Mongolian. Significant differences (p < 0.0001) were all observed for the 3 parameters of heterozygosity  among the 5 ethnic groups. Highest values of Nei genetic distance were always observed at HUI-Uygur pairwise when analyzed with X-STRs or X-Indels separately and combined. Phylogenetic tree and PCA analyses revealed a clear pattern of population differentiation of HUI and Uygur. However, the HAN, Tibetan and Mongolian ethnic groups were closely clustered. Eighteen X-Indels exhibited in general congruent phylogenetic signal and similar cluster among the 5 ethnic groups compared with 16 X-STRs. Aforementioned results proved the genetic polymorphism and potential of the 34 X-markers in the 5 ethnic groups. Identification of the genetic information of populations is an important task for which different types of genetic markers can be used, including autosomal1223565666567868Some Chinese investigators have examined population differentiation and admixture patterns for Chinese ethnic groups and some Central Asian populations with mtDNA or Y chromosomes1034 X-Chromosomal markers (16 X-STRs and 18 X-Indels) of 500 samples from 5 main ethnic groups of China were genotyped for 100 individuals in each ethnic group . The genotyping profiles of control DNA 9947A (0.5 ng) amplified with Panel I (16 X-STRs) and Panel II (18 X-Indels) are listed as p > 0.05), male and female samples were pooled for each ethnic group and used for further investigation. Allelic frequencies of the 34 X-Chromosome markers are listed in Since the 34 studied markers were located on X-Chromosome, the allelic frequency of each marker in each population was calculated in both male and female samples. As no significant differences were observed (p < 0.0001) were observed among the 5 ethnic groups with each kind of heterozygosity. Population heterozygosity varied greatly from locus to locus as seen in Based on the genotyping information, the Observed Heterozygosity (Ho), Expected Heterozygosity (He) and Unbiased Expected Heterozygosity (UHe) of the 34 markers in the 5 ethnic groups were calculated with the GENALEX 6.3 software . HeterozLinkage Disequilibrium (LD) testing of the 34 X-Chromosome markers was explored with Arlequin 3.1 software. No LD was observed neither among the 16 X-STRs nor the 18 X-Indels. Thus the cumulative power of exclusion  of the 16 X-STRs ranged from 0.999985835 (HAN ethnic group) to 0.99999846 (Uygur ethnic group) and the cumulative power of exclusion of the 16 X-Indels ranged from 0.999045398 (HUI ethnic group) to 0.99997081 (HAN ethnic group) among the 5 ethnic groups.et al.A population comparison was made among all testing samples. Ten pairwise ethnic groups were obtained among the 5 ethnic groups. Upon analysis with the two different types of X-Chromosome markers separately and combined, Nei genetic distances of the 10 pairwise ethnic groups were listed in 141458910Comparison of the data from three groups , the groComplementary phylogenetic trees, constructed from specific genetic distances, can easily deduce the evolutionary relationships and origins of different populations1719When the Indels data were combined with the STRs data for phylogenetic analyses, some small changes in population groupings and slight increases in bootstrap support for some nodes of the UPGMA phylogram were observed . The rel16 X-STRs and 18 X-Indels were detected with same PCR components (except for the primer mixes) and PCR conditions in 5 main ethnic groups of China. The genetic data varied for each population and enriched the database of the X-Chromosome markers. Twenty-three private alleles were detected among 4 ethnic groups (except HUI). For the 3 parameters of heterozygosity  of the 34 X-markers in the 5 ethnic groups, significant differences (p < 0.0001) were observed. Nei genetic distances between HUI and Uygur are always the highest, regardless of the markers used for analysis. HAN, Tibetan and Mongolian ethnic groups were closely clustered when analyzed with the 34 X-Chromosome markers. Although Indels are less polymorphic compared with STRs, they provided in general congruent phylogenetic signal and same cluster among the 5 ethnic groups. These results demonstrated that geographic isolation and interactions play significant roles in differentiation of genetic constitution of ethnic groups. However, the observed genetic relationships could be complicated by many factors including the origins of samples, the choice of genetic markers and coverage of reference populations. Thus, great care must be taken when relating one Chinese ethnic group to another, as there may have been intermixture with now formally extinct populations that contributes to current population genetic structure.Human blood samples were collected upon approval of Ethics Committee at the Institute of Forensic Sciences, Ministry of Justice, P. R. China. A written informed consent was obtained for each participant in this study. The main experiments were conducted at the Forensic Genetics Laboratory of Institute of Forensic Science, Ministry of Justice, P.R. China, which is an accredited laboratory by ISO 17025, in accordance with quality control measures. All the methods were carried out in accordance with the approved guidelines of Institute of Forensic Sciences, Ministry of Justice, P.R. China.In this study, peripheral blood samples of healthy unrelated individuals from 5 main ethnic groups  of China were collected. The detailed location information of the 5 ethnic groups is labeled in 34 X-Chromosome markers for this study here were inferred to 18 X-InDels and 16 X-STRs. Detailed information of the amplification system is listed in the xl Genetic Analyzer . Genotyping data were analyzed with GeneMapper v3.2.1 software . Control DNA of 9947A  was used as positive sample.PCR products were then analyzed by mixing 1 μL of each amplified product, with 9 μL of a 18:1 mixture of Hi-Di formamide  and SIZ 500  for capillary electrophoresis. Electrophoresis was performed on AB 3130ST values, were assessed using GENALEX 6.3 software. For an easier visualization of the observed genetic distances, PCA analysis was reformed with GENALEX 6.3 software also. The significance level was based on 999 random permutations. Phylogenetic tree based on genetic distances was conducted with MEGA6 software using the Unweighted Pair Group Method with Arithmetic mean (UPGMA) method. Phylograms were created for STR and Indel marker data separately and also for combined datasets. The reliability of phylograms was estimated by bootstrapping 2000 replicates over loci and the extended majority rule consensus trees were inferred.Analyses of genetic parameters including allelic frequencies, observed and expected heterozygosities, Hardy-Weinberg equilibrium testing were performed with GENALEX 6.3. Exact tests for differentiation between female and male were performed with Arlequin. LD analysis was performed using the SNP Analyzer v2.0How to cite this article: Zhang, S. et al. Population genetic study of 34 X-Chromosome markers in 5 main ethnic groups of China. Sci. Rep.5, 17711; doi: 10.1038/srep17711 (2015).


