Given the existence of plenty of river valleys connecting Southeast and East Asia, it is possible that some inland route(s) might have been adopted by the initial settlers to migrate into the interior of East Asia. Here we analyzed mitochondrial DNA (mtDNA) HVS variants of 845 newly collected individuals from 14 Myanmar populations and 5,907 published individuals from 115 populations from Myanmar and its surroundings. Enrichment of basal lineages with the highest genetic diversity in Myanmar suggests that Myanmar was likely one of the differentiation centers of the early modern humans. Intriguingly, some haplogroups were shared merely between Myanmar and southwestern China, hinting certain genetic connection between both regions. Further analyses revealed that such connection was in fact attributed to both recent gene flow and certain ancient dispersals from Myanmar to southwestern China during 25–10 kya, suggesting that, besides the coastal route, the early modern humans also adopted an inland dispersal route to populate the interior of East Asia. Actually, as suggested by GIS-based analysis, river valleys had likely played an important role in populating the interior of South Asia by modern humansIt is generally agreed that the peopling of East Asia resulted mainly from the Late Pleistocene south-to-north migrations, initiated from Southeast Asia by the earliest settlers after they migrated from Africa Unfortunately, so far no genetic trace of this inland dispersal(s) was observed, notwithstanding much progress has been achieved on dissecting the genetic landscapes in East Asians23711121371516174Therefore, if this hypothesized ancient inland dispersal route did exist, Myanmar likely served as the corridor. In fact, the two major rivers  in Myanmar can trace their upstream back to southwestern China. The existence of such river valleys would facilitate the potential population movement northwards into the interior zones. Coincidentally, our recent study has observed the enrichment of a number of new basal mtDNA lineages in southwestern China  and suggested this region likely to be the genetic reservoir of the modern humans after they entered East Asiahttp://www.phylotree.org/) and therefore were named as M82, M83 and M84 here  and South Asian (17.40%) ancestries remains stable in the whole Myanmar population. The East Asian-prevalent haplogroups , Rakhine and Karen show closer relationship with Tai-Kadai and Hmong-Mien in Southeast Asia, while Naga and Chin show closer affinity to Austro-Asiatic and Tibeto-Burman populations in northeast India. This pattern plausibly reflects different population dynamics that the populations had suffered during their southward migration from East Asia and subsequently peopling scenarios32Intriguingly, even only Tibeto-Burman populations from Myanmar were analyzed in this study, which, as suggested in the historical records, could trace their origin back to western China3218Since these basal lineages likely represent the genetic relict of the initial settlers in Southeast Asia, the trace of the suggested Pleistocene inland dispersal, if really existed, would then be witnessed by these lineages. Meanwhile, the observed high diversity of the basal lineages in Myanmar seems to echo with the previous observation in southwestern Chinain situ. Indeed, by showing the positive significant Moran's I values for small geographic distances and negative significant values for large distance classes  are also found in southwestern China  online. rn China , which irn China , is obsern China . Haplogrrn China , likely In this report, by extensively dissecting the matrilineal composition in Myanmar populations  at a high molecular resolution, our results show that more than a half of their maternal components belong to previously defined eastern Eurasian haplogroups, an observation in agreement with their linguistic affinity (Tibeto-Burman) and the historical records32Significantly, our study has identified a number of novel and basal lineages in Myanmar, notwithstanding the fact that the Myanmar populations under consideration all have suggested Tibeto-Burman ancestry. Given the ancient coalescent ages  are also observed in southwestern China, suggesting certain direct but previously unknown genetic connections between Myanmar and mainland China. Further analyses reveal that haplogroups M24, M90 and M91 in southwestern China were the results of recent gene flow from Myanmar, likely occurred during the expansion of Pyu populations at about 200 BCEThis genetic connection finds additional support from the archaeological records. For instance, the carved stones, mainly observed in Paleolithic cultures in Yunnan of China, have also been found  in Anyathian sites near the border of Yunnan and MyanmarIn summary, the enrichment of a number of basal lineages in the Myanmar population indicates that this region likely served as one of the differentiation centers of the initial settlers after they reached southeastern Asia. Intriguingly, consistent with their geographic affinity between Myanmar and southwestern China, direct genetic connection exist between both regions which was attributed to recent gene flow from Myanmar to southwestern China  and certain ancient dispersals from Myanmar to southwestern China ranged from 25–10 kya . This observation finds further support from the archaeological records and well explains the enrichment of basal lineages in southwestern China observed in our previous studyTo dissect the matrilineal landscape in Myanmar, a total of 845 individuals, sampled from 14 Myanmar populations representing the major ethnic groups in the country including Burmese, Chin, Rakhine, and Naga, were considered in the current investigation. The experimatal protocol were approved by the Ethics Committee at Kunming Institute of Zoology. Informed consent was obtained from each individuals before the study. The relevant information of the ethnic populations as well as the sample locations were summarized in HhaI, 5176AluI, 9820HinfI, 12406HpaI, 13262AluI, 14465AccI, were tested following the methods of Yao et al. (2004)114152262526272864344http://www.phylotree.org/) and our recently suggested haplogroup schemeProcedures for the extraction of genomic DNA, PCR amplification and DNA sequencing of mtDNA segment(s) have already been well described elsewhere212To ensure the quality of the complete genome data, our previously proposed quality-control measures, such as independent amplification, detecting errors by phylogenetic analysis and matching or near-matching method were followed6116To facilitate the comparison with the reported data from the neighboring populations and thus to distill the genetic trace left by the Pleistocene inland immigrants, previously published mtDNA data, including 1,524 mtDNAs from mainland Southeast Asia and 3,120 mtDNAs from East Asia, 690 mtDNA sequences from northeast India and 302 from Bangladesh, were taken into account. In addition, the recently published Myanmar data (116 Barma and 155 Karenwww.fluxus-engineering.com/sharenet.htm). Phylogenetic tree of haplogroup was reconstructed manually based on complete sequences and confirmed by mtphyl software. Contour maps of spatial frequencies of haplogroup were constructed using Kriging linear model of Surfer 8.0 . Spatial analysis was performed using the PASSAGE software packet. Moran's I metrix was applied in correlogram analysisA genetic distancesThe PCA and CA were performed based on haplogroup frequencies by using Statistical Package for the Social Sciences (SPSS) 16.0 software. Reduced median network for each basal haplogroup was constructed manually and checked by the program NETWORK 4.510 (Q.-P.K. and Y.-P.Z. designed the research; L.-N.L., L.-Q.Y., C.-L.Z. and S.-F.W. colleted the samples; Y.-C.L., H.-W.W. and J.-Y.T. collected the data; Y.-C.L., H.-W.W., J.-Y.T. and C.-L.Z. performed the experiments; Y.-C.L., H.-W.W., J.-Y.T., Q.-P.K. and Y.-P.Z. analyzed data; Y.-C.L., Q.-P.K. and Y.-P.Z. wrote the paper.Accession numbers: All of the sequences obtained in the present study have been deposited into GenBank, with accession numbers KP345975-KP346066 for whole mtDNA genomes and KP346067-KP346911 for control region sequences.Supplementary informationDataset 1Dataset 2


Plasmodium vivax merozoite surface protein 3α (PvMSP3α) is conserved and has been proposed as a potential candidate for a malaria vaccine. The present study aimed to compare sequence diversity in PvMSP3a block II at a local microgeographic scale in a village as well as from larger geographic regions (countries and worldwide). Blood samples were collected from asymptomatic carriers of P. vivax in a village at the western border of Thailand and PvMSP3α was amplified and sequenced. For population genetic analysis, 237 PvMSP3α block II sequences from eleven P. vivax endemic countries were analyzed. PvMSP3α sequences from 20 village-level samples revealed two length variant types with one type containing a large deletion in block I. In contrast, block II was relatively conserved; especially, some non-synonymous mutations were extensively shared among 11 parasite populations. However, the majority of the low-frequency synonymous variations were population specific. The conserved pattern of nucleotide diversity in block II sequences was probably due to functional/structural constraints, which were further supported by the tests of neutrality. Notably, a small region in block II that encodes a predicted B cell epitope was highly polymorphic and showed signs of balancing selection, signifying that this region might be influenced by the immune selection and may serve as a starting point for designing multi-antigen/stage epitope based vaccines against this parasite.Block II of  Plasmodium falciparum—a species responsible for the majority of malaria-related deaths. However, vaccine research for P. vivax has trailed far behind [P. vivax is the most widespread human malaria parasite and it causes 50–70 million infections annually [P. vivax malaria further highlights the difficulty for controlling this parasite and emphasizes the need to develop integrated control strategies including vaccine for this parasite [Vaccine is a long-term hope to combat malaria—a major infectious disease responsible for more than half a million deaths annually around the world. The alarming signals of artemisinin resistant parasites seemingly to follow the same path initially laid down by chloroquine-resistant parasites across international borders in Southeast Asia further urge the development of vaccines against malaria , 2. Vaccr behind , 4. Yet,parasite .P. falciparum [P. vivax  have also been characterized. Antigenic diversity in these genes [P. falciparum merozoite surface protein 3 (PfMSP3) gene [Several antigens have been proposed as potential vaccine candidates for lciparum , and these genes –14 has sse genes –17, sincP3) gene , 19. FurP3) gene .MSP3 in P. vivax is a family of 11 members with a complex evolutionary history [MSP3α and 3β, have been widely used as population genetic markers for typing P. vivax isolates based on polymorphisms depicted by PCR/RFLP analysis [PvMSP3α gene sequences have observed differential pattern of diversity across different domains of the gene [PvMSP3α is composed of an N-terminal signal sequence, a central alanine rich region and an acidic C-terminus. The alanine rich repeat region of PvMSP3α encodes block I (residues 104–396) and block II (434–687). Block II has been shown to be relatively conserved with non-random variations clustered in two structural motifs; motif I from amino acid position 533 to 538 and motif II from 580 to 587 [LSKLEE and motif II: TAANVVKD/KEATAAKL). All of these alleles have been found equally prevalent in natural P. vivax populations [P. vivax. history , 22. Twoanalysis , 23–25. the gene , 26, 27.residues 4–396 andulations , 26, 27.ulations  and Brazulations . In factulations . All thePvMSP3α has been previously characterized only in a few laboratory adapted strains [PvMSP3α block II in samples from a small village  compared with the larger geographic structures. By studying the extent and distribution of polymorphisms in PvMSP3α block II among P. vivax samples from 11 countries, we hope to understand the evolutionary mechanism underlying the variation patterns. Genetic diversity in block II preferentially revealed large number of rare alleles, and high frequency variants were restricted to specific genetic regions. The prominent allelic forms of block II were extensively shared by diverse P. vivax populations.A conserved pattern of variation in an antigenic sequence has been widely associated with purifying (negative) selection that can be the result of either structural constraints or strong immune directional selection , 31. On  strains , and lim strains , 34 and  strains , 27. ThuP. falciparum and P. vivax infections are more prevalent and notably, we have reported a large number of asymptomatic infections (only detected by expert microscopists and by PCR assay) in that area recently [P. vivax infections at a micro-geographic