The genetic information of the Chinese Tibetan group has been a long-standing research hotspot among population geneticists and archaeologists. Herein, 309 unrelated individuals from two Tibetan groups living in Qinghai Province, China (CTQ), and Tibet Autonomous Region, China (CTT), were successfully genotyped using a new homemade six-color fluorescence multiplex panel, which contained 59 autosomal deletion/insertion polymorphisms (au-DIPs), two mini short tandem repeats (miniSTRs), two Y-chromosomal DIPs, and one Amelogenin. The cumulative probability of matching and combined power of exclusion values for this new panel in CTQ and CTT groups were 1.9253E-27 and 0.99999729, as well as 1.5061E-26 and 0.99999895, respectively. Subsequently, comprehensive population genetic analyses of Tibetan groups and reference populations were carried out based on the 59 au-DIPs. The multitudinous statistical analysis results supported that Tibetan groups have close genetic affinities with East Asian populations. These findings showed that this homemade system would be a powerful tool for forensic individual identification and paternity testing in Chinese Tibetan groups and give us an important insight for further perfecting the genetic landscape of Tibetan groups. Critical samples from crime scenes may contain only small amounts of highly fragmented and damaged DNA, so forensic scientists make every effort to address this complex problem. Numerous research approaches have emerged to amplify shorter amplicons of the target sequences in the damaged DNA samples from mass disasters or forensic caseworks . Short tTibetan group is one of the nationalities with historic backgrounds, diligence, courage, and wisdom in China. The regions where Tibetans live are near the Tibetan Plateau, which is inlaid with snow-capped highlands and mountains for most of the year, resulting in a relatively geographical closure. Their pronounced historical traditions, distinctive culture, and genetic characteristics made them a long-term focus to explore hotspots among population geneticists and archeologists. In recent decades, many scholars’ studies on archeology , languagThus, the present research intends to estimate the effectiveness of the new homemade six-color fluorescence multiplex system in Tibetan groups and further perfect the genetic landscape of the Tibetan groups from the perspective of the 59 au-DIPs. This study will be a significant contribution toward understanding the genetic background and diversity of Tibetan group.F-statistics (FST) values for these loci among the five East Asian populations from the 1000 Genomes Project database were less than 0.06.The genetic markers have been carefully selected and constructed in a new homemade six-color fluorescence multiplex panel . The 2 YBloodstain specimens of unrelated healthy individuals examined in the present study were gathered from the Tibetan group in Qinghai Province, China (CTQ), and the Tibetan group in the Tibet Autonomous Region, China (CTT), with the sample sizes of 155 and 154, respectively. The present research was conducted according to the ethical guidelines of the Southern Medical University and Xi’an Jiaotong University Health Science Center and further authorized by the Ethical Committee of the Xi’an Jiaotong University Health Science Center .2 was used as template in a reaction mix with 10 µl total volume, comprising 2 µl of Master Mix , 1 µl of Primer Mix, and 7 µl of nuclease-free water. The cycling conditions were as follows: an initial denaturation step of 5 min at 95°C; followed by 2 cycles of 94°C for 10 s and annealing at 63°C for 90 s; and then 23 cycles of 94°C for 10 s, annealing at 60°C for 90 s, and extension at 60°C for 90 s; and then final extension at 60°C for 15 min. Then, 1.0 µl PCR product was mixed with 0.5 µl of an internal lane size standard and 8.5 µl of Hi-Di deionized formamide. The mixtures were denatured at 95°C for 3 min, cooled immediately for 3 min, and then genotyped on the Applied Biosystems® 3500xL Genetic Analyzer (Thermo Fisher Scientific) for electrophoretic separation and detection. Finally, GeneMapper ID version 3.2 software (Thermo Fisher Scientific) was utilized to determine the genotyping results.Bloodstain samples were amplified via the GeneAmp PCR system 9700 . About 1.0 mmThe 26 comparison population data from five different geographic regions  were downloaded from the 1000 genomes database. The statistical analysis information used in this study is summarized in p values of the LDs (p > 0.05/1830 = 0.00002732). The p values of the HWE exact tests are displayed in p values ranged from 0.0300 to 1.0000 and from 0.0180 to 1.0000 in the CTQ and CTT groups, respectively. Some slight deviations for HWE were observed at loci rs10581929 (HWE-p = 0.0320), rs35828751 (HWE-p = 0.0300), rs3830338 (HWE-p = 0.0450), and rs3833559 (HWE-p = 0.0300) in the CTQ group, and at loci D1S1656 (HWE-p = 0.0110), rs3059221 (HWE-p = 0.0310), rs66739142 (HWE-p = 0.0280), and