Xenopus gilli, an endangered frog, hybridizes with the African clawed frog, X. laevis, in South Africa. Estimates of the extent of gene flow between these species range from pervasive to rare. Efforts have been made in the last 30 years to minimize hybridization between these two species in the west population of X. gilli, but not the east populations. To further explore the impact of hybridization and the efforts to minimize it, we examined molecular variation in one mitochondrial and 13 nuclear genes in genetic samples collected recently (2013) and also over two decades ago (1994). Despite the presence of F1 hybrids, none of the genomic regions we surveyed had evidence of gene flow between these species, indicating a lack of extensive introgression. Additionally we found no significant effect of sampling time on genetic diversity of populations of each species. Thus, we speculate that F1 hybrids have low fitness and are not backcrossing with the parental species to an appreciable degree. Within X. gilli, evidence for gene flow was recovered between eastern and western populations, a finding that has implications for conservation management of this species and its threatened habitat.The Cape platanna,  However, gene flow between species also poses risks by eroding species boundaries8, disrupting adaptively evolved complexes of alleles10, promoting the exchange of genetic variation associated with disease11, influencing pathogen emergence12, and facilitating species invasion14. As such, hybridization has important implications for biodiversity conservation.Gene flow (introgression) between species may facilitate adaptive evolution through the exchange of beneficial genetic variation. This expedites the colonization of specialized ecological nichesXenopus); 28 of 29 species are polyploid, and all of these are probably allopolyploid16. When backcrossed in the laboratory, there is variation among F1X. gilli-laevis hybrid females with respect to whether or not their progeny are polyploid17. Laboratory studies indicate that in some crosses (X. gilli-X. laevis and X. laevis-X. muelleri) F1 hybrid males are sterile, but female F1 hybrids are fertile18. F1X. gilli-X. laevis hybrid females are capable of backcrossing with either parental species, and both sexes of the F2 backcross generation can be fertile17. Thus there exists the possibility that gene flow among Xenopus species could occur in nature. At least three Xenopus hybrid zones are thought to exist21, and hybrids in each of these zones may have the same ploidy level as the parental species , habitat near the town of Kleinmond, and habitat near the town of Pearly Beach  marker and 13 nuclear DNA (nDNA) markers. Genetic samples were collected from within managed (west) and unmanaged (east) portions of the range of X. gilli. Samples were analyzed from both locations that were collected shortly after management began, and then in the same areas again 20 years later. We expected that if introgression was occurring in the populations during this time period, it would be more pronounced in the east population. If efforts to minimize hybridization in the west were successful, we expected more evidence of gene flow in the samples collected soon after management began as compared to more recently. However, in both localities and both sampling times, we found no evidence of shared mitochondrial haplotypes or nuclear alleles between these species, suggesting that the F1 hybrids have low fitness and are not backcrossing with the parental species to an appreciable degree, despite potential fertility of F1 females17. Within X. gilli, we recovered evidence of gene flow between east and west populations, and found genetic diversity to be higher in the unprotected eastern population. These findings have implications for management and conservation of this endangered habitat specialist.To further investigate the effect of hybridization on gene flow between 28. The 2013 collection included X. gilli and X. laevis individuals from the same or geographically close (within 5 km) sites as the 1994 collection, and both sampling efforts used funnel traps. Animal sampling protocols approved by the Institutional Animal Care and Use Committee at Columbia University and work was performed in accordance with all relevant guidelines and regulations for animal experimentation, in accordance with laws for studying wildlife in South Africa and with appropriate collection permits from the Chief Directorate of Nature Conservation and Museums, and was approved by the Animal Ethics Committee at the University of Cape Town and the Stellenbosch University Research Ethics Committee: Animal Care and Use. Samples were obtained east and west of False Bay for both species and for both time periods  based on dorsal and ventral patterning, shape of head, and overall size44. Because this study aimed to explore genetic effects of backcrossed hybrids, for both time points, we intentionally excluded individuals whose intermediate morphology  indicated that they were F1 hybrids .Genetic samples analyzed in this study were collected either in 1994 or in 2013. Some of the samples from the earlier collection were also analyzed in two earlier studiesX. gilli and X. laevis individuals, respectively, using primers from ref. 16S). Exons of 13 nDNA genes ranging from 333–770 bp in length were sequenced for 20–41 X. gilli and 11–31 X. laevis individuals using paralog specific primers (primers are from ref. BCL9), BTB domain containing 6 (BTBD6), Chromosome 7 Open Reading Frame 25 (C7orf25), Fem-1 Homolog C (FEM1C), Microtubule Associated Serine/Threonine Kinase Like (MASTL), Mannosyl-oligosaccharide glucosidase (MOGS-1), Nuclear Factor, Interleukin 3 Regulated (NFIL-3), protocadherin 1 (PCDH1), phosphatidylinositol glycan anchor biosynthesis class O (PIGO), protein arginine methyltransferase 6 (PRMT6), Ras association domain family member 10 (RASSF10), SURP and G-patch domain containing 2 (SUGP2), and zinc finger BED-type containing 4 (ZBED4). A table of sample IDs and which loci were amplified for which samples is available in the Appendix. In the phylogenetic analysis of individual genes (discussed below), we used as an outgroup a sequence from X. tropicalis from the genome assembly version 9.0 on Xenbase47. When possible, we also included orthologous and homeologous sequences from X. laevis from the genome assembly version 9.1 on Xenbase47, which was identified using BLAST48; this was not possible when a homeologous sequence was not identified, which could be due to gene loss or missing data in the genome sequence. Sequence data were aligned using MAFFT49 and corrected by eye. Coding frame was estimated using the ‘minimize stop codons’ option in Mesquite v.3.0450, and alignments were trimmed to begin at the first position and end at the third position of the reading frame.DNA was extracted from tissue samples using Qiagen DNEasy tissue extraction kits , following the manufacturer’s protocol, or a phenol-chloroform protocol. A fragment of the mtDNA genome was amplified and sequenced for 36 and 33 52 with default parameters. Each individual’s allelic sequences for each locus were used in subsequent population genetic, clustering, and gene tree analyses. Thus, for each nuclear locus, an individual frog was represented by two sequences, each corresponding to one allele.We calculated the phase of nDNA alleles (i.e. haplotypes) using the ‘best guess’ option of PHASE53. Substitution models were selected based on the Akaike Information Criterion using MRMODELTEST v.254, and xml files were prepared for BEAST using BEAUTI (part of the BEAST package). For each nDNA locus, we ran two Markov chain Monte Carlo runs for 25 million generations. For the mtDNA, the model selected by MRMODELTEST2 (GTR+Γ) failed to converge on stable parameter estimates, and we therefore instead used the simpler HKY+Γ model, and ran two chains for 50 million generations. For each analysis, convergence of parameter estimates on the posterior distribution was assessed using TRACER v.1.555 based on an effective sample size (ESS) value >200 and inspection of the trace of parameter estimates against the MCMC generation number. Based on this, for all phylogenetic analyses the first 25% of the posterior distribution was discarded as burn-in. Then, using TREEANNOTATOR, we produced consensus trees from the post-burn-in posterior distribution of trees.Gene trees were estimated for each phased nDNA exon and the mtDNA alignment using BEAST v1.8.356. We trimmed the dataset to include only  nDNA genes with all populations sampled (see Genetic clusters section for details) and included only individuals sampled for all genes. All X. laevis individuals were considered to be the same species , and we separated the east and west X. gilli populations into separate species , and X. tropicalis was considered its own species. We set a simple HKY+Γ model joined for all data partitions (so that convergence of parameter estimates could be reached), assumed a strict molecular clock joined for all data partitions, and allowed the underlying gene tree structure to vary across data partitions. We ran the 8 chains for 170 million generations and removed 50 million generations as burn-in.We also estimated a species tree  using the multi-species coalescent model of *BEAST57 to estimate individual assignment probabilities to genetic clusters using best-guess phased nDNA alleles on a subset of individuals. Three loci lacked data from the east X. gilli 1994 population , so we excluded them from STRUCTURE analysis. We also excluded individuals with >50% missing data for the remaining 10 loci. This resulted in a dataset of 13, 8, 11, and 6 X. gilli individuals from the following localities and years respectively: east 1994, east 2013, west 1994, and west 2013, where east and west refer to the sampling locations relative to False Bay. This analysis also included 9, 12, 6, 4 X. laevis individuals from east 1994, east 2013, west 1994, and west 2013 respectively. We used the admixture model of STRUCTURE and assumed no correlation between alleles at different loci. We ran the Markov chain Monte Carlo for 20 million generations, following a two million generation burn-in. We tested a number of clusters (K) ranging from 1–8, with 5 replicate analyses for each setting of K. To correct for label switching and to average assignment probabilities across runs, we used CLUMPP v.1.1.258. We first computed the D statistic, following recommendations in the CLUMPP manual58, to decide on the particular algorithm to employ for maximizing similarity across runs. We then used the ad hoc method of ref. K).We used STRUCTURE v.2.3.4X. laevis and X. gilli but did detect shared haplotypes and alleles between populations of X. gilli and between populations of X. laevis. Because X. gilli is of conservation concern, we evaluated the fit of data from this species to three evolutionary models . In the second (ongoing migration) model, X. gilli population divergence was followed by ongoing symmetrical migration between the populations. Under the ongoing migration model, shared alleles would be due to ILS or migration, and some of the alleles shared due to migration could have been exchanged millions of years ago. In the third (secondary contact) model, X. gilli population divergence was followed by a period of no migration and then by a period during which symmetrical migration occurred between east and west populations. Under the secondary contact model, shared alleles would again be due to ILS or migration, but alleles shared due to migration could only have been exchanged recently.As discussed below, our analyses did not detect mitochondrial DNA haplotypes or nuclear alleles that were shared between els Fig. . In the T, which is the time