scale.The present study was conducted in a small village Suan Oi in Tha Song Yang district of Tak province located at western border of Thailand , which irecently , 36. Asyrecently , thus prPlasmodium species identification was carried out by species-specific rDNA based primers following method described in Snounou et al., 1993. Twenty four samples showing single P. vivax infections were included in sequencing analysis.Finger-pricked blood samples were obtained from all the residents of Suan Oi during mass blood surveys. The presence of malaria parasites in some of the blood samples from participants were confirmed by microscopic examination of Giemsa-stained blood films and by PCR . GenomicWritten informed consent was obtained from the participants or guardians. This study was approved by the Institutional Review Boards of Pennsylvania State University and Thai Ministry of Public Health.PvMSP3α in P. vivax samples was amplified using primers described previously [PvMSP3α present in all samples were extracted for analysis.eviously . AmplifiPvMSP3α block II sequences generated in the present study were compared and analyzed together with the sequences retrieved from GenBank (http://www.ncbi.nlm.nih.gov/genbank/) and Plasmodb (http://plasmodb.org/plasmo/) database. Totally, 237 sequences were derived from 11 parasite populations, which included 52 samples from Thailand  and haplotype diversity (Hd) . Local don index  and the software .fluxus-engineering.com) using the median joining algorithm [Phylogeographic clustering of the isolates was evaluated by Maximum Likelihood (ML) tree in MEGA6  using Talgorithm .To examine departure from neutrality, we estimated the numbers of synonymous substitutions per synonymous site (dS) and of nonsynonymous substitutions per nonsynonymous site (dN) using the Nei and Gojobori method  as impleRm) according to the four-gamete test by Hudson & Kaplan [datamonkey.org) [ρ) and mutation rate (θ) were calculated using LDhat package [We used an array of methods to detect recombination signals viz; RDP, MaxChi, GENECONV, GARD and minimum number of recombination events  , which i package .PvMSP3α gene displays enormous genetic diversity in P. vivax populations and consequently has been used as a molecular marker for differentiating field parasite strains [PvMSP3α gene on a microgeographic scale, we collected P. vivax samples from asymptomatic carriers in a small village Suan Oi (~500 residents) in western Thailand (PvMSP3α gene was successfully amplified in 22 of 24 P. vivax samples. Two PCR length variant types A (1.9 Kb) and C (1.1 Kb) were observed, whereas type B (1.5 Kb) was not observed in the tested samples [The  strains . To inveThailand  during m samples . Two of PvMSP3α, PCR products of 20 samples were sequenced and aligned with the Sal I reference gene. PCR fragment type C contained a ~750 bp deletion in block I, whereas block II was relatively conserved in all 20 samples. PvMSP3α block II in the 20 samples had 28 single nucleotide polymorphisms (SNPs), 24 of which were parsimony informative sites (SNPs observed in more than one sequence). Among these SNPs, 15 were non-synonymous mutations which changed 15 amino acids (12 as parsimony informative). Nucleotide diversity of block II was 0.013 and haplotype diversity was 0.800 with 10 haplotypes/allelic forms of block II.In order to determine the details of sequence diversity of PvMSP3α block II sequences from the Suan Oi isolates with 32 publically available PvMSP3α sequences from Thailand. A total of 85 mutations were observed in 32 sequences, of which 59 were singletons. These sequences differed from the Suan Oi samples in the excess number of singletons. While singletons could be potential consequences of sequencing errors or population expansions [LSKLEE and motif II: TAANVVKD/KEATAAKL) [We compared the genetic diversity of the pansions , low frepansions . Twelve EATAAKL) .PvMSP3α block II from 11 parasite populations. The sequences included 52 obtained from Thailand and 185 of worldwide isolates retrieved from the GenBank and Plasmodb database  with an overall allelic diversity of 0.9724 (0.816–0.977 country-wise). Fifteen block II alleles were shared between populations and 85 were population specific. Both shared and population-specific alleles were observed in each population. Interestingly, all the population-specific alleles were singletons in five out of the 11 populations . PopulatST showed a modest genetic structure (0.083), which means population genetic differentiation accounting for only 8% of the total variations in the gene. The highest degree of population differentiation was observed between Thail and Mexico  and the lowest between Mexico and China .Genetic differentiation between worldwide populations estimated using FPvMSP3α block II sequences. Phylogenetic relations between isolates revealed three robust clusters  based on sequence variations. Group I included 21 sequences from only four countries , whereas Group II and III included 112 and 104 sequences from all the 11 populations, respectively (MSELEK and TAANVVKD), whereas group III contained both alleles of motif I (LSKLEE/MSELEK) and only mutated allele in motif II (KEATAAKL).Absence of population structuring was further supported by ML analysis of the 237 ectively . High-frectively . Group Iin vitro studies have localized a B-cell epitope in motif II . Moreover, in silico B Cell Epitope Prediction server [The rate of synonymous substitutions was found significantly higher than the rate of non-synonymous substitutions in worldwide sequences as well as in each population (data not shown), which indicates purifying/negative selection on block II. This observation was further supported by the significant negative values of Tajima’s D and Fu and Li’s F* tests observed in worldwide sequences. The Tajima’s D and Fu and Li’s F* values were positive in eight populations, but the deviation was significant only in Peru and Venezuela populations . Interesn server  also prePvMSP3α. Since recombination rates estimated by different methods vary with the number of sequences, rate of recombination, and the number of recombination sites, we analyzed all 237 sequences together using five different tests for detecting recombination events. Among phylogenetic approaches of recombination detection, RDP, GENECONV and GARD tests failed to identify any breakpoints, while MaxChi identified one recombination breakpoint. In contrast to these phylogenetic approaches, 13 recombination events were identified by population genetics based estimator using DnaSP. We assumed that singletons might have influenced the DnaSP results, but singleton-free data  produced similar results. Recombination is also evident by eye, since any combinations of dimorphic alleles of the two structural motifs were observed among 237 sequences, implying that recombination in block II has taken place. This was further supported by the recombination event observed by DnaSP in the region between the two motifs (data not shown). Moreover, the estimated recombination rate (ρ = 0.06) was higher than mutation rate (θ = 0.0375) leading to a ρ/θ ratio of 1.6. Recombination to mutation ratio exceeding 1 signifies that the recombination is more prevalent in the dataset than mutation.Intragenic recombination has been repeatedly reported as a prominent evolutionary force in maintaining genetic diversity in P. falciparum that identified considerable diversity in the haplotypes used for designing MSP3-LSP and MSP119 vaccines [P. vivax since its antigens are still understudied and it has been observed that many genes encoding vaccine candidates in P. vivax show different patterns than P. falciparum [PvMSP3α, being relatively conserved with restricted variations has been proposed as a good vaccine candidate, but defining immunological relevance of the region is required. We analyzed PvMSP3α block II in 11 P. vivax endemic countries worldwide to highlight the extent and distribution of polymorphisms and the potential mechanisms generating these variation patterns. Block II was found less diverse compared to other vaccine candidate genes and many mutations were singletons. The pattern of variation was extensively shared by diverse P. vivax populations suggesting functional/structural constraint on block II, however, each population maintained different allelic forms of block II.Analyzing the diversity of gene encoding antigens and the mechanisms involved in the maintenance of such variation is a necessary step for prioritizing vaccine candidates and monitoring their efficacy . The impvaccines –61. Signlciparum , 61–64. PvMSP3α block II of worldwide P. vivax populations, 66% (104/158) of them were singletons. Singletons and low-frequency alleles are generally excluded from diversity analysis of vaccine candidate genes [PvMSP3α block II in each population was found lower than that of the full-length PvMSP3α gene [P. vivax populations, e.g., PvMSP1 (θπ = 0.027) [PvMSP7C, PvMSP7H and PvMSP7I  [PvMSP3β (θπ = 0.0367) [PvMSP5 (θπ = 0.0375) [PvMSP8 and PvMSP10 (θπ = 0.0033 & 0.0022) [PvMSP3α block II is relatively conserved among many merozoite surface proteins in P. vivax populations. Additionally, nucleotide diversity in block II showed a non-random pattern, as peaks of nucleotide diversity were restricted to certain regions of the block II. A similar trend was observed when samples from different geographical regions were analyzed, suggesting of functional/structural constraint, since block II is rich in alanine heptad repeats that are predicted to form coiled coil structures possibly needed for the functioning of PvMSP3α gene [Though a large number of SNPs were observed in te genes  to avoidte genes , 65. AltP3α gene , 27 as w= 0.027) , PvMSP7C& 0.043) , PvMSP3β 0.0367)  and PvMS6% 104/15 of them  0.0022) . This reP3α gene .Clustering analysis of block II sequences revealed a general lack of geographic structure. However, three robust clusters, each comprising of mixture of sequences from diverse populations were also observed. Moreover, highly frequent non-synonymous SNP-based haplotypes were shared by multiple populations irrespective of their geographical locations, which suggest either extensive gene flow between populations, or independent convergence of variations due to their functional/structural importance. The phylogenetic grouping was found to be based on the type of sequence variations especially influenced by the presence/absence of dimorphic alleles of two structural motifs, suggesting of selective pressure on these motifs.ST value in worldwide samples was 0.09 with the highest estimate was observed between Thailand and Mexico as 0.2559, P<0.05. Moreover, an FST value of 0.012 between Asian (n = 139 sequences) and American (n = 98) samples analyzed in this study is remarkably low as compared to the FST estimates previously observed between Asian and American P. vivax populations using mitochondrial DNA (FST = 0.15–0.50) [ST = 0.228) [ST values [Antigenic genes are generally expected to be under diversifying/balancing selection, genes under such selective pressure could show lower levels of genetic differentiation between populations than the one expected by genetic drift alone. Accordingly, the overall F15–0.50)  and sile= 0.228) . This miT values , a pattein vitro studies is localized in this region. Moreover, both alleles were predicted as B cell epitopes with >75% specificity. Blocking this region might prevent merozoite invasion of reticulocytes, and inclusion of both alleles in a vaccine design might be able to induce immune responses recognizing both alleles. However, immunological studies are required to assess the immunogenicity and protection-inducing capability of both alleles in natural P. vivax infections in diverse population backgrounds.Though recombination seems to play an important role in generating new genetic variants, polymorphisms in block II are largely clustered in particular regions. Moreover, the pattern of amino acid changes is shared by diverse populations, possibly due to purifying selection that might be acting on alanine heptad repeats for structural conservation. However, a small region in block II encoding motif II showed evidences of balancing selection. Particularly, two alleles of this motif seem to maintain intermediate frequencies in the study populations. This motif might be involved in host pathogen interaction since one of the epitope identified by PvMSP3α compared to the full-length PvMSP3α as well as many other merozoite surface proteins provides a strong support for vaccine development based on block II. An important observation is the common and extensively shared pattern of polymorphisms among diverse populations, which increase the possibility of formulating vaccines effective against worldwide P. vivax populations. Though conserved regions in malaria antigens are generally not highly immunogenic and protective [P. vivax infections [P. vivax infections.Conserved pattern of amino-acid variations in block II of otective , 73, antfections . This stS1 Table(DOC)Click here for additional data file.


Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high . Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father–son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database. Further, Y-chromosome DNA analysis is important in genetic genealogy and for community genetic purposes such as personal ancestry identification, as well as for the identification of male lineages and inferring paternal genetic ancestry for judicial and investigative purposes [Kayser et al., −2 per locus per generation) mutation rates were identified and termed rapidly mutating (RM) Y-STRs. Furthermore, in this previous study and a subsequent study [Ballantyne et al., Haplotypes generated from conventional Y-STRs, such as the widely used 17 markers included in the commercially available AmpFlSTR® Yfiler® PCR Amplification Kit  (subsequently referred to as Yfiler), suffer from two main limitations: (1) their inability to conclusively resolve some male lineages due to identical haplotypes arising in individuals that are not of common descent because of recurrent mutation, and (2) their inability to differentiate between paternally related males due to the moderately low mutation rate of the loci tested. In general, Y-STRs with much higher mutation rates than those of the conventionally used loci are expected to overcome or at least reduce both limitations. In a previous comprehensive Y-STR mutation rate study where nearly 200 Y-STRs were investigated in almost 2,000 father–son pairs confirmed by autosomal DNA analysis [Ballantyne et al., However, to fully explore the potential for the RM Y-STR set in differentiating unrelated as well as related males for various purposes, much more data are needed. Therefore, the International RM Y-STR Study Group, a worldwide collaboration between 52 laboratories, was formed. Group members, chosen based on pre-existing practical experience in Y-STR analysis, genotyped under quality-controlled conditions the 13 RM Y-STRs in 14,644 males including 12,272 unrelated males from 111 worldwide populations and 4,744 closely related males. To compare the RM Y-STR set with conventional Y-STRs, Yfiler data were gathered in a subset of 7,784 unrelated men from 65 worldwide populations as well as in most of the male relatives analyzed for RM Y-STRs. The large collection of RM Y-STR haplotypes presented and explored in the present study enables a better understanding of their value to differentiate related and unrelated male individuals and populations, and additionally provides a suitable reference database for future use of RM Y-STRs in forensic, genealogical, anthropological, and population genetic studies.http://www.yhrd.org), hence having proven experience in Y-STR analysis. Each of the 52 contributing laboratories within the International RM Y-STR Study Group genotyped a selection of their own in-house population sample sets, each consisting of seven to 634 individuals  across 111 defined population samples per laboratory. Population sets were to include only unrelated males. Three population samples were notable exceptions—the Biaka Pygmy group and the Bhutanese Lhokpu and Mönpa language groups. These populations, and particular sample sets used here, are known to contain high numbers of male relatives (Biaka), or were specifically selected as having extremely low resolution with Yfiler (Bhutan). Because these samples were ascertained differently to the other 108 populations within the study, they were excluded from the continental groups during analysis to avoid bias. The Aboriginal Australian samples used here were also not ascertained completely randomly, as they were selected based on Y-SNP haplogroups known to be authentic for Aboriginal Australians to avoid admixture effects as described elsewhere [Taylor et al. With a worldwide coverage in mind, contributing laboratories were identified and recruited based on their previous submission of Y-STR population data to the Y-chromosome Haplotype Reference Database  for the most commonly used panel of 17 Y-STRs. Relative pairs were also analyzed, with 2,339 newly genotyped father–son pairs, 30 brother pairs, and three uncle–nephew pairs, for which relationship had previously been confirmed by autosomal DNA analysis; this dataset of male relatives was supplemented by 156 previously published relative pairs [Ballantyne et al., A subset of 7,784 individuals from 65 populations were additionally genotyped with the AmpFThe organizing laboratory  provided genotyping protocols, allelic ladders, and tools for allele calling to all participating laboratories, and organized a quality control exercise prior to population data generation (for details see below). The 13 single- or multicopy RM Y-STR markers  were amplified in three multiplex PCRs, as described in Supp. The commercially available AmpFlSTR® Yfiler® PCR Amplification Kit (Life Technologies) targeting 17 conventional Y-STRs was used and allele calling was performed according to the manufacturer's instructions.To ensure genotyping consistency between the laboratories, all participants received allelic ladders prepared by the organizing laboratory, and six blind control DNA samples previously genotyped by the organizing laboratory. Genotyping of population samples was only allowed after a participating laboratory demonstrated the correct genotyping of these six blind control DNA samples at all 13 RM Y-STRs. If a participant reported erroneous genotypes, screenshots were requested and submitted to the organizing laboratory for an evaluation of the possible cause of error(s). Once the cause of error was identified, three additional blind control DNA samples were provided and genotyped by the participant. If these samples were typed correctly, the laboratory was allowed to type their population samples. In cases of unexpected results such as previously unknown alleles or microvariants, screenshots were requested and submitted to the organizing laboratory for inspection. Independent genotyping, and in some case DNA sequencing, was performed to resolve differences in genotyping of certain samples. Samples with missing data from more than one marker were excluded from data analysis to prevent low quality samples affecting genotype and haplotype distributions. Further, any differences observed between relative pairs were confirmed through duplicate, independent PCR amplifications, and genotyping, and in some cases, DNA sequencing.Additional quality control was performed for the multicopy markers DYF403S1a+b and DYF399S1, whereby a subset of population sample electropherograms for these markers were sent to the organizing laboratory by all participants for blind confirmatory genotype scoring.The nomenclature of the RM Y-STRs was updated to comply with the guidelines of the International Society of Forensic Genetics—ISFG [Gusmão et al., FST genetic distances, and analysis of molecular variance (AMOVA) were calculated with Arlecore v3.5.1.3 [Excoffier and Lischer, http://www.yhrd.org. Only single-copy markers were utilized in network construction, due to the inability to assign multicopy genotypes to individual loci. Testing of statistical significance (t-tests) was performed in SPSS v17.0. Theoretical estimates for the rates of relative differentiation for each Y-STR panel were calculated using the formula P(k < 0) = 1 − P(k = 0) = 1 − eNm− [Ballantyne et al., N represents the number of markers and m represents the average mutation rate of the set of markers obtained from the sampling from the posterior distribution.Haplotype diversities, average number of differing loci between pairs of individuals, pairwise −8). Of the 102 haplotypes that were nonunique within this dataset , 90 were shared between two males, 10 between three males, and two between four males. All nonunique haplotypes were shared between males of the same geographic region, and all but six were from the same sampling population. The six nonunique haplotypes shared between different populations involved males from Argentina (Chubut and Rio Negro regions), Greenland (from two separate samples of Inuit), Czech Republic (from two sample sets), Cologne and Warsaw, and Bhutan (the Lhokpu and Mönpa tribes), respectively. As a result, the proportion of haplotypes shared between populations was exceptionally low at 0.05%, and the proportion of haplotypes shared within populations was slightly higher at 0. 8%.The RM Y-STR set applied here generated exceptionally high haplotype diversity in the worldwide sample set analyzed with 12,156 unique  haplotypes observed in 12,272 unrelated samples from 111 worldwide populations  alleles, which can challenge interpretation even in single source samples due to a lack of single base resolution with POP-4 during capillary electrophoresis. To determine the effect of removing this potentially problematic marker from the RM Y-STR panel, the entire dataset was additionally analyzed without DYF403S1a+b. The global haplotype diversity slightly decreased from 0.9999985 with the full 13-loci RM Y-STR set to 0.9999981 with the 12-loci set. This translated to an increase in the number of nonunique haplotypes from 116 to 152 in the 12,272 samples—a decrease of zero to five haplotypes per regional population, with an average of 0.32 fewer haplotypes within each of the 111 populations. Populations most affected by the omission of DYF403S1a+b were Aboriginal Australians (from 96 to 92 haplotypes among the 100 men tested), and Bhutan (from 56 to 51 haplotypes among the 78 men tested). However, the vast majority of the populations tested  were not affected by the removal of DYF403S1a+b, with the haplotype diversities remaining the same. Therefore, and because of the extra care in quality control employed for this marker, all following data analyses were performed based on the complete 13-loci RM Y-STR set.Due to its highly multicopy nature, DYF403S1a+b caused concerns with some study group members regarding genotyping accuracy, so that additional quality control was performed for this marker (as well as for DYF399S1), as described in FST values  demonstrated that 99.98% of haplotype variation was within populations, extremely high for Y-chromosome markers [Willuweit and Roewer, FST was as low at 0.00017. Between the regional groups, the average pairwise FST value was only 0.000127, with a maximum value of 0.00058 observed between Aboriginal Australians and Middle Eastern populations. Even when considering individual populations, the highest pairwise FST value observed was only 0.02815 between the Bhutan