Lutjanus purpureus is a marine species fish commonly found associated with rocky seabeds and is widely distributed along of Western Atlantic. Data on stock delineation and stock recognition are essential for establishing conservation measures for commercially fished species. However, few studies have investigated the population genetic structure of this economically valuable species, and previous studies  provide an incomplete picture. The present study used a multi-locus approach  to elucidate the levels of genetic diversity and genetic connectivity of L. purpureus populations and their demographic history. L. purpureus has high levels of genetic diversity, which probably implies in high effective population sizes values for the species. The data show that this species is genetically homogeneous throughout the geographic region analyzed, most likely as a result of dispersal during larval phase. Regarding demographic history, a historical population growth event occurred, likely due to sea level changes during the Pleistocene.The Caribbean snapper  Due to the apparent lack of vicariant barriers in marine environments, processes leading to genetic isolation are rare compared to terrestrial continental environments . In addiLutjanus purpureus (Poey 1866) is a marine fish found in rocky and sandy seabeds, usually associated with substrate, at depths of up to 160 meters )http://www.aoml.noaa.gov/phod/graphics/dacdata/seasonal_brazil.gif), which may facilitate connectivity in the region studied. Additionally, data collected for drifters show that floating objects can cross the study region in a little more than a month  [L. campechanus [O. chrysurus [L. purpureus [L. purpureus. Significant negative values for the tests of neutrality were measured for a large portion of the loci used here, which may be due to deviations from neutrality caused by population expansion  , L. camppechanus , O. chryhrysurus  and L. purpureus ,7. The pion (e.g.). It is ion  [O. chrysurus [Lutjanus synagris (Linnaeus 1758) .The Bayesian Skygrid analysis clearly showed a growth curve with the effective population size increasing ten-fold  beginning approximately 150 thousand years ago, congruent with a glacial maximum period . Similarer 1830) , O. chryhrysurus  and LutjHowever, we used a phylogenetically derived mutation rate, which can sometimes result in erroneous inferences of demographic events, hence the age estimate of the population expansion event should be interpreted with caution, because of time-dependence of molecular clock . For spA region . HoweverL. purpureus maintain high levels of genetic diversity, probably a reflex of the large effective population size of the species. However, these findings should not be understood as evidence of a lack of overfishing effects on the snapper populations, as this activity is recent. Additionally, given the large effective population size of this species, the effects of this activity on genetic diversity may not be detectable.The results obtained here, show that L. purpureus is genetically homogeneous across the region studied, which may contribute to the high levels of genetic diversity present there [sensu Dawson et al. [L. purpureus, have an initial larval dispersal period and spawn in the open ocean [In addition, nt there . It is an et al. ). Neverten ocean ,71. ThusS1 File(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableThe highest probability values and lower variance when K = 1.(DOCX)Click here for additional data file.


Trichuris muris, is intimately associated with its house mouse host, we studied its population genetics across the European house mouse hybrid zone (HMHZ) which has a strong central barrier to gene flow between mouse taxa. T. muris has a direct life cycle and nonmobile free stage: if these traits constrain the parasite to an intimate association with its host we expect a geographic break in the parasite genetic structure across the HMHZ. We genotyped 205 worms from 56 localities across the HMHZ and additionally T. muris collected from sympatric woodmice (Apodemus spp.) and allopatric murine species, using mt‐COX1, ITS1‐5.8S‐ITS2 rDNA and 10 microsatellites. We show four haplogroups of mt‐COX1 and three clear ITS1‐5.8S‐ITS2 clades in the HMHZ suggesting a complex demographic/phylogeographic history. Microsatellites show strong structure between groups of localities. However, no marker type shows a break across the HMHZ. Whipworms from Apodemus in the HMHZ cluster, and share mitochondrial haplotypes, with those from house mice. We conclude Trichuris should not be regarded as an ‘intimate’ parasite of the house mouse: while its life history might suggest intimacy, passage through alternate hosts is sufficiently common to erase signal of genetic structure associated with any particular host taxon.Host‐parasite interaction studies across hybrid zones often focus on host genetic variation, treating parasites as homogeneous. ‘Intimately’ associated hosts and parasites might be expected to show similar patterns of genetic structure. In the literature, factors such as no intermediate host and no free‐living stage have been proposed as ‘intimacy’ factors likely constraining parasites to closely follow the evolutionary history of their hosts. To test whether the whipworm,  It has been suggested that the short generation time, and therefore potential for rapid divergence, of parasites relative to their hosts means the genetic structure of ‘intimate’ parasites can be used to resolve recent divisions in host populations (review in Whiteman and Parker Mus musculus musculus (hereafter Mmm) and western Mus musculus domesticus (hereafter Mmd), after having diverged for around 500,000 years in isolation  is a gastrointestinal nematode parasite of murine rodents  and rats (Rattus sp.) are also identified as T. muris , found to carry the western T. muris type, were included in their study . Within each T. muris type, no genetic distinction according to host species was found. It should be noted, however, that the marker used in this study, the ITS1‐5.8S‐ITS2 region of the ribosomal DNA (rDNA), is highly conserved and may simply not be variable enough to pick‐up potential host race formation in sympatric hosts – this might require more polymorphic markers such as microsatellites   Do the ITS1‐5.8S‐ITS2 rDNA lineages found in the HMHZ correspond to the western and eastern clades previously suggested across Europe; (3) Does T. muris show population structure at the murine species/locality level? (4) What is the inferred role of other sympatric murine host species in the transmission of T. muris across the HMHZ?To test which of these two scenarios best fits n et al. . We addrTrichuris muris worms were collected from house mice at 56 localities across a transect (125 km long on South‐North axis and 140 km wide on Western‐Eastern axis) stretching from northeastern Bavaria (Germany) to western Bohemia (Czech Republic) between 2007 and 2013  from the same HMHZ area, from one Mmd captured in Northern Spain, and from one Apodemus sp. and one Rattus rattus from Southeastern France (Table S1). DNA was extracted from 214 worms, one nematode individual per host  using the DNeasy® DNA isolation kit . DNA was eluted in 40 μL of buffer AE. In general, DNA was extracted from male or immature T. muris worms. When no male or immature worms were available, we isolated the DNA from the upper part of a female body (stichosome) to avoid extracting the DNA from eggs contained in the posterior part of the body.