of separation between the X. gilli populations and a parameter θ, which is the population polymorphism parameter of the ancestral and both descendant populations. The second and third models have an additional parameter m, which is the number of individuals in each population that are replaced per generation by individuals from the other population (east vs west), divided by the product of four times the effective population size of each population. The third model includes another parameter τ, which is the proportion of T going back in time from the present that secondary contact began. Thus, the ongoing migration and isolation models are special cases of the secondary contact model in which τ = 1, or τ = 1 and m = 0, respectively. We note that several assumptions of these models are undoubtedly violated  but we made them nonetheless so we could complete the simulations (see below) within a reasonable amount of time, and because of the relatively small size of the dataset.All models include a parameter 60. In this approach, the likelihood is approximated by the natural logarithm of the number of simulations for which the sum of four summary statistics from a simulation (discussed next) were within ±ε % of the sum of the observed four summary statistics from actual sequence data, divided by the number of simulations, where ε = 25. The value of ε determined how close the simulations must match the observed data in order to contribute to the likelihood, and was selected based on a compromise between the computational efficiency of the likelihood estimation and the accuracy of the estimate60. For the ongoing migration model and the secondary contact model, 40,000 simulations were performed for each combination of parameter values we considered. For the isolation model, no simulations had summary statistics within ±ε % of the observed; thus, 1,000,000 simulations were performed in order to achieve an upper bound for the likelihood estimate. The likelihood of the data over all combinations of the following parameter value intervals were estimated: T: every 1,000,000 generations in the interval of 0–20,000,000 generations; θ: every 0.001 units in the interval of 0.001–0.01 and every 0.01 units in the interval of 0.01–0.1; τ: every 10% in the interval of 10–100%; m every 0.1 units in the interval of 0–1 and every integer in the interval of 1–10.The approximate likelihood of combinations of values for these parameters was estimated using rejection sampling62. These four summary statistics include the number of sites with a derived polymorphism (i) in the west population of X. gilli only, (ii) in the east population of X. gilli only, (iii) shared between the west and east populations of X. gilli, or (iv) fixed in either the west or in the east population of X. gilli. The simulations used a fixed value for the mutation rate equal to 2.69e−9 substitutions per site per generation, which was estimated based on the average synonymous divergence between a randomly selected X. gilli sequence and an orthologus sequence from X. tropicalis, and assuming a divergence time of 65 million years for the separation of these lineages63, and a generation time of one year. Each locus had a mutation rate scalar based on synonymous divergence to X. tropicalis that accommodated variation among loci in the rate of evolution. To minimize the influence of natural selection on the polymorphism data, summary statistics and likelihood calculations were based only on variation at synonymous positions. Confidence intervals were estimated using the profile likelihood method  units from the maximum).We used the sum across loci of four summary statistics described by ref. FST (with significance computed by a permutation test) was quantified for the same data used in the STRUCTURE analysis using ARLEQUIN v3.5.2.264. Nucleotide diversity (π) of each locus was calculated using the pegas package in R66. We then calculated a mean value of π across loci for each of the eight populations, weighting the estimate by gene length for each locus. Confidence intervals were obtained by bootstrapping the weighted π values 5000 times.We performed various analyses to assess whether the genetic diversity varied among these species, over time, or among populations east and west of False Bay. Pairwise 67, which involves downsampling data to the smallest number of samples in each population across all nuclear loci for which there were data. This analysis was performed with the same data as used in the STRUCTURE analysis. For X. laevis populations there was one exception; the prmt6 locus had only four sampled alleles for the X. laevis west 1994 population, thus we did one run with all of the data  and another run excluding prmt6 . For all X. gilli populations, the smallest number of sampled alleles was eight. We generated confidence intervals by bootstrapping of the allelic diversity measurements 5000 times.Because allelic diversity is influenced by sample size, we using the program HP-RARE to calculate rarefied estimates of allelic diversity68. We built models for the estimated values of nucleotide diversity (π) and allelic diversity independently with diversity values measured for each locus, using time (1994 or 2014), location (east or west) and species (X. gilli or X. laevis) as fixed effects  and considering locus as a random effect. For each parameter of both models, we also used lme4 to compute confidence intervals with the confint function.To statistically evaluate differences in genetic diversity over time, location and species, we constructed linear mixed models using the R package lme4X. gilli and X. laevis clustered in reciprocally monophyletic clades .In the mitochondrial and 13 nuclear gene trees, alleles from des Fig. . No indione Fig. . This anX. laevis individuals were assigned to a single genetic cluster at K = 2–8, indicating a lack of allele frequency clustering, which is consistent with gene flow across the population range. The X. gilli samples were assigned to two clusters corresponding to sampling location (east and west) at K = 3–8, indicating differences in allele frequencies, which is consistent with restricted gene flow between them .Using simulations and summary statistics, we evaluated the fit of the lnL of each model follows a χ2 distribution with degrees of freedom equal to the difference in the number of parameters in each model , this difference in model likelihoods follows a mixture of 69. The secondary contact model is thus not supported over the ongoing migration model (p = 0.08), but the ongoing migration model is supported over the isolation model (p = 0.009). Overall then, these results support an inference of gene flow between X. gilli populations, but fail to discern substantial temporal heterogeneity in the level of gene flow.Nested models can be compared by assuming that twice the difference between the θ: 0.002 (0.001–0.003) and m: 0.7 (0.1–2) individuals/generation. The maximum likelihood estimate for T was 8,500,000 generations; the 95% CI was unable to be estimated because it exceeded the boundaries we tested , suggesting low statistical power to estimate this parameter. Comparisons to similar parameters estimated for African clawed frogs by other studies using other methods70 suggest that these estimates are biologically plausible. Our intuition that the shared identical alleles between east and west X. gilli populations are due to ongoing migration is thus supported, with caveats that several model assumptions, discussed below, are violated to some degree.The maximum likelihood parameter estimates and 95% confidence intervals for the ongoing migration model were FST was measured in all pairwise comparisons of the east and west populations of X. gilli . For X. gilli, between time points within each location (east or west), FST was not significantly different from zero . For X. laevis, pairwise comparisons of east and west populations, within the same year (1994 east to 1994 west or 2013 east to 2013 west) and between time points (1994 east to 2013 west and 2013 east to 1994 west), had intermediate FST values that departed significantly from zero . But within locations comparing time points (1994 east to 2013 east and 1994 west to 2013 west), FST was not significantly different from zero .In line with results from STRUCTURE analysis, a high π, the effect of species was significant with X. laevis higher than X. gilli by 0.00073 substitutions per site (95% CI: 0.00027–0.00119). The effect of location was significant with π lower in the west than the east population by 0.00091 (95% CI: 0.00045–0.00137). The effect of time of sampling was not significant, with the 2013 samples being lower by 0.00016 but the 95% CI of this difference spanning zero (−0.00062–0.00030). Similar results were recovered for allelic diversity, with X. laevis having higher allelic diversity than X. gilli , the west being less diverse than the east , and no significant effect of sampling time .Both nucleotide diversity and allelic diversity did not change drastically over time, but within species, both statistics were higher in the east population than the west Fig. . In the X. laevis and X. gilli found no evidence of widespread genetic introgression28, a result that seemed to be at odds with the incidence of morphologically and genetically identified hybrids in this and other studies71. In this study, we analyzed many of the samples from refs X. laevis and X. gilli was not detected in mitochondrial DNA or in any of 13 nuclear loci 74 and this presumably acts to some degree as a prezygotic barrier to hybridization. However, an observation is that at high densities, Xenopus individuals amplex indiscriminately , potentially overriding some prezygotic barriers. In some ponds, X. gilli individuals can be outnumbered by X. laevis 3:141, and indiscriminate amplexus could mean X. gilli males  are outcompeted for access to females. This may be why hybrids are occasionally seen, but the extended period of divergence between these species (~14 my) appears to have resulted in strong post-zygotic barriers preventing introgression.The lack of introgression is despite the continued identification (based on morphology) of a low frequency of putative 5. In California tiger salamanders , for example, some loci are fixed for foreign alleles from the introduced barred tiger salamander (A. mavortium), whereas other loci exhibit no sign of introgression6. That the barred tiger salamander was introduced only 60 years ago suggests that mosaicism of genomic introgression arose rapidly  also exhibit substantial geographic differences in X. gilli allele frequencies between these populations  (95% CI: 4.8–13.4), which is the same as the estimate obtained here using our coalescent modeling approach. This estimate is older than the 1 my divergence time estimated by the *BEAST analysis .The ity Fig. , this reity Fig.  may be a10. In an attempt to reduce gene flow between species, three conservation actions were implemented in the mid-1980s. A wall was built around one impoundment in CoGH25, populations of pure X. gilli were translocated to areas without X. laevis40, and X. laevis were manually removed from CoGH78. Removal of X. laevis ceased in 2000, but resumed in 201178. The same management efforts have not been conducted for the population of X. gilli east of False Bay, most of which resides in non-protected areas41.Hybridization and introgression has the potential to threaten species survivalX. gilli41 and fewer hybrids , Acacia mearnsii (Black Wattle), and Hakea sericea (Silky Hakea)80. Continued efforts to conserve and restore coastal fynbos habitat both inside and outside of protected areas43, such as removal of invasive vegetation, restoration of native vegetation, and removal of X. laevis, stands to benefit X. gilli. This is particularly important in the east population of X. gilli near Kleinmond where genetic diversity is highest and the population resides on private land.This study suggests that patterns of gene flow within Supplementary Table 1Supplemental Figures 1 and 2