Lhokpu and Argentinian Wichi from Salta Province (data not shown). Across all population comparisons, an average pairwise FST value of 0.000826 was obtained.The θ values for all regional groups and for almost all populations were exceptionally low Table , indicatFST values  analysis was performed on Slatkin's linearized ues Fig. . The majues Fig. . The difues Fig.  did not ues Fig. , or 25/pWhen considering the utility of the RM Y-STR set for future applications in forensic, genealogical or anthropological genetic studies, it is informative to compare its properties to those of conventional Y-STRs, such as the 17 loci included in the Yfiler kit. Hence, RM YSTR and Yfiler haplotypes were compiled for 7,784 individuals across 65 populations as a subset of the global dataset presented here. In every diversity measure applied, the 13 RM YSTR set provided enhanced estimates relative to the 17-loci Yfiler set. Global haplotype diversity was increased from 0.99995 with Yfiler to 0.999997 with RM Y-STRs; the total number of haplotypes detected was increased from 6,975 to 7,714, and the number of singletons from 6,469 to 7,647. All regional groups showed more haplotypes and higher diversity estimates with RM Y-STRs relative to Yfiler, that is, haplotype diversity increased on average by 0.00226 and the number of singletons on average by 77  Table .N = 13). Notably, the level of outside-population matches compared with within-population matches was markedly higher for most populations for Yfiler haplotypes, whereas strikingly reduced for RM Y-STRs  demonstrates the power of the RM Y-STR panel in male lineage differentiation. For RM Y-STRs, no haplotype sharing was observed between different populations sampled from the same countries, such as the five Indian, two Japanese, three Korean, three Austrian, two Belgian, three German, two Italian, two Polish, three US American, and two Hungarian populations, except for the two Czech populations. Further, almost no RM Y-STR haplotype sharing was observed between populations from the same geographic region such as the densely sampled continent of Europe except for Cologne and Warsaw. In contrast, many Yfiler haplotypes were shared between populations within countries, and between populations within regions such as Europe  (Table n = 558 per category) showed that the RM Y-STRs still result in decreased diversity in endogamous and rural populations compared with urban populations .The observation of RM Y-STR haplotype sharing within, but  not between, populations might indicate that RM Y-STRs allow the detection of unknown  paternal relationships in populations. If true, more shared RM Y-STR haplotypes would be expected between putatively unrelated individuals sampled from rural areas, where male relatives often stay in the region (and thus can be sampled), and especially from endogamous populations, than among putatively unrelated individuals sampled from urban areas, where male relatives tend to not to stay (and thus cannot be sampled). To test this hypothesis, all populations were designated according to their urbanization status—each set of males was classified as either urban , rural , mixed , or endogamous . In support of the RM Y-STR set's ability to detect hidden paternal relationships, we indeed see significantly lower haplotype diversity and lower proportions of unique haplotypes in the endogamous group than in the rural group than in the urban group . For the Yfiler set, it is estimated that a mutation at one or more of the 17 Y-STR loci will occur with a probability of 0.047  per meiosis, which for the RM Y-STR set is more than fourfold higher at 0.195 (95% CI 0.177–0.21). Notably, for the recently released PowerPlex Y-23 kit (Promega), which targets all 17 Yfiler markers together with two of the 13 RM Y-STR markers and four additional Y-STRs, this value is 0.092 (95% CI 0.077–0.107), nearly twofold higher than for Yfiler, but more than twofold lower than for the RM Y-STR set studied here.In addition, we tested the value of RM Y-STRs for differentiating male relatives. On a theoretical level, the rate of relative differentiation per meiosis can be calculated using the mutation rates of each locus within a genotyping panel , confirmed the observed allelic differences by independent genotyping (and some by sequencing), and combined these new data with those from the 156 relative pairs separated by one to 20 paternal genetic transfers (or meioses) described previously [Ballantyne et al., It should be noted, however, that only for the 2,378 father–son pairs investigated here, was the sample size large enough to allow reliable conclusions, whereas for more distantly related males, future studies need to deliver more data. However, as the chance of mutations increases with each meiosis, it is expected that the more distantly related men are, the higher the chance that they will have different RM Y-STR haplotype; a trend that can be seen in our data Fig. . The impRemoving DYF403S1a+b from the RM Y-STR panel resulted in the nondifferentiation of 84 relative pairs that could be differentiated when DYF403S1a+b was included; the overall relative differentiation rate dropped to 26%. However, the difference only occurred in pairs with fewer than five meioses separating the individuals—in all other pairs, there were sufficient mutations at other RM Y-STR loci to allow differentiation (data not shown).As pointed out previously [Ballantyne et al. The large worldwide dataset compiled, presented, and analyzed here demonstrates the exceptional value of the RM Y-STR panel for differentiating male lineages on a global and regional scale. Many of the current limitations of Y-chromosome applications are reduced with this RM Y-STR set, providing increased utility and effectiveness to the genetic analysis of male populations and lineages. In particular, this 13 RM Y-STR set provides near-complete paternal lineage differentiation in general populations as well as in populations with otherwise reduced Y-chromosome diversity, due to peculiarities in population history or cultural practices. The effects of this near-complete male individualization will be of great benefit to numerous fields using the Y-chromosome genetics to investigate male lineages, such as in genealogical studies , population genetic and genetic history studies , and in forensic applications . Moreover, the immense value of this RM Y-STR set to differentiate between both close and distant male relatives will have beneficial effects on these same fields, especially in forensic genetics, providing increased confidence that haplotype matches between unknown samples such as those from crime scenes and reference samples such as those from suspects are due to individual identity, rather than relatedness.On the other hand, however, the extreme degree of RM Y-STR haplotype diversity highlights the limitations of the current approach for placing a statistical weight on Y-STR haplotype matches by using empirically derived haplotype frequency estimates obtained from Y-STR haplotype reference databases. Although already noted with the currently used Y-STR sets such as Yfiler, the problem that an observed haplotype is not present in the large and growing reference database, and how to deal with rare haplotypes in obtaining frequency estimates and estimating match probabilities for which no consensus has been reached yet [Krawczak, To assist future studies utilizing RM Y-STRs, complete dataset of RM Y-STR and Yfiler haplotypes obtained from the 12,272 individuals analyzed here is made available via Supp.


Colletotrichum truncatum is an extremely important fungal pathogen. It can cause diseases both in humans and in over 460 plant species. However, little is known about its genetic diversity within and among populations. One of the major plant hosts of C. truncatum is pepper, and China is one of the main pepper-producing countries in the world. Here, we propose the hypotheses that geography has a major influence on the relationships among populations of C. truncatum in China and that infections in different populations need to be managed differently. To test these hypotheses, we obtained and analyzed 266 C. truncatum isolates from 13 regions representing the main pepper-growing areas throughout China. The analysis based on nine microsatellite markers identified high intrapopulation genetic diversity, evidence of sexual recombination, and geographic differentiation. The genetic differentiation was positively correlated with geographic distance, with the southern and northern China populations grouped in two distinct clusters. Interestingly, isolates collected from the pepper-breeding center harbored the most private alleles. The results suggest that the geographic populations of C. truncatum on peppers in China are genetically differentiated and should be managed accordingly. Our study also provides a solid foundation from which to further explore the global genetic epidemiology of C. truncatum in both plants and humans. Colletotrichum are common in the environment and important plant pathogens. Many species in this genus can cause not only anthracnose and blights on the aerial parts of growing crop plants but also postharvest rots (Bailey and Jeger http://faostat.fao.org)    marker analysis of C. graminicola isolates from turf grass revealed a high degree of genetic similarity among isolates recovered from the same host : the internal transcribed spacer (ITS) region and genes encoding glyceraldehydes-3-phosphate dehydrogenase (GAPDH), a part of actin (ACT), chitin synthase 1 (CHS-1), beta-tubulin (Tub2), and histone3 (HIS3). The primers used and the PCR conditions followed the description by Damm et al. (C. truncatum (CBS 151.35) and other two C. truncatum stains  as well as several closely related species . The tubes were frozen in liquid nitrogen, and a Mixer Mill  was used to grind the frozen mycelia to a fine powder. DNA was extracted using a 2% CTAB (cetyl trimethylammonium bromide) method (Murray and Thompson C. truncatum for this study (Table2O) were included in each step of the analyses, to eliminate potential contamination.Based on the patterns of polymorphism described previously  in populations was examined using the Multilocus1.3b  was run by GenAlEx6 to calculate the Nei's unbiased genetic distance (Nei K) and test for admixture  was estimated, 10 replicated runs of K = 1–13 were carried out after a burn-in period of 100 000 generations followed by a run length of 100 000 generations. The number of