® 96 Tissue Kit (Qiagen) following the manufacturer's instructions. All mice were genotyped either at 1401 ‘diagnostic’ X‐linked and autosomal SNP markers spaced approximately 1.86 Mb apart across the genome as part of a parallel study  was calculated to place any mouse on a linear scale from Mmd (HI = 0) to Mmm (HI = 1) depending on the count of musculus alleles at assayed loci .Host DNA was extracted from spleen and/or tail using DNeasyμL of Multiplex PCR™ Kit (Qiagen), 0.5 μmol/L of each primer, 2 μL of DNA and double distilled water added to the final volume of 20 μL. PCR started with an initial activation step at 95°C for 15 min, followed by 35 cycles of denaturation at 92°C (94°C for ITS region) for 60 sec, annealing at 51°C (52°C for ITS region) for 90 sec, extension at 72°C for 60 sec, and a final extension at 72°C for 10 min. PCR products were directly sequenced in both directions by VIB Genetic Service Facility . We were aware that ITS1‐5.8S‐ITS2 rDNA may generate problematic chromatograms after sequencing because this marker is present in multiple potentially divergent copies in the genome. However, previous work on T. muris using cloning .A portion of the mitochondrial cytochrome oxidase I (COX1) gene (1143 bp) was amplified using newly designed primers Trichuris_Cox1_F: CAGGAAATCACAAAAAAATTGG and Trichuris_Cox1_R: GAAAGTGTTGGGGYAKAAAAGTTA. The nuclear ITS1‐5.8S‐ITS2 rDNA region was amplified using NC2 and NC5 primers . For development of new microsatellite markers, we chose those contigs containing ideal repeats . Primers were designed using the online tool Primer3Plus (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi), and the forward primers were labeled fluorescently. Amplification success and polymorphism of the loci were initially tested on 10 samples from different localities (see more details in Table S1). A final set of 10 loci was amplified in three multiplex PCRs designed according to the dye and length of the products to avoid overlapping of loci with same labels (Table S2). The multiplex PCRs were carried out in a final volume of 10 μL in a Mastercycler‐ep‐Gradient‐S (Eppendorf) with a mix containing 5 μL of the Multiplex PCR™ Kit (Qiagen), 0.25 μmol/L of each primer, 1 μL of DNA and double distilled water. PCR started with an initial activation step at 95°C for 15 min, followed by 30 cycles with denaturation at 94°C for 60 sec, annealing at 58°C for 90 sec, extension at 72°C for 60 sec, and a final extension at 72°C for 10 min. Length of PCR products was analyzed on ABI3130xl Genetic Analyzer . The reaction mix for the fragment analysis composed of 0.4 μL of Gene Scan‐500LIZ Size Standard , 12 μL of formaldehyde, and 0.8 μL of PCR product was denatured at 95°C for 5 min before capillary electrophoresis. Gene Mapper v3.7 (Applied Biosystems) was used for identification of peaks and detection of allele lengths.We first used three primer pairs originally designed for h) and nucleotide diversity (∏) were calculated in DnaSP v5 . A haplotype data file was generated and haplotype diversity  sequences of the nuclear ITS1‐5.8S‐ITS2 rDNA region (969 to 976 bp long) could be read from direct sequencing. Among these 39, when the chromatogram of an individual appeared to have divergent copies of the marker, i.e., several peaks starting to appear on the chromatogram at the microsatellite motif of the ITS1 and/or ITS2 regions, one copy was much stronger and could easily be distinguished from the other(s) which appeared as very low baseline noise. We thus report here the ‘strong’ sequences of these 39 individuals. Sequences were aligned in Geneious 7.1.7  with the ITS1‐5.8S‐ITS2 rDNA sequences of n et al.  making rBayesian inference of phylogenetic relationships among mitochondrial and nuclear sequences was performed in MrBayes_v3.2.0 (Ronquist and Huelsenbeck T. muris worms were genotyped at 10 microsatellite loci (Table S2). All loci consistently amplified in all samples. To analyze the genetic structure at locality level, we performed population genetic analyses for localities with at least five genotyped individuals. We tested Hardy–Weinberg equilibrium using GENEPOP 4.3 (Raymond and Rousset Ho) and expected (He) (Nei's unbiased estimator) heterozygosities and pairwise FST values in FSTAT 2.9.3 (Goudet FST was transformed to FST / (1 − FST) as suggested by Rousset , were plotted using Structure Harvester  and then localities ranked according to average HI of host individuals in that locality (from 0 toward 1). Additionally, one model‐independent exploratory approach, Discriminant Analysis of Principal Components (DAPC), was applied to the microsatellite dataset, using the adegenet package  was 0.790. Maximum p‐distance between different haplotypes was 3.4%, minimum was 0.1% and average was 1.5%. Analyzing the pair of worms from the same host individual showed identical haplotypes  (Table S1). The mt‐haplotype network showed two big and three small haplogroups  is found in this clade and fall into six haplotypes . The second clade (posterior probability = 1) corresponded to the “Eastern continental Europe (ECE)” clade of Callejón et al.  grouped together haplotype HMHZ_H1 which belongs to an individual from the western side of the HMHZ with a T. muris haplotype from Croatia from A. flavicollis and a T. muris haplotype from Spain from A. sylvaticus. We thus call this the pan European (PE) clade. Plotting membership of the three ITS1‐5.8S‐ITS2 rDNA clades on a map, sequences of the first two clades (WCE and ECE) from T. muris in the HMHZ did not show any geographic break at the host contact zone . jModeltest suggested the HKY substitution model. The Bayesian phylogenetic reconstruction Fig.  providedn et al.  which grHo) was 0.257 and expected heterozygosity (He) was 0.354 . Most populations showed significant deviations from Hardy–Weinberg equilibrium except HOHE1, OBIL, and WOHL (Table S3). Pairwise FST values between populations with ≥5 individuals ranged from 0.050 to 0.580 (Table S3) with the overall value of 0.271. No significant genetic isolation by geographical distance was observed between localities   suggesting important role of genetic drift  and long‐distance migrations (low genetic differentiation between distant populations).All microsatellites were polymorphic, ranging from two to 11 alleles per locus (Table S2). There was no evidence of linkage disequilibrium (not shown), although many tests within locality samples were not possible because of locally fixed alleles. Worms from the same host individuals (six pairs) showed genotypes differing in average at five loci (range 3‐8). The average value per population for observed heterozygosity (K) from 1 to 15, increased asymptotically  and the resulting estimates of cluster membership are shown in Figure K and without respect to the host hybrid index (HI): Individuals from localities with similar host HI such as PILG and OTTM grouped in different clusters even at low K . Other localities such as PAST and PILG, despite being close to ‘pure’ Mmd and Mmm separated by 99 km cluster together even at high K. T. muris from Apodemus individuals caught in the HMHZ differ no more from house mouse T. muris taken from different localities, and never form an exclusive cluster.Likelihoods for STRUCTURE analyses for increasing numbers of genetic clusters . The first twenty PCA axes and five discriminant functions were retained and explained 93% of the total variance. As with the STRUCTURE analyses, clustering roughly corresponded to groups of localities  are predictive of the parasite's genetic structure across the European house mouse hybrid zone, or if, in contrast; (2) the frequency of T. muris passage through alternative hosts is sufficient to break any potential signal of intimate association with the house mouse host.We set out to compare two alternative hypotheses: whether (1) the characteristics of the host–parasite interaction between T. muris population genetics are consistent with previous studies. Pairs of T. muris from six mice had different nuclear, but similar mitochondrial, genotypes. This is consistent with accumulation of individual host worm load through repeated low‐level infection  is a better explanation of the data than hypothesis (1). That is: although T. muris seems to have a suite of traits making it a good candidate for an intimate host relationship, in nature it does not form an intimate relationship with house mice.At a broader scale we show that, while there is plenty of signal of structure within our T. muris clades which correspond in part to the previously suggested WCE and ECE clades of Callejón et al.  