We found gene introgression from cultivated rice to its in situ conserved wild relative populations, which has considerably altered their genetic structure and relationship. The level of introgression is associated with the spatial distances of the wild populations from cultivated rice fields. The findings are based on genetic analyses of six wild populations and two sets of rice cultivars using two types of molecular markers. Crop-wild introgression alters the genetic integrity of wild populations. Thus, appropriate measures should be taken for effective in situ conservation of wild relatives of crop cultivars. in situ and ex situ conservation of crop wild relative (CWR) species. However, introgression of crop alleles into CWR species/populations may change their genetic structure and diversity, resulting in more invasive weeds or, in contrast, the extinction of endangered populations. To determine crop-wild introgression and its consequences, we examined the genetic structure and diversity of six wild rice (Oryza rufipogon) populations under in situ conservation in China. Thirty-four simple sequence repeat (SSR) and 34 insertion/deletion markers were used to genotype the wild rice populations and two sets of rice cultivars (O. sativa), corresponding to the two types of molecular markers. Shared alleles and STRUCTURE analyses suggested a variable level of crop-wild introgression and admixture. Principal coordinates and cluster analyses indicated differentiation of wild rice populations, which was associated with the spatial distances to cultivated rice fields. The level of overall genetic diversity was comparable between wild rice populations and rice cultivars, but a great number of wild-specific alleles was detected in the wild populations. We conclude based on the results that crop-wild introgression can considerably alter the pattern of genetic structure and relationships of CWR populations. Appropriate measures should be taken for effective in situ conservation of CWR species under the scenario of crop-wild introgression.Maintaining genetic integrity is essential for  Daucus carota ssp. sativus) with their wild relatives (D. carota ssp. carota) resulted in adjacent wild relative populations genetically similar to cultivars, which stimulated the evolution of more aggressive weeds in carrot fields (Raphanus sativus) to wild radishes (R. raphanistrum) in California, which caused the extinction of local wild radishes (Dioscorea rotundata) to wild yams (D. abyssinica and D. praehensilis) might be the reason for the local extinction of wild yams in West Africa (Sustaining genetic diversity of crop wild relative (CWR) species is essential for crop genetic improvement , althougt fields . The oppAs demonstrated by in situ conservation (Helianthus annuus) caused by introgression of an insect-resistant transgene (Bt) from cultivated sunflower exemplified the potential of enhanced invasiveness in wild sunflowers (Oryza sativa f. spontanea) by introgression of a glyphosate-resistant transgene from crop rice  crops to their CWR populations through pollen-mediated gene flow has aroused worldwide biosafety concerns because of the potential environmental impact and increased difficulties for CWR ervation ; Lu 2016rop rice , 2014.Oryza rufipogon; referred to as W hereafter) is the wild ancestor of cultivated rice (O. sativa) and is extensively distributed in the tropics and subtropics of Asia  and japonica rice variety (Nipponbare) were originally developed to identify indica and japonica traits of rice  and Gaozhou  as separate references. We attempt to address the following questions in this study. (i) Does introgression occur from cultivated rice to its wild progenitor? (ii) Does crop-wild introgression affect genetic relationships and structure of the recipient W populations? (iii) Does crop-wild introgression alter genetic diversity of the recipient W populations? The generated knowledge will be useful for understanding the role of crop-wild introgression in shaping the genetic structure and diversity of CWR populations, and for designing strategies for effective 2 in Gaozhou of Guangdong Province in China were included for this study . Seed samples of all rice cultivars were donated by the Guangdong Academy of Agriculture Sciences (Guangzhou) and the Chinese National Gene Bank (Beijing).Leaf samples were collected from different individuals of the six W populations  and placed separately in zip-lock plastic bags containing silica gels for dehydration. The dry leaf samples were stored in a freezer at −20 °C until used. Two independent sets of rice cultivars (CV) were included in the analyses. The first set (coded as CV-1) contained 35 cultivars collected from southern China, including Guangdong, Guangxi and Fujian provinces see. The secThe total genomic DNA was extracted from leaf samples using the modified CTAB protocol described by http://www.gramene.org) and 34 InDel primer pairs developed by see . PCR reaction was performed in ABI  Thermocycler using the following programme: a denaturation period of 4 min at 94 °C followed by 28 cycles of 30 s at 94 °C, 30 s at 55 °C and 40 s at 72 °C, and then 7 min at 72 °C for final extension. Reactions were carried out in a volume of 10 μL containing 1× buffer, 2.5 mM each of dATP, dCTP, dGTP and dTTP, 10 μM of SSR or InDel primer, 50 ng of genomic DNA and 0.6 unit of Taq polymerase (TaKaRa Inc.). PCR products were separated in 4 % denaturing polyacrylamide gel (PAGE). The electrophoretic bands were revealed using a silver staining procedure. The gel was washed twice with ddH2O and then placed into a 200–400 mL solution of 0.1 % AgNO3 solution to stain for 10–15 min. The stained gel was washed twice by 200–400 mL ddH2O, and then placed into a 1.5 % NaOH solution (about 200–400 mL) containing 0.4 % formaldehyde for about 10 min to obtain visible DNA bands. Photographs were taken from the PAGE gels using a digital camera (Sony Inc.).Thirty-four SSR primer pairs from the rice genome  marker ladder . The dimorphic InDel markers were also co-dominant and their electrophoretic banding patterns were scored as a homozygous AA or BB genotype, or a heterozygous AB genotype following the standard of kΣamin/D) among W populations and CVs either at the 34 SSR loci (CV-1) or 34 InDel loci (CV-2) were calculated, respectively, using the software Microsatellite Analyser (MSA) version 4.00  was evaluated from 1 to 8 and 10 replicates were run for each K. Each run had a burn-in period of 100000 and 200000 iterations after burn-in. The software CLUMPP ver. 1.1.2 was used to find the optimal alignments of the 10 replicates with the ‘FullSearch’ algorithm (https://web.stanford.edu/group/rosenberglab/distructDownload.html). The highest log-likelihood value indicated the closest test to the actual number of genetically distinct populations. If an individual’s genotype profile indicated admixture, it would be assigned to a population with the same colour code. The W populations that were introgressed with the cultivar population would then be determined.The software STRUCTURE version 2.3.4 was used to determine the population genetic structure and admixture  to estimate genetic relationships and structure among W populations, using rice cultivars as a reference. The scatter plot was generated based on the values of correlation coefficient of the first two principal coordinates. A UPGMA dendrogram was constructed based on the pairwise genetic distance  to estimver. 6.5 .see . However, the proportion of shared alleles of the two sets of rice cultivars with their corresponding W populations varied considerably. Noticeably, the E-population nearby rice fields showed the highest proportion of shared alleles with the two sets of rice cultivars, with ~41 % with CV-1 (SSR markers) and ~64 % with CV-2 (InDel markers), respectively. On the other hand, the C-population far from rice fields showed the lowest proportion of shared alleles with CV-1 , although the proportion of shared alleles with CV-2  was similar to other wild populations  with the corresponding W populations at the 34 SSR and InDel loci was calculated, compared with the shared alleles among W populations. In general, a greater proportion of pairwise shared alleles was detected among W populations than that between the W populations and the two sets of rice cultivars , likely ulations .see . Interestingly, the E-population showed a relatively high frequency of alleles that were abundant in rice cultivars (CV-2) at a few SSR loci, indicating possible introgression of cultivar alleles to this W population. On the other hand, all the cultivar abundant alleles were not detected in the C-population, suggesting no or extremely limited introgression of cultivar alleles to this population. In addition, most of the InDel alleles were shared by the rice cultivars (CV-2) and W populations, without a clear pattern see . This result suggested that the dimorphic InDel markers had limited uses for detecting introgression in rice.To gain more detailed information about the shared alleles between the Gaozhou W populations and rice cultivars from southern China (CV-2), we analysed the frequency of abundant SSR alleles (>15 %) at various loci of the rice cultivars in each of the W populations. Our results showed that many of the SSR alleles detected in the rice cultivars with high frequencies (up to 91 %) were also present in the W populations, although a large number of W-specific alleles (105) and cultivar-specific alleles (61) were detected. Apparently, these SSR alleles detected in the rice cultivars (CV-2) with high frequencies were present in W with considerable variation among populations K = 3) and InDel (K = 3) data matrices   among W populations. Noticeably, the lowest values of genetic differentiation were observed between the E-population and the rice cultivars based on both SSR and InDel data matrices  of the pairwise genetic distance or genetic differentiation (Fst) with the geographic distance of the W populations, suggesting no isolation-by-distance pattern in terms of genetic divergence of the W populations.Pairwise nd CV-2) . The levmatrices , suggestA) and effective alleles (Ae), observed heterozygosity (Ho), expected heterozygosity (He), Shannon Index (I) and Nei’s unbiased genetic diversity (Ho = 0.81) of the E-population was considerably greater than that of all other W populations, although the level of its genetic diversity was comparable with the other W populations. On the other hand, genetic diversity of the C-population showed the lowest value in nearly all the parameters, except for the observed heterozygosity. Noticeably, a much greater number of wild-specific alleles was detected in the W populations than that of crop- specific alleles in rice cultivars (Ho) that show a similar trend with the highest value in the E-population see . In addition, no specific alleles to rice cultivars or wild rice populations were detected see .We included data matrices from SSR and InDel markers to estimate genetic diversity of the W populations using the two sets of rice cultivars (CV-1 and CV-2) as references. All the 34 selected SSR loci were polymorphic in W populations and rice cultivars. A total of 326 alleles were recorded in all samples with the highest number of alleles per locus at RM228  and the lowest at RM29, RM130, RM16, RM348, RM598 and RM245 . Results of other diversity parameters, such as the number of alleles  showed a relatively distant relationship with rice cultivars