rs67365630 (HWE-p = 0.0180) in the CTT group. After conducting the Bonferroni correction (0.05/61 = 0.0008), there were no deviations from HWE at these loci.For the two studied Tibetan groups,  the LDs  for all  the LDs  and high the LDs , which sobs values of 59 au-DIPs ranged from 0.0571 (rs3833559) to 0.2401 (rs10546179 and rs1611025); from 0.5126 (rs3833559) to 0.6547 (rs10581929); from 0.3453 (rs10581929) to 0.4874 (rs3833559); from 0.2885 (rs3833559) to 0.3750 ; and from 0.2839 (rs3833559) to 0.5548 (rs10546179 and rs1611025), with median values of 0.1738, 0.6199, 0.3801, 0.3732, and 0.4839, respectively. The PE, PD, PM, PIC, and Hobs values of the two miniSTR loci D1S1656 and D3S1358 were 0.6727, 0.9496, 0.0504, 0.8094, and 0.8387, as well as 0.3942, 0.8650, 0.1350, 0.6556, and 0.6774, respectively. The cumulative PM (CPM), cumulative PD (CPD), and combined PE (CPE) values of the 59 au-DIPs in the CTQ group were 2.8296E-25, 1-2.8296E-25, and 0.9999863, respectively. After adding the two miniSTRs, their CPM, CPD, and CPE values reached to 1.9253E-27, 1-1.9253E-27, and 0.99999729, respectively.Herein, the allele frequencies and other forensic parameters are summarized in obs values of the 59 au-DIPs were from 0.0772 (rs10556197) to 0.2878 (rs66739142); from 0.5351 (rs10556197) to 0.6570 (rs3059221); from 0.3430 (rs3059221) to 0.4649 (rs10556197); from 0.3006 (rs10556197) to 0.3750 ; and from 0.3312 (rs10556197) to 0.5974 (rs66739142), with median values of 0.1819, 0.6106, 0.3894, 0.3719, and 0.4935, respectively. The PE, PD, PM, PIC, and Hobs values of the D1S1656 and D3S1358 loci were 0.7349, 0.9297, 0.0703, 0.8022, and 0.8701, and 0.5153, 0.8441, 0.1559, 0.6482, and 0.7532, respectively. The CPM, CPD, and CPE values of the 59 au-DIPs in the CTT group were 1.3742E-24, 1-1.3742E-24, and 0.9999919, respectively. After adding the two miniSTR loci, their CPM, CPD, and CPE values reached to 1.5061E-26, 1-1.5061E-26, and 0.99999895, respectively.A total of 138 alleles were detected at the 59 au-DIPs and the 2 miniSTRs in the CTT group . The insFST values and DA distances of pairwise populations based on the 59 au-DIPs were used to perform genetic relationship analyses from these diverse genetic distances. The results showed  and DA distance (DA = 0.0012) were between the CTQ and CTT groups. The second smallest FST value and DA distance were between the CTQ group and the CHB population  from East Asia, and then between the CTT group and the CHS population  from East Asia. The maximum FST values  and DA distances  were found between the Tibetan groups and Esan in Nigeria (ESN) from Africa. In the heatmaps of FST (the upper right part of the picture) and DA distance (the bottom left part of the picture) values (The s showed  that the) values , the colFST values of the pairwise populations at the same 59 au-DIPs in the novel panel. The 28 populations involved were divided into two primary branches, one of which was dominated by seven populations from the African region and another included the other 19 populations from East Asia, South Asia, America, and Europe regions, as well as the CTQ and CTT groups. The CTQ and CTT groups formed a sister clade with the other five populations from East Asia. The basically analogous phylogenetic branch distributions also were observed in the radiation tree, which was established based on the allelic frequency data applying the Neighbor-Joining method  also revealed the genetic relationships among the CTQ group, the CTT group, and 26 other reference populations. The various color modules in the trees represented the populations from different geographic regions. g method .The PCA plots based on the population level  and indiIn K = 2–7) were distinguished by different colors  to 83.07%  in the African seven populations. The predefined appropriate ancestral information component was three, which was determined by the online Structure Harvester program  to 66.53% (ASW), from 74.68% (TSI) to 65.87% (GBR), from 68.85% (CTT) to 54.67% (CDX), and from 68.92%  to 59.4% , whereas the American ancestral composition had the largest value in Peruvian in Lima, Peru , followed by the Mexican Ancestry in Los Angeles, California (MXL); Colombian in Medellin, Colombia (CLM); and Puerto Rican in Puerto Rico (PUR) populations . At present, the East Asian ancestral component proportions in the CTQ and CTT groups were 61.98% and 68.85%, respectively , were summarized in In values and locus-by-locus of FST values of these 59 au-DIPs for pairs of regional populations are summarized in In value >0.131  was found in the African and American combination at the rs10579944 locus with the FST value of 0.7298. The rs144378883 (In = 0.1732), rs3076465 (In = 0.1767), rs3083268 (In = 0.1415), rs34731870 (In = 0.1770), rs35713041 (In = 0.1579), and rs66739142 (In = 0.1517) loci also showed higher In values (>0.131) with higher FST values of 0.5389, 0.5248, 0.5336, 0.5617, 0.4462, and 0.5957 between the African and American