T. muris carried by the two taxa of house mice diverged during a period in their history where passage in alternate hosts could not break their association – for example, as the two mice taxa spread in opposite directions round the Mediterranean and Black seas during their colonization of Europe , breaking down the association between worm strains and host taxa such that eastern and western worm clades no longer strictly match eastern and western mouse clades. (2) The same scenario but T. muris is originally carried by two taxa of woodmice, which colonized Europe before the arrival of synanthropic house mice: A. sylvaticus and A. flavicollis are closely related woodmouse species which recolonized the Western Palearctic (and thus the HMHZ area) from different glacial refugia, A. flavicollis from the Italo‐Balkanic area and A. sylvaticus from the Iberian Peninsula  clade of T. muris may be a remnant of distinctive strains brought by either the house mouse or the rat, which colonized Europe even later , and thus a considerable life history investment by the parasite. An important question is: if a locality used by one host taxon (and therefore ‘infected’ with the free‐living stage of the parasite) lapses from host use, how often will it be repopulated  while ‘infection’ is still possible? If the duration of the free‐living stage of the parasite is long relative to this period of interhost‐taxon locality usage turnover then, that duration may be long enough to break host–parasite intimacy. Similar logic applies to descriptions of host as having ‘low vagility’: If host vagility is sufficient in the context of potential host taxa distribution (sympatric/parapatric) to allow interhost locality usage turnover faster than the free‐living stage of a parasite looses its infective potential then, that vagility may be sufficient to break intimacy.Given the potential importance of parasites as biotic factors affecting evolutionary process, and the consequences of parasites moving between hosts  are judged to have partly overlapping ecological niches, but are rarely trapped simultaneously from the same farm building . This might suggest low passage rates between alternate hosts, but these same locations are almost always found to be occupied by at least one host , suggesting the period during which sites remain unoccupied is short, allowing parasite transmission through the free‐living stage in feces.How then can generalist parasites form host races when their hosts are sympatric , each of which shows a break in genetic structure across the HMHZ  stage, the oocyst, and has been found in a range of vertebrates   and genetic (as FST / (1 − FST)) distances among populations.Figure S2. Log probability of data L(K) as a function of K (mean ± SD for 10 replicates).Figure S3. Inference of the number of clusters in the DAPC analysis.Click here for additional data file.Table S1. Localities and their location, sample ID, host hybrid index (HI), host grouping based on hybrid index (Group), mitochondrial DNA haplotypes (mt hap), samples used in microsatellite analysis (Micro) and ITS1‐5.8S‐ITS2 haplotypes.Table S2. Characteristics of 10 microsatellite loci and measures of genetic diversity )Table S3. Hardy‐Weinberg equilibrium test  and pairwise FST values among populations .Click here for additional data file.


Pseudocheirus occidentalis) in Busselton, Western Australia. Using spatial autocorrelation analysis, we found positive genetic structure in continuous habitat over distances up to 600 m. These patterns are consistent with the sedentary nature of P. occidentalis and highlight their vulnerability to the effects of habitat fragmentation. Pairwise relatedness values and Bayesian cluster analysis also revealed significant genetic divergences across an artificial waterway, suggesting that it was a barrier to gene flow. By contrast, no genetic divergences were detected across the major road. While studies often focus on roads when assessing the effects of artificial linear structures on wildlife, this study provides an example of an often overlooked artificial linear structure other than a road that has a significant impact on wildlife dispersal leading to genetic subdivision.The fragmentation of habitats by roads and other artificial linear structures can have a profound effect on the movement of arboreal species due to their strong fidelity to canopies. Here, we used 12 microsatellite DNA loci to investigate the fine-scale spatial genetic structure and the effects of a major road and a narrow artificial waterway on a population of the endangered western ringtail possum ( Crotalus horridus) and bighorn sheep (Ovis canadensis nelsoni). Since inbreeding and reductions in genetic diversity increase the risk of extinction of isolated populations [Roads and other artificial linear structures, such as railways, powerline corridors, and artificial waterways are thought to inhibit movements of some animals, leading to the fragmentation of populations, increased inbreeding, and loss of genetic diversity . In a reulations , it is cPseudocheirus occidentalis Thomas 1888) is a medium-sized nocturnal marsupial endemic to the southwest of Western Australia, the only biodiversity hotspot on mainland Australia [Strictly arboreal species are thought to be more vulnerable than the majority of terrestrial species to the impacts of artificial linear structures without canopy connections because many of them tend to avoid descending to the ground , 7. The ustralia , 10. Stuustralia . They alP. occidentalis has gone through a dramatic decline in numbers and range due to anthropogenic factors such as habitat destruction and fragmentation and the impact of introduced predators [P. occidentalis [Over the last few decades redators , 12. Theredators , and suidentalis .P. occidentalis are supported by the presence of fine-scale genetic structure. We also investigated whether a road and artificial waterway without canopy connection were associated with genetic divergences. Given the limited movements across artificial linear structures, a strong reluctance to traverse on the ground, and lack of evidence that the species voluntarily swims [In this study, we used microsatellite markers to investigate whether the previously reported small home ranges and limited dispersal in ly swims , we predAgonis flexuosa) is supporting a high density of P. occidentalis [This study was conducted in Locke Nature Reserve and surrounding campsites, 9 km west of Busselton, Western Australia , where the habitat dominated by peppermint trees . Sample sizes within each block ranged from 10 to 32 . Sample collection followed the methods developed by P. de Tores and reported by Clarke [Between March 2010 and November 2012, we captured a total of 145 adult possums by using a specially modified tranquiliser dart gun with darts containing a dose of 11–12 mg/kg of Zoletil 100y Clarke . A thin y Clarke  or 90% eWe extracted DNA from each sample using Qiagen DNeasy Blood and Tissue kit  following the manufacturer’s instructions. Concentration and quality of each DNA sample were then determined using a NanoDrop ND-1000 spectrophotometer . We used 12 species-specific microsatellite markers  following PCR conditions described by Wilson et al. . GenotypAR) and Nei [H) within Fstat v.2.9.3 software package [FIS) statistic. Significantly positive FIS values indicate a deficit of heterozygotes relative to a random mating model, while negative results indicate an excess of heterozygotes. Genotypic disequilibrium was tested between each pair of loci within each study block. FIS values and tests for deficits in heterozygotes and genotypic equilibrium were calculated using the Fstat v.2.9.3. Tests for differences in genetic diversity and FIS among study blocks were performed using Friedman’s ANOVA and Wilcoxon rank tests with the R v.3.0.1 statistical package [The presence of null alleles was assessed for each locus using Microchecker v.2.2.3 . Genetic and Nei ’s estima package . Deviati package .n = 69) to investigate how the genetic similarity of individuals changed over geographical distance within continuous vegetation . The result from this analysis would also tell us whether distances that were the same as the widths of the road and artificial waterway were large enough to cause genetic divergence in continuous vegetation. We performed SA analyses using GenAlEx v.6.501 [r) were calculated and plotted over a range of distance classes increasing at 100 m intervals to obtain autocorrelograms. Secondly, because estimates of r are influenced by the size of distance classes [r for a series of increasing distance class