based on PCoA data. A similar pattern of genetic relationships between W populations and cultivars was also found based on UPGMA cluster analyses. Notably, the E-population was clustered together with rice cultivars in the dendrogram constructed based on SSR data matrix, although no significant correlation between the genetic and spatial distances was detected based on the Mantel test. In addition, the pairwise Our results did not show considerably high genetic diversity of the E-population, which has answered our third question raised earlier about genetic diversity of W populations. That is crop-wild introgression did not alter overall genetic diversity of the W populations. However, the studied W populations contained a much greater number of wild-specific alleles, suggesting relatively abundant and unique genetic variation in the W gene pool in Gaozhou. The unique genetic variation in wild rice germplasm can be explored further for rice breeding as reported previously . GeneticO. rufipogon can produce highly fertile hybrids from natural hybridization, owing to their close genetic relationship and high compatibility genes. Given that transgenic rice may be eventually released for commercial production in China  to the protected areas. However, genetic impact of natural introgression from their crops on the protected W populations was not taken into consideration for this type of in situ conservation, where rice cultivars are extensively planted near the conservation site. Results from our study clearly indicated that crop-wild introgression could considerably alter the genetic structure and relationships of the protected wild rice populations, particularly those occurring in the close vicinity of cultivated rice fields. In other words, extensive crop-wild introgression may significantly change the genetic integrity and structure of CWR populations, even though they are under in situ conservation. Therefore, questions concerning the long-term impact of crop-wild allelic introgression on genetic integrity/diversity and evolutionary destiny of CWR populations and species in agroecosystems should be further studied for the effective in situ conservation.It is widely accepted that genetic resources harboured in CWR populations or species are extremely valuable for crop breeding. However, due to habitat changes and disturbances by human activities, effective ifficult ; Lu 2013O. rufipogon) populations that are under in situ conservation, although such introgression seemed not to change their overall genetic diversity. Our findings have broad implications for the in situ conservation of endangered CWR species/populations, particularly for those that are genetically and spatially close to their domesticated counterparts. The in situ conservation strategy for CWR species/populations only by means of a physical isolation  to interrupt disturbances from outside sources may not be sufficient, particularly at the sites with extensive cultivation of their domesticated species. Probably a buffer zone with properly determined spatial distances or other types of cultivated crops can be established to limit crop-to-wild allelic introgression for maintaining the genetic integrity of the in situ protected W populations. In addition, restoration of ecological habitats that are suitable for CWR populations/species may also be useful for in situ conservation, as proposed by Crop-wild gene flow and allelic introgression considerably alters the genetic structure, relationship and integrity of the wild rice  collected from southern China  for SSR (simple sequence repeat) marker analysis.Table S2. Name of the rice cultivars (CV-2) collected from Gaozhou and its neighbouring regions in Guangdong Province, China, for InDel (insertion/deletion) marker analysis.Table S3. Name of the 34 SSR primer pairs and their location on chromosomes used in this study (http://www.gramene.org).Table S4. Name of the 34 InDel primer pairs and their location on chromosomes used in this study from Table S5. Frequencies of shared alleles by cultivated rice  and wild rice (O. rufipogon) populations (A–F) at 16 SSR loci. ‘-’ indicates no relevant allele detected.Table S6. Frequencies of shared alleles by the populations (A–F) of perennial common wild rice (Oryza rufipogon) and rice cultivars at different InDel loci. ‘-’ indicates no relevant allele detected.Table S7. Genetic diversity parameters for wild rice (Oryza rufipogon) populations (A–F) in Gaozhou compared with those of cultivated rice controls (CV-2 and CV-1) from southern China based on InDel fingerprints.This research is supported by the Nature Science Foundation of China , National Program of Development of Transgenic New Species of China (2016ZX08011-006) and ‘Gan-Po Talent 555’ Project of Jiangxi Province, China.B.-R.L., C.L. conceived and designed the experiments. X.J., Y.C., P.L., X.X.C., B.-R.L. performed the experiments. X.J., Y.C., B.-R.L., J.R., C.L. analysed the data. X.J., Y.C., J.R., B.-R.L. wrote the manuscript.None declared.Supplementary MaterialsClick here for additional data file.


Branchiomma, with special emphasis on a taxon so far identified as Branchiomma bairdi. This species, originally described from Bermuda, has frequently been reported as an invader in the Mediterranean, the Atlantic and the Eastern Pacific, but recent observations have raised some taxonomic questions. Samples of this taxon were collected from five sites in the Mediterranean Sea, two sites in the original distribution area of B. bairdi in the Gulf of Mexico and four localities in the east Pacific and Atlantic Oceans where B. bairdi has been reported as invasive. The molecular results revealed a conspicuous genetic divergence (18.5% K2P) between the sampled Mediterranean populations and all the other ones that led to a re-evaluation of their morphological characters. The latter showed that the Mediterranean and extra-Mediterranean populations also differ in some discrete morphological and reproductive features. Consequently, the Mediterranean samples were re-designated as B. boholense, another non-indigenous species originally described from Philippines. Branchiomma bairdi and B. boholense differ in body size, development and shape of micro and macrostylodes, size of radiolar eyes and body pigmentation. Genetic diversity was high in B. boholense from the Mediterranean as well as in B. bairdi from the Gulf of Mexico, but low in B. bairdi populations outside their native range. The phylogenetic analysis revealed the presence of connections between the Mediterranean localities as well as between native and introduced B. bairdi populations that focus the attention on the Panama Canal as important passage for the introduction of the species from the Gulf of Mexico to the north-east Pacific Ocean.This study was performed to analyse the genetic and morphological diversity of the sabellid annelid genus  Introductions of marine non-indigenous species (NIS) have occurred for centuries, but in the last decades the increasing maritime traffic and aquaculture activities have favoured the spread of marine species, with irreversible and devastating impact on global biodiversity –7. UnderBranchiomma Kölliker, 1858. Several species in this genus have been reported outside of their natural expected distribution ranges . A total of 123 sequences were generated using either of the two primer combinations  and then bidirectionally sequenced using the forward and reverse primer M13 for the Mediterranean and Mazatlán populations. For the sequencing of the samples collected from all the other populations, the new set of primer was utilized. Cycle sequencing was performed using BigDye Terminator version 3.1 in a 10 μl reaction containing 5 μl of 10% trehalose, 2 μl of 5X BigDye buffer, 1 μl of BigDye, 1 μl of each primer (or primer cocktail) and 1 μl of the PCR products. After clean-up of the sequence reactions using Zymo Sequencing Clean-Up Kit, the sequences were analysed with an ABI PRISMinations .Forward and reverse sequences were combined and manually edited with Sequencher v. 4.8 . The sequences were aligned using the algorithm CLUSTAL W implemented in MEGA 5  and trimThe levels of genetic polymorphism for each studied population were estimated by the number of haplotypes (Nh), haplotype diversity (h) and nucleotide diversity (π) using DNAsp 5.10.01 .ST among all populations was calculated using ARLEQUIN v. 3.5.2.2 [Bispira manicata  [Bispira serrata Capa, 2007 [Branchiomma sp. [B. bairdi [® KX894904.1; KX894907.1; EU835657.1; HQ015126.1; KP254646.1). Branchiomma sp. sequences belong to specimens collected in Australia (Darwin Harbor) and Hawaii  [B. bairdi was collected in Florida [Pair-wise F 3.5.2.2 . The sig 3.5.2.2 . The gen 3.5.2.2 . A Bayes 3.5.2.2  using th 3.5.2.2 . The Baye, 1878) , Bispirapa, 2007 , Branchiomma sp.  and B. by, Oahu) , while BFor this study, detailed observations of the external morphology of specimens collected from different localities were undertaken using light microscopy. For each locality, 15 adult individuals were examined and for all the specimens, the total body length , the trunk length  and the radiolar crown lengths were measured. Moreover, some morphological features such as length of radiolar tips, shape of stylodes, dorsal lips and number of rows of teeth above the main fang of thoracic and abdominal uncini were analysed. The number of thoracic and abdominal chaetigers and the ratio of crown-trunk lengths were also measured.All the analysed morphological features were photographed using a digital camera attached to the stereo- and compound microscopes . Thoracic uncini were drawn with the aid of a camera lucida. Due to the variation in size and morphology of uncini along the body segments and within the tori, their morphology was analysed considering the dorsal most uncini of second thoracic chaetiger.A one-way analysis of variance (ANOVA) was performed by using STATSOFT STATISTICA v. 6.0 to test for significant differences in the mean value of the crown/trunk length ratio between Mediterranean and extra-Mediterranean samples. Pearson correlation analysis was carried out on the number of chaetigers and the values of body length in order to evaluate the relationship between the two measurements taken.The sequencing of the 493 base pair fragment of the COI gene in 123 individuals identified 11 haplotypes. These haplotypes derived from 44 polymorphic sites of which 43 were parsimony informative. Most polymorphisms were the results of synonymous changes (43 of the 44 sites) while five mutations produced amino acid replacements (non-synonymous changes). Seven of the 11 identified haplotypes were location private, six of which were represented by a single individual. Four haplotypes were shared by individuals collected from distinct populations and sampling sites, two of which resulted the most common haplotypes: haplotype 1 was present just in the Mediterranean Sea and was shared by 35 individuals belonging to all the sampled Mediterranean populations, while haplotype 9 was shared by 67 individuals collected from different populations in both the Pacific Ocean, the Gulf of Mexico and the Atlantic Ocean .Haplotype (h) and nucleotide (π) diversity were quite different within populations . HaplotyST and average K2P genetic distance analysis evidenced a great molecular divergence between the Mediterranean populations and those sampled both in the Gulf of Mexico, Atlantic Ocean and Pacific Ocean  . Group 1a bairdi  and usedThe MJ network of haplotype showed results highly concordant with the phylogenetic analysis. The analysis distinguished two groups of haplotypes, separated by 33 mutations . HaplogrThe two groups of populations that molecular analysis showed to be genetically divergent, also showed slight morphological