genetically homogeneous clusters (K) was identified by following the method developed by Evanno et al.  was used to calculate the genetic differentiation with the GenALEx6  was calculated for each population and the entire sample. The IA values for seven populations and for the total samples were significantly higher than the simulated data sets obtained assuming panmixia .The index of association , among populations within regions, and among individual isolates within populations respectively. All three sources of variation were significant (P < 0.01)  and genetic differentiation (Rst) among geographic populations were positively correlated , with a correlation coefficient of 0.367.The IA value of QY population did not support random recombination, but the PrCP (0.472) was lower than most other populations, consistent with the host diversity playing a role in pathogen genotype diversity. Other parameters such as the number of genotypes and the total number of alleles also showed a similar pattern. The only population showing more genotypes, higher allelic richness, and lower linkage disequilibrium than the QY population was the WC population. However, the WC population had a lower than expected number of private alleles.The QY population, which came from a chili pepper seed-breeding center in Qingyuan in Guangdong Province in southern China, harbored the most private alleles (11). After excluding the influence of sample size, we found that the number of private alleles in QY population was still significantly greater than expected  if we asC. truncatum in China and found evidence of recombination in C. truncatum populations on chili in China. We also uncovered that the Chinese C. truncatum populations could be clustered into two distinct genetic groups, which correspond to the geographic boundary between southern and northern China. Below, we discuss the relevance of our results in the management and prevention of anthracnose in chili peppers.In this study, we analyzed the population genetics of C. truncatum populations on chili peppers in China and suggested that substantial sexual recombination occurred in these populations. Similarly, isolates of C. gloeosporioides from strawberry revealed a low level of linkage disequilibrium as would be expected in sexually recombining populations , likely through both windborne and seedborne due to the movement of contaminated seeds.Although there was significant genetic differentiation among populations within both the southern and the northern regions, clonal dispersal was observed between adjacent geographic areas . Here, tStatistically significant genetic isolation by geographic distance was found in our analyses. Such a result suggests that geography can act as a partial barrier to prevent the homogenization of the pathogen populations in China. However, as has been found in many fungi, human activities such as international travel and trade can be a significant factor in facilitating fungal dispersals (Khankhet et al. C. truncatum in China. We found a high level of genetic variation and evidence for recombination in C. truncatum populations on chili in China. We revealed that the Chinese C. truncatum populations were clustered into two distinct genetic groups, one in southern China and the other in northern China. Although the north/south divide contributed the most to the observed genetic variation, within both the southern and the northern regions, subtle genetic differences were still identified among local populations. Whether other plant fungal pathogens follow a similar geographic pattern remains to be examined. The knowledge of population structure of C. truncatum could have a significant impact on pathologists and breeders to screen chili pepper germplasms for new sources of resistance genes. C. truncatum is worldwide distributed and has many hosts. The relationship among global geographic populations of C. truncatum and from different hosts remains to be explored.In this study, we analyzed the population genetics of


The global distribution of J2-M172 sub-haplogroups has been associated with Neolithic demic diffusion. Two branches of J2-M172, J2a-M410 and J2b-M102 make a considerable part of Y chromosome gene pool of the Indian subcontinent. We investigated the Neolithic contribution of demic dispersal from West to Indian paternal lineages, which majorly consists of haplogroups of Late Pleistocene ancestry. To accomplish this, we have analysed 3023 Y-chromosomes from different ethnic populations, of which 355 belonged to J2-M172. Comparison of our data with worldwide data, including Y-STRs of 1157 individuals and haplogroup frequencies of 6966 individuals, suggested a complex scenario that cannot be explained by a single wave of agricultural expansion from Near East to South Asia. Contrary to the widely accepted elite dominance model, we found a substantial presence of J2a-M410 and J2b-M102 haplogroups in both caste and tribal populations of India. Unlike demic spread in Eurasia, our results advocate a unique, complex and ancient arrival of J2a-M410 and J2b-M102 haplogroups into Indian subcontinent. Population history of India has been under investigation to study the earliest settlement of anatomically modern humans Out of Africa and later to study the demographic episodes of Eurasia1215769Previous studies have established that J2a-M410 and J2b-M102 represent the Y-chromosomal component associated with demic diffusion of Neolithic farmers in North Africa and Eurasia from Mesopotamia (Iraq and Syria)14151719The population of the sub-continent shows an outstanding biological and cultural variation, which has been shaped by geographical, ecological, social and linguistic factors171871718Out of 3023 samples, 355 chromosomes belonged to J2-M172 HG . We coul18We found moderate occurrence of J2a-M410 and J2b-M102 (0–8%) in different populations inhabited in different parts of India . We obseIn NW and South India we found the highest variance and oldest TMRCA (time of most recent common ancestor) of J2a-M410 haplotypes, whereas in North, West and Central India the TMRCA  was compAmong linguistic groups, our results indicate that Indo-Europeans (IE) and Dravidians (DR) have received the J2a-M410 influx approximately at the same time. However, our results indicate a very late influx and rare presence of the J2a-M410 HG in Austroasiatics (AA). In eastern region, J2b-M102 exhibits few dense focal points in contour map due to high frequency among some AA groups like Asur caused by founder effect. Comparing the TMRCA and diversity of J2b-M102 haplotypes between different linguistic groups  suggestsMDS  plot based on Rst values for HG J2a-M410 shows that Indian populations do not form a single cluster but are closer to the Central Asian and European populations, while Caucasians (populations from Caucasus) form a separate cluster . HoweverJ2-M172 is a predominant HG in West and Central Asia. Populations living West from India show high frequency, subclade variation and presence of paragroups. Worldwide subclade diversity and distribution of J2a-M410 suggest its spread from West and Central Asia into India through NW corridor . The spacul-de-sac valleysVarious studies have given evidences to support the influence of Neolithic from Near East on Indian subcontinent (in Mehrgarh) dated around 10.5 KYA The AMOVA results found that geographically distant populations have higher Fst values . Lower F1636Absence or negligible presence of classical markers of Eurasian demic diffusion in India advocates against it to be the sole explanation of J2a-M410 and J2b-M102 distribution in the subcontinent. High variance, haplotype diversity with no sharing haplotype, geographically pronounced phylogeny and seemingly autochthonous origin of sublineage J2a-M68, suggest towards the antiquity of the HGs. Dispersal of J2a-M410 and J2b-M102 from Near East to NW region and further eastwards of the subcontinent seems to have unique and complex history of various known and unknown possible events. Regardless of the complexity of dispersal, NW region appears to be the corridor for entry of these haplogroups into India. Remarkable presence of J2a-M410 among tribal groups inhabited in remote geographical regions strongly dismisses the earlier belief of it to be caste-specific.We studied a total of 3023 Y- chromosomes belonging to 77 different Indian populations. Of these, 1102 individuals were from different primitive tribes, 362 individuals were from nomadic tribes, 856 individuals were from different castes and 541 individuals were from different tribes. These individuals were affiliated to different linguistic groups  distributed all over India. In addition to Indian samples, we also analysed 39 samples of Thapa population from Nepal. Details of populations studied are given in the lSTR® PCR amplification kit . Detailed method of genotyping is given in Text S1. J2-M172 Individuals were resolved for downstream biallelic markers and assigned to the HG J2-M172 subclades .Biallelic Y chromosome markers defining HG J-M304 and its subclades  were genotyped following the hierarchy of the Y chromosome phylogeny. Samples were genotyped through Sanger sequencing using BigDye™ Terminator cycle sequencing kit . For Y-STR genotyping, every second or third sample from each population was randomly selected based on the frequency of the subclades of HG J-M304 in the respective population. Out of 355 J-M304 possessing Y chromosomes, 158 were genotyped for 17 Y-STR markers with AmpFwww.fluxus-engineering.com). Equal weights were given to each locus. One Steiner tree was selected and shown . HG isofrequency maps were generated using Surfer v 8 , following the Kriging procedure. We excluded two DYS385 loci from all current analyses due to technical issue. We have also filtered out some highly deviating haplotype samples from any age estimation analyses. The deviating samples were detected by determining their high mutational distance from the modal haplotypes. The selected samples used for TMRCA analyses are listed in Population diversity indices, Rst values and AMOVA  based Fhttp://www.R-project.org/).TMRCAs and the times of individual population splits were calculated using the Bayesian approach implemented in BATWING, considering exponential growth from a constant-size population model65How to cite this article: Singh, S. et al. Dissecting the influence of Neolithic demic diffusion on Indian Y-chromosome pool through J2-M172 haplogroup. Sci. Rep.6, 19157; doi: 10.1038/srep19157 (2016).