sizes. Decreasing r with increasing distance interval class indicates significant positive spatial structure, and the distance interval class at which r is no longer greater than zero represents the limit of detectable genetic structure [r using 1000 replicates. We also conducted 2-Dimensional Local Spatial Autocorrelation (2DLSA) analysis using GenAlEx. This analysis investigates uniformity of spatial autocorrelation over the study area by estimating local autocorrelation (lr) by comparing each individual with its nearest neighbours [lr were made using the nearest five individuals with statistical significance determined using permutation tests.We performed a Spatial Autocorrelation (SA) analysis on the samples collected from the nature reserve only  based on allele frequencies across all loci. Analyses were based on an ancestry model, which assumes admixture and correlated allele frequencies with study blocks used as prior information about the origin of the samples. A burn-in period of 100,000 and 1,000,000 Markov Chain Monte Carlo (MCMC) iterations were used for 10 replicate runs for each number of clusters (K) ranging from 1 to 10. We then determined the most likely value of K using the ΔK method of Evanno et al. [K clusters from 10 replicate runs was calculated and re-organised using Clumpp v.1.1.2 [v.1.1 [Population structure across the whole study area was assessed using pairwise  v.2.3.4 . This meo et al.  implemeno et al. . Each in v.1.1.2  and visu2 [v.1.1 .The effects of artificial linear structures were then examined by comparing mean relatedness values between pairs of individuals on the same side of the road or waterway with relatedness values between pairs of individuals on opposite sides of the road or waterway. The effect of the road was examined without data from individuals on the other side of the waterway (2B and 2D) to remove the effect of the waterway, and the effect of the waterway was examined without data from those on the other side of the road (1B and 1D) to remove the effect of the road. We calculated pairwise relatedness values using the method of Queller and Goodnight  with GenFIS values were significantly different from zero, indicating that the groups of possum in all blocks were in Hardy-Weinberg Equilibrium. There was no evidence of genotypic disequilibrium between any pair of loci within any study block based on 9240 permutations after correcting for multiple comparisons by using an adjusted P value of 0.000108. There were also no significant differences in allelic richness , gene diversity  or FIS  among study blocks . Since there was no consistent pattern in the presence of null alleles , all loci were retained for further analysis. No y blocks .r) was significantly positive up to 100 m, and it intercepted zero at 347 m  pairs of blocks. These pairs of blocks occurred on the same and opposite sides of Caves Road, as well as the same and opposite sides of the artificial waterway . Pairwiswaterway .K method clearly identified K = 3 as the most likely number of genetic clusters , the bricillata ), the Eues meles ), the soatifrons ), and thlcensis, ). All ofTrichosurus vulpecula) found positive spatial structure up to distances of 896 m and 454 m for males and females, respectively [Phascolarctos cinereus) has also been recorded to disperse up to 10.6 km [P. occidentalis is driven by males, which is based on observations of only a few individuals [P. occidentalis even compared with other arboreal marsupials, highlighting their high susceptibility to the impacts of habitat fragmentation.SA analysis on another species of possum, the common brushtail possum  occurring on different sides of roads and streams, Marsh et al. [Propithecus tattersalli) were a river and geographical distances rather than a road. On the other hand, Estes-Zumpf et al. [Brachylagus idahoensis) possibly due to the greater mobility of this species. These results, together with our own, indicate that different types of linear barriers can have different levels of permeability to the movement of different species, and the permeability of barriers also depends on the mobility of the species. Therefore, it is important to assess the negative impacts of a wider range of artificial linear structures especially in areas where endangered sedentary species occur.In a study investigating genetic divergence among red-backed salamanders  were considerably smaller than the others, lowering the reliability of the FST value. The pairwise FST values showed inconclusive results across the road. For example, FST values between blocks 1A and 1B and blocks 1-2C and 1D were found to be significant; however, FST values between blocks 1B and 2A and blocks 1D and 2A were non-significant despite the presence of a road and greater distances between these blocks. This inconsistency may have been caused by the small sample sizes in some of the study blocks and/or the fine scale of the genetic divergence examined in this study. Individual-level analyses, such as pairwise relatedness analyses and Bayesian cluster analyses, have been suggested to be more appropriate than population-level analyses, such as estimated pairwise FST, for studies examining fine-scale genetic divergence because combining individuals within populations can lead to loss of fine-scale information [ocks see , and it ormation . Given tAll blocks had high levels of genetic variation and did not deviate from Hardy-Weinberg equilibrium, suggesting that the possums in these blocks are not experiencing a high level of inbreeding despite the presence of two barriers. This is probably because blocks were still connected with other patches of habitat outside our study area and they were not completely isolated. The different cluster membership in the Bayesian cluster analysis for blocks 2B and 2D may also be explained by these connections to other patches. Blocks 2B and 2D were isolated by the waterway from the other blocks, and were separated from each other by a mostly cleared campsite. Given the exceptionally highly sedentary and arboreal nature of this species , the limP. occidentalis is expected to decline dramatically in the future due to the current increasing pressure of habitat clearing and impacts of climate change [Caves Road has not caused a detectable genetic divergence among possums yet, but it is restricting their movements and causing mortality , which ae change . Even thP. occidentalis have limited dispersal, making them highly susceptible to the impacts of habitat fragmentation. Indeed, we found evidence to suggest that an artificial waterway has been limiting the gene flow of P. occidentalis, resulting in genetic divergence within spatial scales of hundreds of metres. By contrast, a busy road does not seem to have had a detectable impact on population structure, though future impacts cannot be ruled out. While studies investigating negative effects of artificial linear structures on population structure tend to focus on roads, our study provides an example where an artificial waterway may be posing significantly greater genetic impacts on an endangered arboreal species than a major road. We therefore urge for more research to be conducted on the impacts of artificial linear structures other than roads, and their impacts be mitigated where significant.Both telemetry data from a previous study  and gene