differences. As shown in Moreover, significant differences (p<0.0001) were detected in the values of crown/trunk length ratio between Mediterranean and non-Mediterranean samples . The valThe morphological features of Mediterranean and extra-Mediterranean specimens are listed in Specimens from all the extra-Mediterranean material showed a green  and brownish colouration (in fixed specimens) with interramal small brownish spots along the body and an orange-red pigmented crown in some specimens . Their rB. boholense specimens from its original area of distribution (Indonesia) are shown. Microstylodes appeared digitiform and long as rachis width or slightly longer, while macrostylodes were 2–3 times longer than microstylodes and tongue-like (flattened) in shape  observed between the two groups of populations were similar to those reported for other congeneric polychaete species [This study revealed significant genetic differentiation in the mitochondrial gene COI between Mediterranean and extra-Mediterranean  species ,79–85. TB. bairdi [B. boholense for our Mediterranean material originally identified as B. bairdi. Indeed, Mediterranean specimens showed morphological features comparable to those reported for specimens from Indonesia, reviewed in this paper  population were previously explained as adaptation to a new environment [B. bairdi [B. bairdi were often detected, and never observed in the Mediterranean taxon presumably identified in this study as B. boholense.It is worth mentioning that biological data on reproduction and population dynamics concerning the Mediterranean form previously referred as . bairdi ,30 couldironment ,30. Resuironment ,45, whilBranchiomma bairdi and B. boholense are difficult to distinguish and it is likely that in the past the two species have been frequently misidentified. According to Tovar-Hernández et al. [B. bairdi, whereas B. boholense has flattened tongue-like macrostylodes and thoracic uncini with one large tooth. However, recently, Keppel et al. [B. boholense two rows of teeth can be present, suggesting that this character cannot be considered for the separation of the two taxa.As already reported by different authors ,37,41,42z et al. , the mail et al.  and CepeB. bairdi with the syntype of B. boholense giving some useful suggestion about the main differences separating the two taxa.Our morphological observations on Mediterranean and extra-Mediterranean specimens as well as on individuals from Indonesia integrated the results reported by Cepeda & Rodríguez-Flores  that comB. boholense, showed mainly flattened, tongue-like macrostylodes, short microstylodes (as long as rachis width or slightly longer) and small brownish radiolar eyes (covering less than 1/2 of side of radiolar rachis). By contrast, specimens of B. bairdi had cylindrical, strap-like macrostylodes, long microstylodes (notoriously longer than rachis width) and big red-pigmented radiolar eyes (covering more than 3/4 of side of radiolar rachis). Moreover, both species showed more than one row of teeth over the main fang. Nevertheless, in B. bairdi the main fang appeared followed by 2–3 rows of teeth, while in the Mediterranean taxon as well as in specimens from Indonesia , only present unpaired basal stylodes  and showing plasticity in the majority of the other studied Branchiomma species. But the relation of presence-absence of this feature according to juvenile or adult stages, or regenerating worms have not been determined.The slight difference observed between the morphology of the Mediterranean material and the syntype of z-Flores , could bz-Flores , was prostylodes . HoweverBranchiomma in the Mediterranean present different reproductive modes and discrete morphological differences, including body size. Capa et al. [Branchiomma are greatly homoplastic and polymorphic, and Keppel et al. [Branchiomma have been reported as alien worldwide, but it appears that many additional NIS remain undetected for this family, in multiple geographic regions around the world. Consequently, for early detection programs of invasive species we recommend the combined use of molecular and morphological approaches for Branchiomma species. It would work at least for B. bairdi, B. boholense, B. bombyx, B. luctuosum and other seven unnamed, but alien species included in Capa et al. [As this study shows, two invasive species of a et al.  already l et al.  emphasiza et al. . HoweverBranchiomma bairdi and B. boholense within the Mediterranean basin.On the basis of both molecular and morphological analysis the present knowledge led to reconsider the distribution of B. bairdi reported for the Italian coast should probably represent B. boholense [B. boholense seems to have been present in the Eastern basin since the early 1900s [B. bairdi was actually collected in the islands of Malta and Formentera [Both species are present in the Mediterranean area, although, following the results of this study, the previous records of oholense ,29,30,87ly 1900s , recentlly 1900s –27 and ily 1900s . By contrmentera ,41, and rmentera , 40.B. bairdi in the western side of the basin and its records at Canary and Madeira Islands [B. bairdi from the Atlantic Ocean throughout the Gibraltar Strait. By contrast, considering the Indo-Pacific origin of B. boholense and that the first findings of the species are from the eastern side of the basin [The presence of  Islands ,42 is cohe basin ,34, the Branchiomma species, with the Mediterranean taxon (presumably B. boholense) showing high levels of genetic diversity, and, otherwise, the introduced Pacific and Atlantic B. bairdi populations exhibiting low levels of genetic diversity.The mitochondrial genetic diversity observed within populations of the examined species are comparable to those reported for other polychaetes ,89. HoweDifferent ranges of genetic diversity have been documented for invasive species according to their life history traits and to the different type of introductions ,90–93. AB. boholense observed in the Mediterranean basin is in accordance with the second hypothesis. Indeed, the expansion of this species across the Mediterranean is very recent with possible recurrent introduction events recorded through different years and in localities that are wide apart across the basin [Styela clava Herdman, 1881 [Styela plicata  along the Italian coasts [The pattern of genetic diversity of (presumably) he basin ,26,34,37an, 1881  and Styen coasts .B. boholense, which showed a short life span, associated with a fast growth, early maturity and a rapid generation turnover ([B. bairdi). Moreover, this species displays a great ecological plasticity and adaptability in colonizing different habitats and substrates. In particular, among the examined sites around Ischia Island the species was collected also at the Castello Aragonese vent’s system, a venting area where intense CO2 emissions lower the seawater pH up to 6.6 values [Amphiglena mediterranea) or through genetic adaptation (e.g. the nereidid Platynereis spp) [Branchiomma boholense seems to acclimate to the CO2 vent conditions like A. mediterranea, as the vent-inhabiting populations are not genetically distinct from nearby non-acidified populations. The ecological plasticity of the species coupled with the high genetic diversity observed might in turn enhance its potential of invasion. A connection between high intrinsic genetic diversity and high ecological plasticity was observed also for introduced populations of the Mediterranean species Sabella spallanzanii  in Australian waters [This pattern of increased within-population genetic diversity may be also associated to the life history traits of urnover  ,96,97. Bn waters , where in waters .B. bairdi. As expected, populations sampled from the original distribution range of the species (Tampa Bay and Veracruz) showed very high values of diversity mainly linked to their higher size and stability compared to the introduced ones. Indeed, these populations belong to a taxon with a huge Caribbean distribution, described for Bermuda in 1885, but successively found in Florida [A different scenario of within-population genetic diversity was observed for the studied native and introduced populations of  Florida  and in V Florida –102.B. bairdi populations  and their monomorphic condition denoted the occurrence of recent bottlenecks from a single introduction event that accounted for the total loss of the native genetic variability observed. This is also consistent with the recent age of introduction of B. bairdi in the southern Gulf of California [By contrast, the low values of diversity observed in the studied introduced lifornia  as well lifornia ,68 and Mlifornia . The reclifornia .B. bairdi can be due to the presence of asexual reproduction [Moreover, Madeira, Galapagos and Hawaii are island populations and, as already observed in other studies ,105, sucoduction  as well,oduction ,107. Nevoduction ,108.ST and K2P analysis evidenced a genetic similarity among the Mediterranean populations as well as among the extra-Mediterranean ones, suggesting the presence of a certain relatedness among the Mediterranean populations and between native and introduced B. bairdi populations as well.Pair-wise FB. boholense as well as between the native and introduced populations of B. bairdi. As regards the Mediterranean basin, the occurrence of two haplotypes shared by different populations suggested the presence of a low but existing gene flow between these populations likely mediated by local vessels rather than by natural dispersion of the species as supported by the in vitro short pelagic larval phase of the species [B. bairdi and individuals introduced in the Pacific Ocean and in Madeira Island was demonstrated by the fact that these populations share the same haplotype detected in native populations. This genetic structure corroborates the hypothesis that the introduction of B. bairdi both in the East Pacific and in the East Atlantic occurred from the Caribbean as already suggested by Tovar-Hernández et al. [B. bairdi samples   but also a single individual from the Galapagos Islands (introduced range). We have no information about the underlying causes for the genetic split, but the wide geographic distribution of this haplotype underlines its invasive potential.The presence of connections between the different populations of the same species was demonstrated also in the median joining network of haplotypes. The network showed two distinct haplogroups representing Mediterranean and extra-Mediterranean detected mitochondrial haplotypes. In both haplogroups the occurrence of just one haplotype shared by the majority of the individuals confirmed the degree of connections between the Mediterranean populations of presumably  species . The Med species ,110, lik species ,111. Likz et al. ,46 and Rz et al.  for the z et al.  and, as z et al. –117 it i15) Figs . No morpBranchiomma species and the genetic structure of the studied populations, providing reliable information on the distribution range of B. bairdi outside its native range and on the invasion scenario of (presumably) B. boholense in the Mediterranean Sea.In this study, the combined use of both molecular and morphological data allowed to clarify the taxonomic status of the two alien Further studies with the use of other molecular markers and a bigger pool of samples could corroborate our findings and provide a better understanding of two species invasion process for a complete and exhaustive management program.S1 Table(CSV)Click here for additional data file.S2 TableValues that were significant are in bold.(CSV)Click here for additional data file.S3 TableValues that are significant are in bold.(CSV)Click here for additional data file.S4 Table(CSV)Click here for additional data file.