Nothapodytes nimmoniana Graham, one of the extensively harvested medicinal plant species in the Western Ghats biodiversity hotspot, India to assess the effectiveness of protected areas in long-term maintenance of economically important plant species.The harvesting of medicinal plants from wild sources is escalating in many parts of the world, compromising the long-term survival of natural populations of medicinally important plants and sustainability of sources of raw material to meet pharmaceutical industry needs. Although protected areas are considered to play a central role in conservation of plant genetic resources, the effectiveness of protected areas for maintaining medicinal plant populations subject to intense harvesting pressure remain largely unknown. We conducted genetic and demographic studies of N. nimmoniana in four protected and four non-protected areas using 7 nuclear microsatellite loci revealed that populations that are distributed within protected areas are subject to lower levels of harvesting and maintain higher genetic diversity  than populations in adjoining non-protected areas . Furthermore, seedlings in protected areas had significantly higher observed heterozygosity (Ho = 0.630) and private alleles as compared to seedlings in adjoining non-protected areas (Ho = 0.426). Most populations revealed signatures of recent genetic bottleneck. The prediction of long-term maintenance of genetic diversity using BOTTLESIM indicated that current population sizes of the species are not sufficient to maintain 90% of present genetic diversity for next 100 years.The analysis of adults and seedlings of Overall, these results highlight the need for establishing more protected areas encompassing a large number of adult plants in the Western Ghats to conserve genetic diversity of economically and medicinally important plant species. The harvesting of medicinal plants from wild sources to meet pharmaceutical industry needs Nothapodytes nimmoniana (Graham) Mabb., one of the medicinally important tree species distributed in the Western Ghats has become a major source of DNA topoisomerase inhibiting anti-cancer drug, Camptothecine (CPT)  , one of N. nimmoniana populations in protected and non-protected areas in the central Western Ghats of southern India to assess the genetic and demographic effects of harvesting and evaluate the effectiveness of protected areas in the maintenance of long-term viability of N. nimmoniana. The specific objectives of our study were to 1) assess the genetic structure and diversity of N. nimmoniana populations in protected and non-protected areas, 2) investigate any evidence for genetic bottlenecks of populations and 3) analyze demographic data to predict future population sizes to evaluate long-term viability of N. nimmoniana populations in the Western Ghats.We conducted genetic and demographic studies of Nothopodytes nimmoniana was carried out in the central Western Ghats regions of Karnataka, with permission from the Karnataka Forest Department. Tissue sampling was carried out under the supervision of forest officers and used solely for scientific research. The sampling was non-invasive with no impact on the natural growth or regeneration of N. nimmoniana populations in the wild.The field work and tissue sample collection of 2N. nimmoniana was overlaid with protected area map of the Western Ghats to identify populations that are distributed within protected areas (PAs), and based upon the availability of N. nimmoniana within the PA network of the Western Ghats, four study sites namely: Dandeli Wildlife Sanctuary (established in the year 1974), Talakaveri wild life sanctuary (established in the year 1987), Kudremukh wild life sanctuary (established in the year 1987) and Agumbe medicinal plants protected area (established in the year 1999) were selected for the present study  Mabb. (Icacinaceae), (Nothapodytes foetida (Wight) Sleumer and Mappia foetida Meirs is a medicinally important tree species naturally distributed in many parts of the Western Ghats in South India, some parts of Assam, Himalayan foothills, Sri Lanka, Burma and Thailand N. nimmoniana led to severe reduction in population sizes N. nimmoniana is a dioecious species, some individuals are polygamous with male, female and bisexual flowers naceae),  formerlyN. nimmoniana in each quadrat was recorded and expressed as a percentage of the total number of adults harvested for each quadrat t-test. The girth-class distributions of adult individuals in each site were determined. The frequency distribution of the girth-class of adults across the PA and non-PA was statistically evaluated using the nonparametric Kolmogorov–Smirnov test In each of the four PAs and their adjoining non-PAs, 10 quadrats (10 m×10 m) were laid out randomly and data on the number of trees per quadrat, girth of all individuals above 10 cm dbh, and number of harvested or coppicing individuals were recorded. As a measure of regeneration, the number of seedlings and saplings (<1 m height) in each quadrat was recorded. For each quadrat, the number of regenerants (saplings and seedlings) was divided by the number of adults to obtain an index of the regeneration per adult. The number of harvested adults of 2. Leaf samples were air-dried and preserved in silica gel until the extraction of total genomic DNA using a modified CTAB (cetryl trimethyl ammonium bromide) protocol 2, 3 µM of each dNTP, 0.25 µM each forward and reverse primer and 0.5 unit Taq DNA polymerase (Sigma). The thermal cycling parameters were: 94°C for 3 min, 35 cycles of 94°C for 40 s, 58–62°C for 40 s, 72°C for 60 s, followed by a final extension of 5 min at 72°C. The amplified samples were prepared for genotyping by mixing 10 µL of deionized formamide, 0.1 µL of 35–500 bp internal size standards  and 1 µL of PCR product. The mixture was denatured at 95°C for 2 min and immediately placed on ice for a minimum of 5 min and electrophoresed on an ABI PRISM 310 Genetic Analyser (Applied Biosystems) to detect fragment sizes. The electrophorograms were analyzed with the GeneScan 3.7 and GenoTyper 3.7 software programs (Applied Biosystems).Leaf samples were collected from 15–20 adults as well as seedlings selected randomly from four PAs and their adjoining Non-PAs. In each site, the area sampled was approximately 0.5 kmThe following genetic diversity measures were calculated for all eight populations and also separately for adults and seedlings: Allelic distribution, private alleles , the mean number of alleles per locus (averaged across 7 loci) and per population were calculated using GENEPOP 3.2a We calculated the total genetic differentiation among population as Fst: which assumes an Infinite Allele Model (IAM) He>Heq) was tested using Wilcoxon signed-rank tests and sign test We assumed that, if extensive harvesting of trees in recent years has affected the populations, they should show signatures of recent genetic bottlenecks between adults and seedlings. We also expect that, the populations outside the PAs should experience higher signatures of genetic bottleneck than populations within PA network, as populations within PAs receive more protection and less harvesting than NPAs. We used following approaches to test these predictions: Populations that experienced a bottleneck often exhibit a reduction of allele number and heterozygosity at polymorphic loci, with allele numbers being reduced at a higher rate than heterozygosity N. nimmoniana is sufficient to maintain 90% of present day observed genetic variation over the next 100 years as a measure of long-term genetic viability of the species To assess whether the current population size of N. nimmoniana populations outside PA are experiencing more threat and harvesting pressure.The mean density of adults between populations of PAs and NPAs were significantly different  was generally higher than mean observed heterozygosity (Ho) across all loci (e = 0.725–0.910). The locus NN54 had lowest level of polymorphism with 3–8 alleles per population and expected heterozygosity (HE) ranged from 0.394–0.779 followed by the locus NNT4 (A = 3–10 and HE = 0.504–0.790). The allelic diversity parameters  and mean observed number of alleles (A) per locus (averaged over 7 loci) were highest for the population Dandeli (AR = 6.018 and A = 11.857±4.180) followed by Talakaveri (AR = 5.476 and A = 10.714±4.309), Joida (AR = 5.999 and A = 10.286±3.817) and the lowest values were found in populations Bondikadu (AR = 4.974 and A = 8.143±4.298) and Vadagere (AR = 4.727 and A = 8.571±2.226) (P). The two of the populations, namely Dandeli and Talakaveri had highest number of private alleles with 11 and 13 private alleles respectively  were found at the species level . The meaectively . The meaall loci . The loc1±2.226) . Many alectively . The Neiectively .ST estimate, θ averaged over seven loci was 0.105 . However, the comparison of genetic diversity parameters between adults and seedlings within individual populations revealed significant decrease in genetic diversity parameters (A and HO) in seedlings as compared to adults in some of the populations  for populations of PA was 0.607, significantly higher than (P<0.05) the 0.511 in populations of NPA. Populations from PA also had highest number of private alleles  from PA and NPA’s. We observed that the adults and seedlings from PA’s had significantly (P<0.05) higher observed number of alleles (A) and highest observed heterozygosity compared to NPA populations  from adults to seedlings  and among individuals within groups (9.2%)  . The varn adults  indicatiN. nimmoniana.The population bottleneck analyses of individual populations with adults and seedlings combined together detected evidence for recent population bottlenecks in five out of eight populations  indicating a deviation from mutation drift equilibrium . HoweverN. nimmoniana in Western Ghats is not sufficient to maintain present levels of genetic diversity for the next 100 years.The BOTTLESIM based prediction of future genetic diversity with varying starting population sizes, and assuming current population size as the maximum (100%), the projected genetic diversity continued to decrease over time . The obsN. nimmoniana is similar to other tropical tree species with similar life history and ecological traits N. nimmoniana is widely distributed throughout Western Ghats in peninsular India and expected to maintain moderate to high level of genetic diversity. The mating system of N. nimmoniana is mixed, which consists predominantly outcrossing mating system with some selfing N. nimmoniana  is comparable to other tropical tree species with mixed mating system and bird dispersed seeds N. nimmoniana are lower than those reported in other microsatellite based studies of tropical tree species At the species level, pattern of genetic diversity observed within O) and expected (HE) heterozygosity levels within populations of N. nimmoniana showed significant excess of homozygosity. This could be attributable to non-random mating with high gene exchange among related individuals and selfing due to the mixed mating system of the species. It is possible that reduction in population size due to habitat fragmentation or harvesting may have increased selfing rates and/or mating among closely related individuals resulting in a higher proportion of homozygous individuals in populations.The comparison of observed (HST = 0.105 RST = 0.096), similar to each other and significantly different from zero suggesting low level of population differentiation with significant amount of gene flow among populations (Nm = 2.125). This observation agrees well with corresponding values for woody perennial species with outcrossing or mixed mating system that maintain most of their variation within populations N. nimmoniana, approximately 56% of total genetic variation was found within individuals followed by within populations (36%). This observation is similar to the values of ISSR marker based genetic variation detected in individuals distributed over a broad geographical area N. nimmoniana was higher than corresponding values reported in other microsatellite based analyses of natural populations of tropical tree species Milicia excelsaThe values of inter-population genetic differentiation based on both measures were relatively low (FIS) indicates that alleles within populations were not united at random and that mating between close relatives may play an important role in determining the genetic structure of N. nimmoniana. At the population level, positive and significant FIS values  than the seedlings of the same population. This is because adult plants represent pre-harvesting generation with genetically diverse individuals resulting from larger effective population sizes. Recent human induced disturbance activities such as harvesting and fragmentation may have reduced the effective population sizes contributing to lowered genetic diversity over generations. Over harvesting leads to drastic reduction in population sizes and increased inbreeding, which can further reduce the genetic diversity in seedlings. This scenario was supported by the fact that there was significant increase in inbreeding values in seedlings as compared to adults in many populations  than NPA populations. Although other measures of genetic diversity (A and HE), did not differ significantly between two types of populations, the greater HO, AP, AR and gene diversity indicates that allele frequencies are higher in populations in PA’s. These differences are due to difference in harvesting pressure between populations of PA and NPA. On average, more than 45% of adults were extracted from populations of NPA as opposed to less than 5% of adults extracted from PA. This change in demographic decline might have contributed to uneven distribution of alleles in NPA populations. We also observed that the genetic diversity parameters among adults and seedlings differed between populations of PA and NPAs. There was significant decrease in genetic diversity parameters (HO and A) from adults to seedlings within populations of NPAs  detected evidence of recent genetic bottlenecks in most of the eedlings . This suO and is likely a result of existing low allelic diversity or recent genetic bottleneck. Similar results have been reported for other endangered species N. nimmoniana in the Western Ghats is not sufficient to maintain present observed levels of genetic diversity over the period of next 100 years. This could be attributable to large scale harvesting of wild populations of N. nimmoniana. After the discovery of camptothecine in N. nimmoniana, the species have been largely exploited from the wild leading to reported 20% decline in natural populations of N. nimmoniana in the Western Ghats N. nimmoniana.Our simulation study revealed a faster decline of observed number of alleles (OA) than HN. nimmoniana highlight the effectiveness of protected areas in conserving genetic diversity of economically and medicinally important plant species. We observed that the populations from PAs had significantly high genetic diversity than populations in NPAs. It was further supported by simulation analysis where NPAs are predicted to lose genetic diversity faster than PA populations in future. PAs were also effective in preventing harvesting pressure on populations as evidenced by harvesting of over 45% from NPAs as compared to about 5% adults harvested from PAs.Our results based on N. nimmoniana in Western Ghats is not sufficient to maintain 90% of present genetic diversity over the next 100 years. If population sizes of N. nimmoniana further continue to decline in the wild, most populations may lose nearly 50% of present genetic diversity during the next 100 years. The above results are consistent with recent demographic decline of N. nimmoniana in its natural habitat. The reduction in its habitat and population size may have led to genetic bottleneck and further loss in genetic diversity. The conservation strategy should be oriented towards protecting natural populations of N. nimmoniana in Western Ghats from further overexploitation to sustain the long-term survival of the species. As adults are known to maintain more genetic diversity, we recommend that conservation efforts geared toward protection of adult plants in each population should be implemented to protect the reproductive fitness and evolutionary potential of the species The long-term persistence of population depends on population size of a species. A population with large number of individuals is considered to have more genetic diversity, which increases their ability to adapt to changing environmental conditions File S1Supplementary Table. S1 Table. Summary of t-test statistics for demographic parameters collected for four protected (PA) and non-protected (NPA) N. nimmoniana populations from Western Ghats.(DOC)Click here for additional data file.