Myotis pilosus).China is characterized by complex topographic structure and dramatic palaeoclimatic changes, making species biogeography studies particularly interesting. Previous researchers have also demonstrated multiple species experienced complex population histories, meanwhile multiple shelters existed in Chinese mainland. Despite this, species phylogeography is still largely unexplored. In the present study, we used a combination of microsatellites and mitochondrial DNA (mtDNA) to investigate the phylogeography of the east Asian fish-eating bat , Sichuan Basin (SCB) and North and South of China (NSC), respectively. The most recent common ancestor of M. pilosus was dated as 0.25 million years before present (BP). Population expansion events were inferred for populations of Clade C, North China Plain region, Clade B and YunGui Plateau region at 38,700, 15,900, 4,520 and 4,520 years BP, respectively. Conflicting results were obtained from mtDNA and microsatellite analyses; strong population genetic structure was obtained from mtDNA data but not microsatellite data. The microsatellite data indicated that genetic subdivision fits an isolation-by-distance (IBD) model, but the mtDNA data failed to support this model.Phylogenetic analyses showed that M. pilosus. Spatial genetic structures of maternal lineages that are different from those observed in other sympatric bats species may be as a result of interactions among special population history and local environmental factors. There are at least three possible refugia for M. pilosus during glacial episodes. Apparently contradictory genetic structure patterns of mtDNA and microsatellite could be explained by male-mediated gene flow among populations. This study also provides insights on the necessity of conservation of M. pilosus populations to conserve this genetic biodiversity, especially in the areas of YTP, SCB and NSC regions.Our results suggested that Pleistocene climatic oscillations might have had a profound influence on the demographic history of  Global climatic oscillations have profoundly influenced species’ distributions and their genetic traits , and as In the context of complex regional conditions in China, studies on animal geographic distribution patterns have provided information on the evolutionary histories following the global Pleistocene climate changes ,4. UsingMyotis davidii; one in the Yungui Plateau region and the other in the Southeast China Hills region  and geographical distances in IBDWS 3.23 [For mtDNA and microsatellite, pairwise genetic distances  and raggedness index (Rag) to test the significance of fit of distribution. When an expansion model could not be rejected, we estimated the time of expansion (t) from τ = 2ut, where τ is calculated as the time to expansion in mutational units, and u is the mutation rate per generation for the whole sequence. The u is equal to μgk, where μ is the mutation rate per nucleotide (see above 10%) and k is the sequence length. The generation time (g) was estimated to be two years [Neutrality tests and mismatch distribution analyses can provide hints to infer population demographic events. Tajima’s D, Fu’s Fs and wo years .The authors declare that have no competing interests.GL conceived and designed the experiments and carried out the molecular genetic analysis, participated in the manuscript’s design, and drafted the manuscript. JF and KS developed the ideas and obtained funding support. AL participated in the design of the study and performed the statistical analysis. JL helped collect samples. KS, DVB and KAM helped draft the manuscript. All authors read and approved the final manuscript.Variable sites of mtDNA for 21 Haplotypes. The Arabic numbers at the top of the figure indicate the variable sites. The “.” indicate agreement with haplotype 1 depicted in the first row. The “-” indicate the insertions or deletions. The “︱ → ←︱”indicate the saved first repeat from 267 to 347 with 81 bp. Sites 267 and 347 are not variable sites.Click here for fileGenBank accession numbers for mtDNA haplotypes and outgroups used in the present study.Click here for fileResults of POWSIM simulations assessing the statistical power of microsatellite loci to differentiate populations with Fst = 0.005. The results show the proportion of simulations out of 1,000 significant with a critical value of 0.05.Click here for filePopulation genetic differentiation  and gene flow  for Myotis pilosus. Additional file 4a for mtDNA and 4b for microsatellite“-” from upper-right indicates the gene flow is infinity. Statistically significant results are indicated by asterisks: * P < 0.05.Click here for fileResults of mismatch distributions for all populations, three lineages and three geographical units. The full circles show observed values whereas the open circle represents expected values.Click here for file


Fontainea picrosperma, a subcanopy tree endemic to the rainforests of northeastern Australia, is of medicinal significance following the discovery of the novel anti-cancer natural product, EBC-46. Laboratory synthesis of EBC-46 is unlikely to be commercially feasible and consequently production of the molecule is via isolation from F. picrosperma grown in plantations.Successful domestication and plantation production requires an intimate knowledge of a taxon’s life-history attributes and genetic architecture, not only to ensure the maximum capture of genetic diversity from wild source populations, but also to minimise the risk of a detrimental loss in genetic diversity via founder effects during subsequent breeding programs designed to enhance commercially significant agronomic traits.PIC = 0.429; PID = 1.72 × 10−6) to investigate the partitioning of genetic diversity within and among seven natural populations of F. picrosperma. Genetic variation among individuals and within populations was found to be relatively low , although there was marked differentiation among populations (PhiPT = 0.248). Bayesian, UPGMA and principal coordinates analyses detected three main genotypic clusters (K = 3), which were present at all seven populations. Despite low levels of historical gene flow (Nm = 1.382), inbreeding was negligible (F = -0.003); presumably due to the taxon’s dioecious breeding system.Here we report the use of eleven microsatellite loci  contains supplementary material, which is available to authorized users. Of the more than 1000 drugs of novel chemical structure  approved for use by international regulatory authorities between 1981 and 2010; greater than 60 % were derived from natural products . This isFontainea picrosperma C.T. White (family Euphorbiaceae), a dioecious subcanopy tree endemic to Queensland’s tropical rainforests, illustrates the opportunity for continuing discovery of novel pharmaceuticals from nature and the value in protecting Australia’s mega-diverse rainforest flora.Australia is one of a small number of countries that are considered ‘mega-diverse’, which combined occupy only 10 % of the Earth’s surface, yet support over 70 % of the world’s biodiversity . The troF. picrosperma is of substantial current interest following the discovery of a novel epoxy-tigliane (EBC-46) with putative anti-cancer activity, in this species [http://www.anzctr.org.au). EBC-46 cannot currently be produced by laboratory synthesis on a commercial scale and instead is manufactured for research, preclinical and clinical use by purification from plantation-grown material of F. picrosperma.iane EBC-6 with puiane EBC-6 with puF. picrosperma may be particularly vulnerable to a loss of reproductive fitness arising from inbreeding among similar genotypes situated in close proximity, or departures from random mating due to the disproportionate contributions of particular individuals to fertilisation events, leading to reduced vigour [A more detailed knowledge of the ecology and genetics of this promising species will be critical to its domestication and future commercial drug production from plantations. Acquiring a basic knowledge of the species chromosome structure, such as chromosome number and levels of ploidy will be of future value. However, gaining an understanding of the genetic diversity and structure of natural populations, patterns of gene flow, and the taxon’s mating system is particularly important , 39. ForF. picrosperma from across the natural geographic range of this species. Our aim was to assess the relevance of populations within the context of the species as a whole, whilst simultaneously maximising the capture of available genetic variation from wild individuals. Furthermore, by ensuring maximal genetic diversity in crosses designed to enhance commercially significant agronomic traits, the microsatellite-based technique will provide an important management tool to support subsequent breeding programs used to develop F. picrosperma as a niche tree crop for the commercial supply of EBC-46.In this study, we investigate the population genetic structure within and among natural stands of PIC = 0.429) were found suitable for the analysis of population genetic diversity and structure in F. picrosperma (Table A) of 2.831 (Table AR) decreased to 2.480 alleles per