Keteleeria davidiana var. formosana, disjunctly distributed in northern and southern Taiwan, for investigation of population adaptive divergence in response to environmental heterogeneity. A total of 13,914 SNPs were detected and used to assess genetic diversity, FST outlier detection, population genetic structure, and individual assignments of five populations  of K. davidiana var. formosana. Principal component analysis (PCA), individual assignments, and the neighbor-joining tree were successful in differentiating individuals between northern and southern populations of K. davidiana var. formosana, but apparent gene flow between the southern DW30 population and northern populations was also revealed. Fifteen of 23 highly differentiated SNPs identified were found to be strongly associated with environmental variables, suggesting isolation-by-environment (IBE). However, multiple matrix regression with randomization analysis revealed strong IBE as well as significant isolation-by-distance. Environmental impacts on divergence were found between populations of the North and South regions and also between the two southern neighboring populations. BLASTN annotation of the sequences flanking outlier SNPs gave significant hits for three of 23 markers that might have biological relevance to mitochondrial homeostasis involved in the survival of locally adapted lineages. Species delimitation between K. davidiana var. formosana and its ancestor, K. davidiana, was also examined . This study has produced highly informative population genomic data for the understanding of population attributes, such as diversity, connectivity, and adaptive divergence associated with large- and small-scale environmental heterogeneity in K. davidiana var. formosana.Double digest restriction site-associated DNA sequencing (ddRADseq) is a tool for delivering genome-wide single nucleotide polymorphism (SNP) markers for non-model organisms useful in resolving fine-scale population structure and detecting signatures of selection. This study performs population genetic analysis, based on ddRADseq data, of a coniferous species,  Conifers are reported to have slower evolutionary rate due to reduced levels of nucleotide mutation and large effective population size, but with higher ratio of non-synonymous to synonymous divergence, in comparison with angiosperms  and Cu. lanceolata (China)  and Ca. macrolepis (China)  Beissner var. formosana) are presently disjunctly distributed on northern and southern rocky mountain ridges, respectively, at elevations of 300–600 m and 500–900 m  Beissner that occurs in China   markers for the purpose of investigating population adaptive divergence in K. davidiana var. formosana. We hypothesized the occurrence of IBE as well as IBD because of habitat heterogeneity and geographic isolation, in particular, between northern and southern populations of Taiwan cow-tail fir. The relative importance of geography and environment shaping the patterns of genetic variation was assessed to gain a deeper understanding of how environmental factors influence evolutionary processes. We also aimed to test the selection of SNPs closely associated with environmental variables across populations of K. davidiana var. formosana and to examine whether potential selective outliers link to specific gene functions that may have played significant roles underlying local adaptation. To examine the genetic relationships between populations of K. davidiana var. formosana and K. davidiana, samples of K. davidiana were also genotyped based on ddRADseq.In a previous study based on amplified fragment length polymorphism (AFLP), local adaptation was found in K. davidiana var. formosana were collected, including three northern and two southern populations  and K. davidiana var. formosana (n = 62) using a modified cetyl trimethyl ammonium bromide (CTAB) protocol  and MseI (20 units) and ligated to barcoded P1 and indexed P2 adapters binding to EcoRI and MseI overhangs, respectively. Fragments in the 250–450 bp size range were selected from an agarose gel. Amplified fragment libraries were quantified using quantitative real time polymerase chain reaction (qPCR) and pooled in equimolar amounts for sequencing on the Illumina HiSeq2000 platform  with 36 samples per lane. Sequence reads, 85 bp in read 1 and 90 bp in read 2, were assigned to individual samples based on barcode sequences. The ddRADseq library construction, sequencing, and the following processing of sequence reads were conducted at the Beijing Genomic Institute, China.ddRADseq libraries were prepared following Peterson et al.  with modhttp://hannonlab.cshl.edu/fastx_toolkit/). The two base pairs following EcoRI restriction sites were removed avoiding the effect of GC content asymmetry that may cause problem in the subsequent de novo assembly  reads were removed using FASTX-Toolkit v.0.0.13 (K. davidiana var. formosana and K. davidiana were used to compute number of SNP (in two of three samples in each population) obtained and percentage of polymorphic loci  using a universal spherical model of the Kriging method in ArcGIS . Monthly MODIS images were generated based on a maximum values composite procedures  conducted in 1969–1986  were downloaded from the WorldClim v.1.4 (FST outliers. BAYESCAN estimates the posterior odds (PO), the ratio of posterior probabilities of selection over neutrality. The parameters for running BAYESCAN were a 100 pilot runs of 50,000 iterations and followed by a sample size of 50,000 with thinning interval of 20 among 106 iterations. Any SNP with log10(PO) > 0.5 was considered to have substantial evidence for selection  and a false discovery rate (FDR) of 1% with each run comprising 106 simulations with both “neutral mean FST” and “force mean FST” selected, and removed FST outliers to increase the reliability when calculating the global distribution of FST. Loci that are detected as outliers by both BAYESCAN and FDIST were analyzed with Samβada  against the NCBI non-redundant nucleotide database for potential gene region identification. In BLASTN, an E-value of 0.001 was used as threshold for significant sequence similarity. Pairwise linkage disequilibrium (LD) between outlier SNPs was assessed using a two-locus exact test implemented in ARLEQUIN v.3.5, and significance determined by 10,000 permutations  test (the “ks.test” function of R). Data of 50% missing value across populations was adopted, based on the number of SNP obtained (Supplementary Table HO), and expected (HE) heterozygosity using STACKS. Nei's unbiased HE (uHE) was also calculated  was calculated with the function “allel.rich” of R package PopGenReport. Proportion of shared alleles between species and between populations was calculated with the “pairwise.propShared” function of R package PopGenReport. Index of association (IA)   were significantly different between populations of K. davidiana var. formosana. Population and locus were used as fixed and random effects, respectively, in linear mixed-effects models. Overall difference was tested using the “Anova” function of R package car  estimation using the “lmer” function of R package lme4  and from K = 1 to K = 6 (both K. davidiana and K. davidiana var. formosana) using the default settings, and the best K evaluated with 10-fold cross-validation (CV) procedure. Genetic assignment of individuals was also inferred based on sparse non-negative factorization (sNMF) and least-squares optimization with the “snmf” function of R package LEA .Three data sets, including total, neutral, and outlier, were used for computation of genetic differentiation via analysis of molecular variance (AMOVA) and across population For principal component analysis (PCA), allelic frequency data was first generated with the “makefreq” function of R package adegenet  and IBD (βD) were calculated and tested with 999 permutations.Mantel correlation between geographic distance and environmental heterogeneity was assessed with the “mantel” function of R package vegan . KS test showed no significant differences between 40 and 50% and between 50 and 60% missing data sets in the distributions of π, HO, HE, and uHE across populations. However, the distributions of AR across populations were significantly different between 50 and 60% missing data sets probably due to the significant increase in the number of different alleles per locus in 60% than in 50% missing data set  than the values in K. davidiana  , 0.103 (0.093–0.109), 0.128 (0.119–0.135), 0.096 (0.089–0.101), and 0.103 (0.093–0.109), respectively . HE was lower compared to HO in all populations . The levels of genetic diversity measures  were comparatively higher in the northern than in the southern populations (Table Ps < 0.0001). No difference was found between the three northern populations in all genetic diversity measures. The levels of all genetic diversity measures except HO were also significantly different between the two southern populations . FIS-values were all negative and deviate significantly from zero for all populations either with data included only K. davidiana var. formosana  > 1.0 in either global or between population comparisons indicating strong evidence for selection  = 1,000 were observed when the southern DW41 population was compared to the northern populations. Samβada revealed four outlier SNPs  with log10(PO) = 1,000 in global comparison associated strongly with environmental variables: CC with aspect and TT with number of rainfall days per year for SNP 109734_46 , TT and GG with annual mean temperature for SNP 334591_7 , CC and AA with annual mean temperature for SNP 505960_78 , and GG with aspect and AA with number of rainfall days per year for SNP 522238_59 . No significant LD between these four outlier SNPs was found with two-locus exact test (Supplementary Table Araucaria angustifolia (E = 5E-10), locus 334591  to the clone GQ03405_M22 mRNA of Picea glauca (E = 4E-23), and locus 521876 (outlier SNP found in comparison between the southern neighboring DW30 and DW41 populations) to the mitochondrial large subunit ribosomal RNA gene (LSU rRNA) of Abies homolepis (E = 3E-35). Clone GQ03405_M22 mRNA sequence of Pic. glauca is corresponded with the sequences of mitochondrial cytochrome oxidase subunit 1 (COX1) of larix gmelinii (EF053147.1)Twenty-three outlier SNPs 0.2%) were found using both BAYESCAN and FDIST in global and pairwise population comparisons  and across population FST  were found based on the outlier data. Comparing between populations of the North and South regions, ΦST was 0.425 (P = 0.001) and FST was 0.437 (P = 0.001). Significant AMOVA and FST were also found between populations within the North  and within the South .Both AMOVA and K. davidiana var. formosana only were analyzed, respectively. However, only small amounts of genetic variation were explained by the first two PCs  , but effective genetic clustering was observed. PCA revealed clear distinction between K. davidiana and K. davidiana var. formosana  and the DW30 population of K. davidiana var. formosana (DW30 clade) forming a KD + DW30 clade, which can be easily collapsed to a KD + DW30 + DW41 clade (BSV > 90%) due to a low BSV of < 70%. The individuals of the three northern populations of K. davidiana var. formosana formed a well-supported separate clade with a BSV of >90%.CE was minimized at a Figure . Both LE Figures . AdmixtuE Figure . SimilarF-test) when the total data was used , aspect , and annual mean temperature . Soil pH , annual precipitation , and slope  were also found to significantly explain the outlier genetic variation.Because similar results were found for the forward selection and MMRR analyses based on the total and neutral data sets, only the results based on the total and outlier data sets are reported Tables , 5. All ed Table . When thr = 0.465, P = 0.025). Essentially no correlation was found between genetic variation and environment, between genetic variation