Procambarus clarkii, in China. The sequences of the mitochondrial control region and the proPOx intron in the nuclear genome of samples from 37 sites (35 in China and one each in Japan and the USA) were analyzed. The results of combined scenarios testing and historical records revealed a much more complex invasion history in China than previously believed. P. clarkii was most likely originally introduced into China from Japan from an unsampled source, and the species then expanded its range primarily into the middle and lower reaches and, to a lesser extent, into the upper reaches of the Changjiang River in China. No transfer was observed from the upper reaches to the middle and lower reaches of the Changjiang River. Human-mediated jump dispersal was an important dispersal pathway for P. clarkii. The results provide a better understanding of the evolutionary scenarios involved in the rapid invasion of P. clarkii in China.Identifying the dispersal pathways of an invasive species is useful for adopting the appropriate strategies to prevent and control its spread. However, these processes are exceedingly complex. So, it is necessary to apply new technology and collect representative samples for analysis. This study used Approximate Bayesian Computation (ABC) in combination with traditional genetic tools to examine extensive sample data and historical records to infer the invasion history of the red swamp crayfish,  Introduced species exist in almost every ecosystem in the world, and the proportions of introduced species in biomes, ecosystems, and habitats are increasing . InvasivSource populations and pathways of invasion can be identified by either direct or indirect methods ,7,8. IndApproximate Bayesian Computation (ABC) testing, a recently developed method ,11, has Procambarus clarkii , the red swamp crayfish, is one of the most notorious invasive species in the world [P. clarkii [P. clarkii in China could be the consequence of human-mediated dispersal [he world ,14. It ihe world ,16. The he world ,18. A hi clarkii ,14,15, a clarkii . General clarkii . The sucispersal ,20 and aispersal  in additispersal .P. clarkii using mitochondrial cytochrome c oxidase I (COI), 16S ribosomal RNA (rRNA) and nuclear microsatellites data sets [P. clarkii populations in China have a relatively high genetic diversity. The aim of the present study was to investigate the routes of invasion of P. clarkii in China. The sequences of the mitochondrial control region and the proPOx intron were used to assess the genetic diversity and variation of the population and to analyze networks of their haplotypes. In addition, the mixed mitochondrial control region and proPOx intron sequence data were analyzed using ABC to test statistically distinct scenarios based on the clusters of P. clarkii population. The expected results were (i) detection of variations in the control region and the proPOx intron; (ii) generation of networks of P. clarkii haplotypes and (iii) inference of scenarios and routes of invasion.As we know, the mitochondrial control region is the most variable part of the mitochondrial genome and evolves three to five times more rapidly than does the rest of the mitochondrial genome . It is gata sets , and fouproPOx intron sequences, the nucleotide diversity was 0.00640. Nine haplotypes (GenBank accession numbers: KC556874–KC556882) were found, and their distributions are presented in Based on the data from the mitochondrial control region sequences  that could not be connected with a confidence limit of 95% [As regards the mitochondrial control region, the 291 individual samples comprised 46 unique haplotypes, and the statistical parsimony analysis in TCS  revealed four distinct networks a. Four ht of 95%   sites and 21 sites with alignment gaps. Of the polymorphisms, two were singleton variable sites of two variants, ten were parsimony informative sites of two variants, and four were parsimony informative sites of three variants  sites and 43 sites with alignment gaps were observed. Of the polymorphisms, 14 were singleton variable sites of two variants, 49 were parsimony informative sites of two variants, and two were parsimony informative sites of three variants . For thevariants .D nor Fu and Li’s D* rejected the null hypothesis of neutral evolution ; therefore, variation patterns of the mitochondrial control region and the proPOx intron sequences may be a reliable reflection of the population history of P. clarkii. Moreover, the positive Fs values derived from the mitochondrial control region sequence and the proPOx intron sequence analyses were 0.773 (p = 0.393 > 0.05) and 0.365 (p = 0.572 > 0.05), respectively, indicating that P. clarkii did not experience a significant population expansion.Neither Tajima’s proPOx intron were found both among and within populations . Thus, no pattern of IBD was shown for the crayfish in China.The Mantel test results for matrix correlation between genetic similarity and geographical distances were not significant  were classified into five populations (regions) based on the distribution of the Chinese populations  and the in China  and the in China b. For thFinally, a total of 19 scenarios (#12–30) were included in cluster 3 . HoweverP. clarkii was introduced from the middle and lower reaches of the Changjiang River into the river’s middle and upper reaches, but we did not find evidence for such a transfer.Taken together, the inferred routes of invasion resulting from scenario testing are summarized in P. clarkii in China.With the recent developments in population genetic approaches, researchers can infer the origin, pathways of introduction, mode of establishment and demographic changes associated with the dispersal of invasive species in invaded areas based on information about the population structure and the past demography of populations; such approaches can be combined with traditional genetic tools and historical records . Our stuP. clarkii populations in China might come from unsampled place(s) completely different from the foreign populations collected in this study. It indicated that (i) insufficient source populations were sampled or/and that (ii) Japan introduced P. clarkii multiple times or from multiple places including unexpected places. Besides, of course, genetic variations may have occurred in Chinese populations after invasion, which maybe to some extent contributed to the unsampled source. The unsampled population may to some extent contribute to the ancestral haplotypes detected by the network analyses. Undoubtedly, further sampling in native areas would help to clarify the source area of the genetic clusters. Regardless, the scenario testing for cluster 1 also indicated that the most strongly supported scenario involved introduction routes for the crayfish invading China from Japan. The results were consistent with those obtained from microsatellites, mitochondrial COI and 16S rRNA sequences in our previous study, which suggested that the P. clarkii populations in China might be derived from Japan rather than the USA [The six ancestral haplotypes  detected in various populations in China were shared by the populations in Japan and China. Only the Hap_i1 and Hap_i3 were shared by the populations from the USA, Japan and China. Therefore, Japan rather than the USA was the probable origin of Chinese crayfish populations. The scenario testing for cluster 1 revealed that an unsampled source population was most likely involved in the overall genetic make-up, which meant that some genetic resource of  the USA . More saP. clarkii was introduced from the middle and lower reaches of the Changjiang River into the river’s middle and upper reaches, but no converse transfer was observed. This finding may be resulted from (i) the crayfish habit of swimming against the flow and (ii) that the number of crayfish naturally distributed in the middle and upper reaches of the Changjiang River was very small, and thus, the crayfish was unlikely to be transferred by humans due to commerce. Thus, the results also indicated that human-mediated dispersal was an important dispersal pathway for P. clarkii in China, and the results to some extent showed that the aim of commerce might mostly contribute to the human-mediated dispersal for the crayfish. The human-mediated dispersal might contribute to the complex of dispersal pathways and the gene flows among the populations in the middle and lower reaches of the Changjiang River.The most likely scenario regarding the invasion of cluster 1 and the most strongly supported scenario for the invasion of cluster 2 revealed an unsampled source followed by a serial introduction from Japan into Nanjing, Jiangsu province, China. The original population was then mainly expanded and transferred to other areas in the Changjiang River basin in China. These results are consistent with those previously reported in the literature ,16. HoweP. clarkii is an economically important aquaculture species in China, and frequent aquaculture-related transfers have occurred among different locations. Human activities such as commercial shipping or unintentional carry have most likely facilitated gene flow among populations of P. clarkii and influenced their population genetic structure and diversity [P. clarkii might be much more complex than previously assumed.Scenario testing for cluster 3, exclusively analyzing the admixture populations, showed that no scenario had higher posterior probability. iversity . The traP. clarkii population and between population samples, which, to some extent, might have enhanced the invasion success of P. clarkii. The results are similar to those obtained in a previous study in which AMOVA also showed that many genetic variations occurred among and within the P. clarkii populations based on COI, 16S rRNA and nuclear microsatellites data sets [P. clarkii from the source and invasive areas, and application of additional methodologies, particularly those related to evaluating the evolutionary history of P. clarkii invasion, is needed.Generally, increased genetic diversity and/or multiple introductions could contribute to invasion success . Howeverata sets . FurtherP. clarkii invasion path identification could help support our understanding of why the crayfish mainly distributed in the middle and lower reaches, and further indicate that human-mediated dispersal was one of the important dispersal pathways for P. clarkii in China. It could also help quarantine, control or eradicate P. clarkii in China, and provide a reference for scientists to introduce a new species in the future.Biological invasions could pose a threat to agriculture, natural environments, and human economic activities . PresentP. clarkii distributed in China, and the USA and Japan sites are the alleged initial places of introduction. The Chinese sites were located in open, abandoned fields, and no specific permits were required for the described field studies. The samples from the sites in Japan and the USA were kindly collected by Dr. Jian Gao from Kagoshima University and Dr. Dan Wang from Ohio State University South Centers, respectively, with permission from the property owners. The muscle samples were dissected from the crayfish and were immediately stored in 95% ethanol. After returning to the laboratory, the samples were stored at −20 °С for later DNA extraction.A total of 35 sites were selected in China, and one site