locus (Table AP) were detected within five of the seven populations surveyed. In the east, two were detected in the large, putatively refugial population at Boonjie (n = 45) and one at Topaz (n = 22), while another was resolved in the 17 individuals sampled at Malanda in the central portion of the species distribution. A further three unique alleles were detected in the western populations of East Barron  and Evelyn Highlands . Proportional representations of private allelic richness for each population following rarefaction (PAR) are given in Table Despite an initial screening of 65 labelled microsatellite primer pairs, only 11 moderately polymorphic loci (mean HO) were relatively low across populations, ranging from 0.298–0.487 (mean HO = 0.397) and were more or less concordant with levels of expected heterozygosity (HE)  calculated under conditions of Hardy-Weinberg Equilibrium (HWE) , however individual population values ranged between F = −0.139 to 0.149, indicating a low to moderate excess of either heterozygotes or homozygotes at particular sites (Table Measures of observed heterozygosity (PID = 1.72 × 10−6) with only two individuals from East Barron found to share the same multilocus genotype. The other 216 samples had unique multilocus genotypes. Several microsatellite markers displaying minimal polymorphism   and populations , translating to a low to moderate level of historical gene flow , sufficient to prevent or slow the rate of genetic drift between sites  and ranged from a level of minimal differentiation  between the relatively proximate populations at Topaz and Towalla to a maximum distance  between the two northern, most isolated populations, Gadgarra and East Barron , neither Gadgarra nor East Barron displayed sufficient gene flow (Nm < 1.000) with any of the other populations to prevent genetic drift [Analysis of Molecular Variance (AMOVA) found most (75 %) of the species diversity to reside within populations, with the rest of the variation due to differences between populations (ic drift . ConversNm = 2.122) strongly suggesting that similarity (FST = 0.105) may be linked to their putative status as long-term interglacial refugia , 58 % K2 (orange) and 8 % K3 (blue), compared to 31 % K1, 13 % K2 and 56 % K3 for the eastern group (Boonjie) and 4 % K1, 32 % K2 and 64 % K3 for the central group . Evelyn Highlands and Boonjie therefore have a more uniform but differing spread of the three genetic clusters as compared to the central group, while one of the genetic clusters that is strongly represented in both Evelyn Highlands and Boonjie (K1) is only minimally represented in the plateau group, together providing support to their assignment as putative refugial populations. The Mantel test found that the geographic structuring of F. picrosperma’s genetic variation did not follow a predictable pattern, and no relationship was detected between genetic and geographic distance matrices among populations .Principal coordinates analysis (PCoA) detected a close genetic relationship between individuals within populations due to low levels of diversity Fig. . The firo et al.  method ip > 0.05). However, a significant (p = 0.004) heterozygosity excess at ten of the eleven loci was found in both the Malanda and Gadgarra populations. This data suggests that individuals at these sites are showing effects of disruption to ‘continuous’ populations and are no longer in mutation-drift equilibrium. These effects likely reflect their long-term isolation from populations in the two putative refugial areas (Boonjie and Evelyn Highlands) for this species and may have been further exacerbated by anthropogenic activities such as aboriginal burning since the Last Glacial Maximum and large-scale rainforest clearing in more recent times.Results of the Bottleneck analysis did not detect any signs of recent bottlenecks in five of the seven populations assessed  The overall genetic diversity of F. picrosperma was relatively low but the seven populations sampled from across the natural range were genetically distinct. (2) The levels of inbreeding in the individual populations were negligible despite their current discontinuous distribution and fragmentation. (3) Within the context of the low levels of genetic diversity and weak genetic structure observed for this species, two putative long-term refugial areas were identified in the eastern (Boonjie) and western (Evelyn Highlands) parts of the natural distribution of the species, which align with the refugial rainforest areas of Bartle-Frere Uplands and western Atherton Uplands identified by Hilbert et al. [There are three key findings from this study that are highly relevant not only to the domestication and breeding of t et al. .Fontainea. We investigated the levels and partitioning of genetic variation across the known range of F. picrosperma and found that the seven populations surveyed were genetically distinct despite having uniformly low levels of genetic diversity. This finding was not unexpected as many Australian plant species are characterised by low levels of genetic diversity, often as an adaptation to harsh environmental conditions [F. picrosperma. In essence, these rainforest taxa are highly adapted over long time periods to specific niches provided by the rainforest environment. As a consequence of this specialisation and niche differentiation in an essentially stable local environment, they experience only modest selection pressure during periods of climatic stability and when environmental conditions change, they retreat into the remaining environmental habitat to which they are so well adapted.This is the first study to utilise microsatellites to examine genetic structure in the genus nditions , 51, 55,nditions . For insnditions  found londitions , includiF = −0.003), which despite local populations having been isolated through glacial events, and more recently by anthropogenic habitat fragmentation, would be expected in an obligate outcrossing, dioecious species like F. picrosperma. Even though proximate trees are likely to be siblings or half-sibs, due to the limited dispersal capabilities of F. picrosperma’s relatively large drupaceous fruit, this suggests that deleterious mutations may have been purged over time, as most of the diversity resolved was between individuals within populations, not among populations.In general, the results indicate extremely low levels of inbreeding , but is a genetically more divergent population, probably due to random founder effects. Gadgarra on the other hand, is genetically distinct, most likely as it contains no unique alleles and is somewhat inbred; essentially Gadgarra is a genetically depauperate variation of the plateau form. Despite the fact that the data suggests the presence of these three groups, it is important to highlight that the genetic diversity within F. picrosperma is low and the genetic structure between these three groups is proportionately low. In fact, the pairwise FST values between Evelyn Highlands and Boonjie, Evelyn Highlands and the plateau group, and Boonjie and the plateau group range from only 0.039 to 0.060. However, each value was significantly different from zero (p <0.001) and within the context of the low levels of genetic variation within this species, this is suggestive of the presence of relevant genetic structure.The UPGMA cluster, principal coordinates and STRUCTURE analyses all provide a clear indication about the genetic distribution of this species. When combined with the genetic diversity analysis, the data show that the geographically distant (~28 km), peripheral populations of Boonjie and Evelyn Highlands, are genetically most diverse in comparison to the other populations whilst having elements of similarity, and form two genetically similar groups. Four of the remaining populations  form another genetic group, whereas the population at East Barron is genetically more divergent. We speculate that the populations at Evelyn Highlands and Boonjie represent two, genetically similar races or forms representing the two main refugial areas, where F. picrosperma exist will necessarily involve chloroplast DNA analysis and the sampling of additional individuals sourced along potential gene flow corridors, such as major river