and geography, and between genetic variation and combined effect of environment and geography, based on the total data . Significant IBD (between genetic variation and geography) was also found . When considering both environment and geography, results showed a pattern of strong IBE based on the outlier data .Strong correlation between environmental and geographic distance matrices was found  though between species FST was low but also significant . Nevertheless, distinction between the closely related island and mainland Keteleeria species pair were elucidated using ddRADseq. Three lines of evidence, including PCA  was most closely related to the DW30 population of K. davidiana var. formosana, Keteleeria colonization from China into southern Taiwan cannot be inferred based solely on the NJ tree. Moreover, the disappearance of K. davidiana var. formosana from its historically occupied habitats in central Taiwan      and may have potential for adaptive evolution under natural selection  had significant non-zero K. davidiana var. formosana based on the total and neutral data sets are in accordance with the general realization that conifers typically exhibit low population differentiation due to long distance wind pollination . Spatial environmental heterogeneity and strong IBE based on the outlier data  suggesting that both IBD and IBE may have influenced population divergence within K. davidiana var. formosana. No significant correlation was found between genetic and environmental distances and between genetic and combined effect of environmental and geographic distances based on the total data using MMRR  was found based on the total data, suggesting that IBD could have played an important role in shaping the population genetic divergence of Taiwan cow-tail fir. When the outlier data was used, genetic variation was significantly correlated with either geographic or environmental  distances. In addition, strong IBE was found when considering combined effect of geography and environment . Our results suggest adaptive divergence corresponding to environmental heterogeneity despite strong IBD within K. davidiana var. formosana.Evolutionary theory predicts that population genetic divergence should be correlated with both geographic distance and environmental heterogeneity. Gene flow among populations could follow both IBD and IBE patterns, corresponding to geographic distance and environmental context  and DW41 (61.5°) (Supplementary Table K. davidiana var. formosana.Temperature and precipitation are commonly found to play prominent roles as selective drivers for adaptive variation in various plant species (Manel et al., E-values to specific genes of mitochondrial AOX, COX, and LSU rRNA based on BLASTN search (Supplementary Table K. davidiana var. formosana.Sequences flanking three outlier SNPs that associated strongly with environmental variables were found to have high sequence similarities with low K. davidiana and K. davidiana var. formosana, and assessment of genetic diversity and fine-scale population genetic structure in K. davidiana var. formosana. Unlike AFLP data (Fang et al., K. davidiana var. formosana based on the total data. Significant negative FIS values estimated with SNP data in the present study reflected outbreeding of K. davidiana var. formosana typical of conifers. The present study highlights the separation of environmental variables into separate categories for investigation of the impact on genetic variation. We identified outlier SNPs potentially evolved under selection strongly associated with specific environmental variables that might have played important roles in maintaining metabolic homeostasis and survival underlying local adaptation despite significant IBD. The present study emphasizes the importance of examining population isolation using ddRADseq to understand the spatial distribution of genetic variation across a species range. Our analyses suggest adaptive divergence between allopatrically distributed northern and southern populations and between the geographically neighboring DW30 and DW41 populations attributed to environmental heterogeneity. In addition, differential contributions of seed dispersal and pollen migration might have played crucial roles in shaping the population structure and spatial distribution of genetic diversity across species range of Taiwan cow-tail fir.Genome-wide SNPs obtained using ddRADseq has permitted for the evaluation of species delimitation between S-YH proposed, funded, and designed the research; J-DC, K-MS, S-YH, and Y-CC collected samples; K-MS performed research; S-YH, K-MS, C-TC, and J-DC analyzed data; S-YH and K-MS wrote the paper. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Parasyncalathium souliei (Asteraceae). We sequenced the plastid psbA-trnH and trnL-rpl32 spacer regions for 417 individuals in 36 populations across the geographic range of the species. To estimate the phylogeographic history of this species, a series of population genetic, phylogenetic, molecular dating, and haplotype network analyses were conducted, as were tested for historical demographic expansions. Using occurrence data, species distribution modeling was used to estimate geographic distributions at three time points: the present, the Mid-Holocene and the Last Glacial Maximum. Significant phylogeographic structure was evident  among the 37 haplotypes detected. Four major haplogroups were identified based on phylogenetic analyses. Private haplotypes were restricted to geographically distinct regions that generally corresponded to previously identified biogeographic subregions within the QTP-HDM region. Our results imply Pliocene-Pleistocene diversification of P. souliei and suggest that the species may have been geographically widespread early in its history. This study may provide valuable evidence for phylogeographic regionalization using chloroplast genetic data in a common, widespread endemic species from the QTP-HDM.Biogeographic regionalization can help to better understand diversity in biogeography, conservation, and macroecology. Historical regionalization schemes typically focus on species distributions, often rarely considering the rich context that phylogeographic information can provide. We investigated whether phylogeographic data could help to delineate floristic regions in the Qinghai-Tibet Plateau (QTP)-Hengduan Mountains (HDM) region by analyzing phylogeographic structure in the herb  The Himalayas, especially their core regions, i.e., the Qinghai-Tibet Plateau (QTP) and the Hengduan Mountains (HDM), comprise one of the key high-altitude biodiversity hotspots in the world   how topography/geography and Pleistocene climatic oscillations have shaped the present-day distributions of P. souliei; (2) whether significant divergence and genetic structure exists among P. souliei populations across different floristic regions/subregions; and (3) whether phylogeographic information may be useful in characterizing potential phylogeoregions in the QTP-HDM region.In this study, we employed an integrative approach to identify phylogeographic patterns within Parasyncalathium souliei are not privately-owned and are endangered or protected in the field. A total of 417 individuals representing 36 populations of P. souliei were collected from across the geographic range of the species. Within each population, 3–19 individuals growing at least 40 m apart were collected to maximize sampling of genetic diversity within populations. For most populations, all individuals found were sampled to obtain maximum coverage of P. souliei. Fresh leaves of all individuals were dried in the field using silica gel. Total genomic DNA was extracted using a QIAGEN DNeasy Plant Mini Kit . Two plastid DNA spacer regions (psbA-trnH and trnL-rpl32) were amplified via PCR in 80 μl reactions, each containing 30–40 ng of template DNAs, 50 mM Tris-HCl, 1.5 mM MgCl2, 0.5 mM dNTPs, 2 mM of each primer and 0.75 U of Taq polymerase . The following PCR program was used: one cycle at 94°C for 4 min; 32 cycles at 94°C for 1 min, 55°C for 1 min, 72°C for 1 min; and one cycle at 72°C for 7 min. PCR products were purified with a QIAquick PCR Purification Kit , and were sequenced using an ABI 3730XL automated DNA sequencer . The sequences were deposited in GenBank (accession numbers MH023542-MH023958).All the Material was collected and deposited in the Herbarium of the Kunming Institute of Botany, China. Otherwise, no specific permissions were required for material collection of the nature reserves about sampling and investigate in field of Provincial Forestry Department; the locations of P. souliei is listed under the older synonym Melanoseris souliei). To identify the optimal nucleotide substitution model, MrModeltest 2.3 was used . In addition, to estimate genetic differentiation among P. souliei haplotypes during Pliocene to Pleistocene, molecular dating was performed under a Bayesian approach as implemented in BEAST 1.6 . Population diversity parameters, including number of haplotypes, haplotype diversity and nucleotide diversity were calculated from the haplotype network and phylogenetic analyses using DnaSP 5.0  and total haplotype diversity (HT) were calculated in PERMUT (http://www.pierroton.inra.fr/genetics/labo/Software/Permut/) to evaluate the level of genetic variation; GST and NST were used to estimate differentiation between populations. When NST is compared to GST using U-statistics, a significantly higher NST than GST usually is interpreted as indicative of phylogeographic structure. The differences between GST and NST were assessed using a permutation test with 1,000 random permutations. Samova 1.0 with default parameters  derived from Shuttle Radar Topography Mission (SRTM) data , Arlequin 3.1 , a total of 159 species occurrence points were compiled for species distribution modeling . Nineteen bioclimatic variables were obtained from WorldClim 1.4, with a resolution of 1 km2  , and the Last Glacial Maximum (c. 22 ka) layer. All models were run in MaxEnt 3.2 with 10 replicates  were detected (Appendix MH023959-MH023995), with 15 populations fixed for a single haplotype . This is congruent with the result of the network and the BEAST-based chronogram. Four haplogroups were identified based on the phylogenetic and network analyses , while average within-population diversity was low (HS = 0.290) (Table Hd) was 0.828 and nucleotide diversity (π) was 0.00685. Genetic diversity was relatively low within most populations . The pairwise between-population differentiation (FST) was also highly variable (Appendix Hd) ranged from 0.604 to 0.888, while nucleotide diversity (π) ranged 0.0009–0.0140 , which is usually interpreted as indicative of phylogeographic structure. AMOVA revealed strong population variation among populations, with the highest FST value obtained when samples were divided into four population groups   did not find a statistically significant pattern of isolation by distance for P. souliei.Total plastid genetic diversity in w HS = 0.90  also rejected the historical population expansion of population group 1 despite its unimodal pattern in the mismatch analysis  is higher than average (HT = 0.670). High plastid diversity has also been detected in other QTP-HDM species such as Eriophyton wallichii Benth. (Lamiaceae): HT = 0.983; Thalictrum squamiferum Lecoy. (Ranunculaceae): HT = 0.973; Paraquilegia microphylla (Royle) J. R. Drumm and Hutch. (Ranunculaceae): HT = 0.984; and Chionocharis hookeri (C. B. Clarke) I. M. Johnst. (Boraginaceae): HT = 0.935 , which may result from the topographic complexity of the QTP-HDM. Considering the lack of spatial population genetic structure discernable from Samova suggesting such high inter-population differentiation may have been driven by genetic isolation and drift arising from dispersal limitation and topographic effects during Pleistocene full-glacial periods       and all other haplogroups (which are centered in the eastern part of the range). This implies that populations could have survived in other regions of the range away from the HDM as well, suggesting vicariance of at least some populations. However, the widespread nature of the relatively young haplotypes 1 and 3 .Biogeographical boundaries delineate the basic macrounits of diversity in biogeography, conservation, and macroecology  A. L. Budantzev (Lamiaceae) detected what the authors called the “Ward Line-Mekong-Salween Divide” (Luo et al., Secondly, our results suggest that the Jinsha River may mark an important phylogeographic boundary between what we propose is a new Himalayan phylogeoregion (region III; Figure P. souliei at approximately 29° N latitude (Figure Thirdly, we suggest that the marked division in the geographic distribution of haplotypes between haplogroups 2 and 3 in e Figure . Signifie Figure . The twoe Figure .Compared to qualitative methods of biogeographic regionalization based on the distribution of endemic plant species and quantitative methods that employ hierarchical clustering and ordination of plant communities, such as those of Wu and Wu  and ZhanHS, JZ, and HW conceived and designed this study; NL, TD, and YS analyzed the data, and MM, NL, TD, and YS wrote the manuscript; XH, WS, and DL aided in field collections. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