each from the USA and Japan were selected . The 35 et al. [CRF: 5′-TCGCTGTAAAGTTGAAGAAGTT-3′; CRR: 5′-TTAATCTCTTCATATCTTTAATTAC-3′) were used to amplify ~957–983 bp of the control region in 50 µL polymerase chain reactions (PCRs). The reaction conditions were as follows: 5 cycles of denaturation at 95 °C for 30 s, annealing at 52 °C for 1 min, and elongation at 72 °C for 1 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 1 min, and elongation at 72 °C for 1 min, and a final 10-min extension at 72 °C and cooling to 4 °C. The PCR products were sent directly to Sangon Biotech Co., Ltd.  for purification and sequencing using the forward PCR primer CRF and an internal forward sequencing primer CRF2-F (5′-TCAAGAATATGATAGTAATGTT-3′).Genomic DNA was isolated using a method adapted from Li et al. . The priproPO) of P. clarkii, referred to as the proPOx intron in this study, spans a region of approximately 240–270 bp. The primers designed to amplify the complete proPOx intron region [proPOxF (5′-GAGACGTAATGAGTTCGGGCTGG-3′) and proPOxR (5′-GCTCAGCTGACCATCTGCCTCAA-3′). The amplification reactions were performed in a volume of 50 µL using the following cycling conditions: 94 °С for 5 min, followed by 30 cycles of 94 °С for 30 s, 58 °С for 45 s and 72 °С for 30 s, with a final extension at 72 °С for 10 min. These PCR products were also sent directly to Sangon Biotech Co., Ltd.  for purification and sequencing. Due to polymorphisms in the intron, some of the PCR products were detected as “double peaks” in certain intron fragments in the chromatogram. These “double peak” PCR products were further cloned using the pGEM®-T Easy Vector System . However, the sequences obtained from these clones were contained in those haplotype sequences of corresponding populations obtained by direct sequencing. Thus, in the haplotype analysis, we omitted the proPOx intron sequences that appeared as “double peaks”.An intron of the gene encoding prophenoloxidase  obtained from the 37 populations were aligned using Clustal X 2.1 [All of the sequences  were assessed using DnaSP 5.10 software . AnalysiThe correlation between genetic distance and geographic distance was assessed using IBDWS version 3.21 . The sigproPOx intron were examined using TCS version 1.21 [The evolutionary relationships among the haplotypes produced by the mitochondrial control region and ion 1.21 , and a sScenario testing was applied separately to three different clusters. First, the populations La , Sa  and NJ  were set as cluster 1, and six scenarios were considered. An unsampled population was included in scenarios 3, 4, 5 and 6 to test for an unknown source of the second gene pool. For cluster 2, we classified the samples collected in China (excluding more admixture populations) into five populations/regions . Generally, approximately 120 scenarios could be tested with five populations, but this number can be decreased using the hypotheses emerging from previous genetic studies and the known historical dates of the first record of invasion in a country . Ultimatt1 < t2 < t3 < t4 < 100; 5 < db < 25; 1000 < N < 1,000,000; 1 < Nf < 10,000. The values of the demographic parameters were t, db, N and Nf. The values of the sample sizes that were measured in number of individuals were N and Nf. The values of times that were measured in number of generations were db and t. The default values of prior distributions of the different parameters for the mutation model of the X-linked and mitochondrial DNA sequences were used. Kimura’s two-parameter model of mutation was used [The scenarios were tested using ABC analyses of demography in DIYABC software v1.0.4.46 beta . Prior dwas used  with 10%was used , posteriP. clarkii might be much more complex than previously assumed. Human-mediated jump dispersal was an important dispersal pathway for P. clarkii in China. More sampling data and further analyses are needed to elucidate actual sources and further details of invasion routes.The red swamp crayfish was most likely introduced into China from Japan from an unsampled source and subsequently mainly expanded and transferred to the middle and lower reaches and eventually the upper reaches of the Changjiang River. However, the results of this study also revealed that the dispersal pathways for


DNA (ND5 and ND6 regions) and microsatellite DNA data. Our results suggest an overall European origin of Hawaiian cats with no pattern of isolation by distance between Australian, Malaysian, and Hawaiian populations. Instead, we found low levels of genetic differentiation between samples from Tasman Island, Lana'i, Kaho'olawe, Cocos (Keeling) Island, and Asia. As these populations are separated by up to 10,000 kilometers, we assume an extensive passive dispersal event along global maritime trade routes in the beginning of the 19th century, connecting Australian, Asian, and Hawaiian islands. Thus, islands populations, which are characterized by low levels of current gene flow, represent valuable sources of information on historical, human‐mediated global dispersal patterns of feral cats.Endemic species on islands are highly susceptible to local extinction, in particular if they are exposed to invasive species. Invasive predators, such as feral cats, have been introduced to islands around the world, causing major losses in local biodiversity. In order to control and manage invasive species successfully, information about source populations and level of gene flow is essential. Here, we investigate the origin of feral cats of Hawaiian and Australian islands to verify their European ancestry and a potential pattern of isolation by distance. We analyzed the genetic structure and diversity of feral cats from eleven islands as well as samples from Malaysia and Europe using mitochondrial  Felis catus) are one of the most widespread introduced invasive predators on islands, having strong negative impacts on the island ecosystems , three Hawaiian islands , and seven Australian islands  Is., Tasmania, Flinders Is., Tasman Is., and French Is.). Cocos (Keeling) Island is hereafter referred to as Cocos Island. Trapping, collection of tissue, hair, and blood samples as well as genomic and mitochondrial DNA isolation were conducted as described in Koch et al. . A totalDNA sequences were determined using an ABI 3730 sequencer and analyzed using Geneious 5.6.6  software for mtDNA and Genemarker V1.95  software for nuclear fragment analysis.KP279467–KP279629], European dataset [EF587077.1‐EF587153.1], Table S1A and B). Mitochondrial genetic diversity was based on the number of haplotypes, haplotype diversity (h), and nucleotide diversity (π) using DNASP V5.1  and their significance estimates with 1000 permutations and 1000 bootstraps  using mitochondrial data was calculated in ARLEQUIN 3.5  >200 for each individual parameter. A maximum credibility tree was subsequently produced using TreeAnnotator v1.7.5. FigTree v1.4.0 was used for graphically display the tree.A Bayesian phylogenetic tree was reconstructed using Beast v1.7.5  network was computed using NETWORK version 4.6.1.0 , expected (HE) and observed (HO) heterozygosity as well as significance values for deviations from Hardy–Weinberg equilibrium (HWE). Allele frequencies and FIS coefficients as a measure of the level of inbreeding were calculated using FSTAT 2.9.3  and Tasman Island (TASM) exhibited the lowest genetic diversity with HE = 0.32 and HE = 0.43, respectively  was excluded from population genetic analyses due to the occurrence of null alleles , Cocos Island (Q), and Christmas Island (CIF) . Tasmania (TAS), Malaysia (M), and Lanai'i (L) lay with some overlap between clusters. Tasman Island (TASM) and Flinders Island (FL) were distinct from all other populations . The DACP analysis showed a similar pattern as the PCoA .h = 0.66, TAS h = 0.53, and DHI h = 0.56 . The mtDNA median‐joining haplotype network consisted of 23 haplotypes attributable to three subgroups  to one in populations with small samples sizes . The mean haplotype diversity was 0.39 with highest values observed for Malaysia/Sulawesi (MS) The comparison of potential routes of introduction using the model selection approach supported a separate introduction of cats from Europe to Lana'i and Kaho'olawe and gene flow from Lana'i to Australia/Malaysia/Sulawesi , among populations within groups (32.5%), and within populations (36.45%) indicating similar genetic variability between locations, respectively, within populations  among nine populations was plotted against the geographical distance  was accounted for by geographical distances (P = 0.24). No evidence of recent bottlenecks in any of the feral cat populations (N > 10) was detected.The genetic differentiation , some haplotypes were shared among Hawaiian, Australian, and Asian populations and no evidence for isolation by distance of these geographical distant populations was detected  operated on the “Golden Round” trade route around the world , island populations are surprisingly genetically variable, suggesting rare but multiple invasions from different source populations. The genetic structure and diversity of invasive island populations is dependent on the level of historical and recent gene flow  with potential gene flow among domestic and feral cats.10.5061/dryad.6t066 (http://dx.doi.org/10.5061/dryad.6t066)]. Sequence data of previously published samples from Australia and South‐East Asia are available in GenBank, .The microsatellite data sets supporting the results of this article are available in the Dryad repository [Dryad doi:None declared.Figure S1. Figures illustrating the phylogeographic model selection as applied to the mitochondrial ND5+ ND6 between Europe (EU), Australia and Southeast Asia (OZ‐AS), Kaho'olawe (K) and Lana'i (L).Click here for additional data file.Figure S2. Figures illustrating the phylogeographic model selection as applied to the nuclear DNA data between Australia (OZ), Tasmanan Island and Flinders Island (TASM‐FL) Tasmania (TAS), Dirk HArtog Island (DHI), Christmas Island (CIF), Cocos (Keeling) Island (Q), Kaho'olawe (K) and Lana'i (L).Click here for additional data file.Figure S3. Principal Coordinates Analysis (PCoA) plot indicating genetic distances between individuals from eleven populations.Click here for additional data file.Figure S4. Inference of population structure of ten island populations based on a Discriminant Analysis of Principal Components (DAPC).Click here for additional data file.Figure S5. Phylogenetic tree of cats based on mtDNA haplotypes obtained in this paper together with those of Driscoll et al. 2007, reconstructed by Bayesian inference with 95% highest posterior density (HPD) represented at nodes.Click here for additional data file.Table S1. A. List of sample locations with abbreviations for sample location and region as well as number of specimens and corresponding geographical coordinates. B. List of European mitochondrial dataset published by Driscoll et al. (2007) with accession numbers and abbreviation for sample region.Click here for additional data file.Table S2. Results of the phylogeographic hypothesis model selection as applied to the mitochondrial ND5+ ND6 data for movements between Europe (EU), Australia/Malayisa/Sulawesi (OZ‐AS), Lana'i (L) and Kaho'olawe (K) . The model with the highest mariginal likelihood indicates the model with the best fit to the data (shown in bold).Click here for additional data file.