systems originating from the putative refugia at Evelyn Highlands and Boonjie.It is likely that the genetic relationship between the populations can be explained not so much by linear geographic distance but by their distribution within major river catchments radiating from the putative refugial sites of Evelyn Highlands and Boonjie. However, we also recognise the possibility that our analysis could merely be reflective of a random distribution of the observed genetic diversity. Therefore, future research to test our hypothesis that two refugial races or forms and a plateau race or form of F. picrosperma was comparatively low, high exclusion probabilities (PID) confirm that these markers will be useful in future paternity analyses and breeding programs; the former to determine patterns of gene flow in natural populations that will guide plantation design of this dioecious species, and the latter to ensure maximal genetic diversity is maintained during breeding of commercially significant agronomic traits, both of which are critical aspects of developing F. picrosperma as a niche tree crop for the supply of EBC-46.Knowledge of the genetic structure of source populations, mating system and patterns of gene flow are vital to the efficient establishment and management of seed orchard plantations and the production of improved open-pollinated seed , 7, 39. F. picrosperma individuals with regard to several commercially significant agronomic traits such as growth, fruit production and EBC-46 content, suggesting that the species will be ideally suited for genetic improvement to optimise production. However, even in dioecious species, the genetic diversity of seed orchards can be eroded by a number of factors including a high proportion of ‘selfs’ arising from consanguineous matings between sibs or half-sibs, and departures from random mating due to unequal contributions of individuals to seed crops [F. picrosperma may be particularly vulnerable to losses in reproductive fitness stemming from elevated rates of inbreeding, leading to reductions in both vigour and yield [Significant variation has been observed among ed crops . In factnd yield , 35. Thend yield .ex situ populations was to position individuals in such a way that the possibility of close relatives mating would be small and hope for the best, however new techniques based on the minimisation of the global probability of consanguinity by considering the genetic relationships among trees within the entire planting have been developed [F. picrosperma to establish which seed source and orchard variables are most likely to govern the efficiency of production plantations.Traditionally, the most cost-effective manner of limiting inbreeding in eveloped . MicrosaFontainea picrosperma is a subcanopy tree from the Atherton Tableland in Far North Queensland, Australia and is of considerable scientific and medicinal interest. The species is locally common, yet has a highly restricted range, and in relatively recent times its distribution has been heavily affected by both natural and anthropogenic habitat fragmentation. Using 11 microsatellite markers, we detected low levels of genetic diversity across the species and a population genetic structure influenced by successive cycles of glacial-induced, population expansion and contraction. The observed low levels of heterozygosity are concordant with other species of the region which have undergone similar cycles of contraction and recolonisation.F. picrosperma to be comprised of three distinct genetic races or forms. We hypothesise that these three groups broadly correspond to the existence of two long-term refugial races , where suitable habitat is known to have persisted during times of eucalypt forest expansion, and an intervening plateau race that has recolonised sclerophyllous woodlands during less severe climatic cycles.Despite the limited variation detected in this study, UPGMA cluster, Bayesian and principal coordinates analyses indicated F. picrosperma is of significant commercial interest because it is the source plant from which the novel anti-cancer agent, EBC-46 was discovered. EBC-46 is a complex small molecule natural product that is not readily amenable to laboratory synthesis and as such, manufacture of this drug candidate will be via purification from plantation-grown raw material. Although individual specimens will be selected from the wild to establish plantations based on commercially important agronomic traits, the microsatellite-based method developed here will ensure that maximum genetic diversity is also captured. Furthermore, it will allow for careful management of future breeding programs by ensuring maximal genetic diversity in crosses designed to enhance the commercially important agronomic traits. The complex ecology and distribution patterns of this dioecious rainforest species, as well as its pharmaceutical potential, will ensure that F. picrosperma will be a species of significant interest into the future.Fontainea picrosperma occurs on soils derived from basaltic parent materials at altitudes of 700–1200 m above sea level (asl) and is restricted to an area of approximately 30 × 30 km on the southern Atherton Tableland, Queensland, Australia. Whilst it is geographically restricted and its distribution is fragmented, the species is relatively common at a local scale where suitable habitat exists.F. picrosperma populations selected to cover the geographical range of the species  were amplified using Multiplex PCR Plus Kits (Qiagen). Forward and reverse primers for each multiplex pool were combined in a 10× primer mix using 1–3 μM of each primer, dependent upon PCR product fluorescence intensities. Reactions, with volumes adjusted to 10 μL, each contained 1 μL of 10× primer premix, 3.0 μL of Qiagen Multiplex Buffer (2x), 3.5 μL of ddH2O, and 2.5 μL of template gDNA (10 ng/μL). Amplification was performed using an Eppendorf Mastercycler  with cycling conditions as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 57 °C for 90 s, and 72 °C for 30 s; with a final extension at 68 °C for 10 min. PCR products were separated by capillary electrophoresis on an AB 3500 Genetic Analyser (Applied Biosystems). Fragment sizes were determined relative to an internal lane standard  using GENEMARKER v. 2.4.0  and double-checked manually. Individuals with low or missing peaks were amplified and genotyped a second time.A detailed description of marker development using GS-FLX Titanium chemistry  is given in Agostini et al. . Eleven A), observed heterozygosity (HO), unbiased genetic diversity (HE), and the fixation index (F) as a measure of past inbreeding [AR) and private allelic richness (PAR) for each population were obtained via rarefaction using the program HP-RARE [PIC) and probability of identity (PID), i.e., the chance of individuals sharing the same multilocus genotype, was calculated in CERVUS v. 3.0.3 [Allelic frequencies for each population were generated in GenAlEx v. 6.5  and usedbreeding . Allelic HP-RARE  to compuv. 3.0.3 .F. picrosperma’s distribution. The average pair-wise level of genetic differentiation /FST [D; [FST was performed employing 999 permutations using POPTREE2 [We used a number of methods to analyse the population structure across on  betweenon /FST  to estim/FST [D; ) was calPOPTREE2 . EstimatTo look for genetic relationships within and among populations, the genetic distance matrix  was alsoK (number of clusters) between 2 and 7 were performed, employing a burn-in of 100 000 followed by 500 000 Markov Chain Monte Carlo (MCMC) steps for each run. The geographic location of samples was not used in the clustering analysis. Results across each run were summarised to infer the optimal value of K using the method of Evanno et al. [We investigated the presence of genetically differentiated groups of populations using the Bayesian genetic clustering algorithm implemented in STRUCTURE v. 2.3.4 . An admio et al. , as implo et al. , processo et al.  to detero et al. .To ascertain the likelihood of recent bottlenecks due to severe range contraction and subsequent founder effects, the program BOTTLENECK v. 1.2.02  was emplAll data supporting the conclusions to this study can be found within the manuscript and its additional files.