India is a patchwork of tribal and non-tribal populations that speak many different languages from various language families. Indo-European, spoken across northern and central India, and also in Pakistan and Bangladesh, has been frequently connected to the so-called “Indo-Aryan invasions” from Central Asia ~3.5 ka and the establishment of the caste system, but the extent of immigration at this time remains extremely controversial. South India, on the other hand, is dominated by Dravidian languages. India displays a high level of endogamy due to its strict social boundaries, and high genetic drift as a result of long-term isolation which, together with a very complex history, makes the genetic study of Indian populations challenging.We have combined a detailed, high-resolution mitogenome analysis with summaries of autosomal data and Y-chromosome lineages to establish a settlement chronology for the Indian Subcontinent. Maternal lineages document the earliest settlement ~55–65 ka (thousand years ago), and major population shifts in the later Pleistocene that explain previous dating discrepancies and neutrality violation. Whilst current genome-wide analyses conflate all dispersals from Southwest and Central Asia, we were able to tease out from the mitogenome data distinct dispersal episodes dating from between the Last Glacial Maximum to the Bronze Age. Moreover, we found an extremely marked sex bias by comparing the different genetic systems.Maternal lineages primarily reflect earlier, pre-Holocene processes, and paternal lineages predominantly episodes within the last 10 ka. In particular, genetic influx from Central Asia in the Bronze Age was strongly male-driven, consistent with the patriarchal, patrilocal and patrilineal social structure attributed to the inferred pastoralist early Indo-European society. This was part of a much wider process of Indo-European expansion, with an ultimate source in the Pontic-Caspian region, which carried closely related Y-chromosome lineages, a smaller fraction of autosomal genome-wide variation and an even smaller fraction of mitogenomes across a vast swathe of Eurasia between 5 and 3.5 ka.The online version of this article (doi:10.1186/s12862-017-0936-9) contains supplementary material, which is available to authorized users. Following the out-of-Africa (OOA) migration, South Asia  was probably one of the earliest corridors of dispersal taken by anatomically modern humans (AMH) –3. A remIndia, the second most populous country worldwide, includes a patchwork of different religions and languages, including tribal groups  and non-tribal populations, who mostly practice Hinduism, grounded in a strictly hierarchical caste system, and speak Indo-European or Dravidian languages. Indo-European is often associated with northern Indian populations, Pakistan and Bangladesh, and a putative arrival in South Asia from Southwest Asia ~3.5 ka  has been frequently connected with the origins of the caste system , 12. AltSouth India, on the other hand, is dominated by Dravidian languages, which have been connected to Neolithic dispersals from Southwest Asia , 12, 17,In the last few years, genome-wide (GW) studies have been employed –29. HoweThere is a way forward, despite the current lack of aDNA. The maternally inherited mitochondrial DNA (mtDNA) allows researchers to identify specific lineage clusters (clades or haplogroups) and to correlate them with geography. By applying a reliable mitogenome molecular clock , it is tTo assess the phylogeographic patterns of South Asian mtDNA lineages, we compiled mitogenomes from South Asia and neighbouring regions available in the literature, complemented with samples from the 1000 Genomes Project (1KGP)  and the In order to clarify the phylogeny of haplogroups M, N and R in South Asia, we focused our study on the lineages with recognized or potential likely origin in the Subcontinent, belonging to macrohaplogroups M , R  and N (N1’5). We also studied U2 (excluding U2e due to its West Eurasian origin) in a complementary analysis. We obtained 381 whole-mtDNA sequences from the 1KGP  ka, and R, at 64.5 ka 55.9; 73.2] are younger than the Mount Toba eruption (~74 ka), suggesting a later arrival  ka, suggesting a major expansion at that time. If we consider that a root type of M could have survived for ~10,000 years after it arose (as is evident from modern clades within that age range), it is plausible that re-expansion created a secondary founder effect within M that decreased the overall age estimates. Such a scenario would impact even more on ρ than ML estimates, which is indeed what we see , M18a (9.2 ka), M30d (12.1 ka), R8b1 (11.6 ka) and U2b2 (9.2 ka), all from a southern source; and R30c + 373 (12.4 ka), from the west. An increment in We also see a further increment in the last few millennia. BSPs for M in the west and centre show an increment in the last 2.5 ka  (Table X2 + 153! + 7109 and U1a3a)  . Gene flow at this time was clearly bi-directional, as seen in the expansion west of lineages M5a2a4, U2c1b + 146 and M3a1b + 13105). This is reflected in the genome-wide ADMIXTURE analysis (below), where the autochthonous South Asian component , the overall pattern is straightforward and clinal  ka, overlapping with the founder age of R  gene pool, contrasting with the ASI or autochthonous population of the Subcontinent , 26. HowNe increments at this period are visible in the west and the south, related to the expansion of indigenous M lineages.In fact, we see mtDNA lineages from Southwest Asia start to arrive as early as ~20 ka. This was a time of short-lived relative global warmth following the peak of the last glaciation, which might have triggered population movements in several regions . Some liAfter the first settlement, most attention in genetic studies has been focused on the Neolithic and Bronze Age periods, in part due to potential implications for the spread of Indo-European languages. The earliest Neolithic sites, on the Indus Valley around Mehrgarh in Baluchistan, date to before 9 ka , 86, andAlthough some have argued for co-dispersal of the Indo-Aryan languages with the earliest Neolithic from the Fertile Crescent , 89, othsoma-using Bactrian Margiana Archaeological Complex (BMAC) in Turkmenistan/northern Afghanistan by 3.5 ka and possibly as early as 4 ka. The BMAC came into contact with the Indus Valley civilisation in Baluchistan from ~4 ka onwards, around the beginning of the Indus Valley decline, with pastoralist dominated groups dispersing further into South Asia by ~3.5 ka, as well as westwards across northern Iran into Syria (which came under the sway of the Indo-Iranian-speaking Mitanni) and Anatolia [An influx of such migrants into South Asia would likely have contributed to the CHG component in the GW analysis found across the Subcontinent, as this is seen at a high rate amongst samples from the putative Yamnaya source pool and descendant Central Asian Bronze Age groups. Archaeological evidence suggests that Middle Bronze Age Andronovo descendants of the Early Bronze Age horse-based, pastoralist and chariot-using Sintashta culture, located in the grasslands and river valleys to the east of the Southern Ural Mountains and likely speaking a proto-Indo-Iranian language, probably expanded east and south into Central Asia by ~3.8 ka. Andronovo groups, and potentially Sintashta groups before them, are thought to have infiltrated and dominated the Anatolia , 97, 98.Although GW patterns have been broadly argued to support this view , there hContrary to earlier studies , 100, reThere are now sufficient high-quality Y-chromosome data available  to be aThe other major member of haplogroup R in South Asia, R2, shows a strikingly different pattern. It also has deep non-Subcontinental branches, nesting a South Asian specific subclade. But the deep lineages are mainly seen in the eastern part of the Near East, rather than Central Asia or eastern Europe, and the Subcontinental specific subclade is older, dating to ~8 ka .Altogether, therefore, the recently refined Y-chromosome tree strongly suggests that R1a is indeed a highly plausible marker for the long-contested Bronze Age spread of Indo-Aryan speakers into South Asia, although dated aDNA evidence will be needed for a precise estimate of its arrival in various parts of the Subcontinent. aDNA will also be needed to test the hypothesis that there were several streams of Indo-Aryan immigration (each with a different pantheon), for example with the earliest arriving ~3.4 ka and those following the Rigveda several centuries later . AlthougAlthough these migrations appear to have been male-driven, it might nevertheless be possible to detect a minor maternal signal. For example, haplogroup H2b  for non-autochthonous mtDNA lineages found in South Asia. The colours represent the most likely source for each lineage; branches exclusively with South Asian complete sequences coloured in green, whereas branches that also harbour sequences from other regions in white and with green contour. Ages according to ML estimates. Figure S3. ADMIXTURE analysis for all K values. Information on the populations included in Additional file Figure S4. sNMF analysis of modern populations for all K values. Information on the populations included in Additional file Figure S5. Cross-validation errors for different values of K for ADMIXTURE: (a) considering only modern populations and (b) including the Yamnaya in the analysis. Figure S6. ADMIXTURE analysis including the Yamnaya for all K values. Information on the populations included in Additional file Figure S7. PCA (for PC1 and PC3) of modern populations. Detailed information on the populations included in Additional file Figure S8. PCA (for PC1 and PC2) including the Yamnaya. Information on the populations included in Additional file Table S1. List of complete mtDNA sequences belonging to South Asian autochthonous haplogroups. Table S2. List of non-autochthonous complete modern mtDNA sequences used in our analyses. Table S3. Dataset used for the GW analyses. (a) Modern dataset. Populations marked with three asterisks (***) were added to the dataset exclusively for ADMIXTURE and sNMF runs, thereby being absent from the PCA. Table S4. Putative origin for the uniparental lineages found in the 1KGP South Asian populations. (PDF 7577 kb)BSP for haplogroup M in different regions of the Subcontinent: (a) West, (b) South, (c) Central and (d) East South Asia. CA for PC and PC3 CA for PC and PC3 Additional file 2:Phylogenetic tree of South Asian mtDNA haplogroups. (XLSX 664 kb)