populations, respectively. In the same way, higher In values and FST values were also found at the rs71698233  locus between the African and East Asian populations and the rs1611025 , rs35173752 , rs5833522, and rs6481  loci between the African and European populations and the rs3083268  and rs35173752  loci between the African and South Asian populations, respectively. These loci were thus considered as the promising marker to differentiate African and non-African populations.The e >0.131 . The larThe genetic polymorphisms of these DIPs included in this new homemade panel and its forensic efficiency for personal identification and paternity testing should be validated in relevant populations. So far, these forensic parameters have not been evaluated in Tibetan groups. Here, 155 CTQ and 154 CTT samples were genotyped by this new panel, respectively. The LD analysis and HWE exact testing illustrated that all pairwise loci were independent of each other, and the samples in this survey were representative. The PD and PIC values of the autosomal loci ranged from 0.5126 to 0.9496, and 0.2885 to 0.8094; and 0.5351 to 0.9297, and 0.3006 to 0.8022 in the CTQ and CTT groups, respectively. The autosomal loci in this new panel were also reasonably informative (PIC >0.25) in the two Tibetan groups . This neR, CLUMPP, Distruct, Infocal, Snipper, and so on.A robust population genetic analysis is a prerequisite for understanding the genetic background of Chinese nationalities. To deeply determine the potential admixture level of the Tibetan groups and the genetic relationships with other reference populations, a comprehensive population genetic analysis was performed employing multitudinous bioinformatics analysis softwares, including STRAF, Arlequin, DISPAN program, Genepop, TBtools, PHYLIY, MEGA, Origin, STRUCTURE, FST value and DA distance are still used to summarize genetic variations within and among populations. They have their own unique evolutionary and statistical properties, and the FST value is generally considered to be the population differentiation caused by the difference in genetic structure (DA distance is developed under the hypothesis that genetic differentiation originates from genetic drift and mutation event (First, the allele frequency distributions of the CTQ and CTT groups were most similar to other populations in the East Asia region, and these seven East Asian populations aggregated together in the different subordinate branches of East Asia in the allele frequency heatmap. The main usage of genetic distance is to measure the genetic divergence among species or populations across the world, including the ancestral relationship or differentiation degree . Differetructure , while ton event . In thison event  and demoon event  and archon event  have alron event . While ton event , and theIn, and locus-by-locus of FST values, were selected to determine the level of informativeness provided by these markers about distinguishing different continental populations. Results revealed that there were more loci that could differentiate between African populations and other non-African populations. These DIPs would be used as potential ancestral information markers to be further explored in future forensic application.Whereafter, the tree model is usually applied to explore the biological evolution history in the bioinformatics field, and genetic affinities among populations are generally displayed through phylogenetic trees . Here, tIn this study, the forensic application efficacy of this new self-made six-color fluorescence multiplex PCR system was validated in the CTQ and CTT groups. The obtained results showed that the novel system could be applied well to the individual identification and paternity testing in two Tibetan groups, especially for forensic applications of degraded biological materials. Furthermore, combining the previous research achievements of these two Tibetan groups, the comprehensive overview for the genetic relatedness and population structure of the Chinese Tibetan groups was explored in-depth again, and these findings were of great significance to further reveal the genetic background and structure of Chinese Tibetan groups in different regions.


Phaseolus coccineus) is one of the five domesticated Phaseolus species. It is cultivated in small‐scale agriculture in the highlands of Mesoamerica for its dry seeds and immature pods, and unlike the other domesticated beans, P. coccineus is an open‐pollinated legume. Contrasting with its close relative, the common bean, few studies focusing on its domestication history have been conducted. Demographic bottlenecks associated with domestication might reduce genetic diversity and facilitate the accumulation of deleterious mutations. Conversely, introgression from wild relatives could be a source of variation. Using Genotyping by Sequencing data  from 237 cultivated and wild samples, we evaluated the demographic history of traditional varieties from different regions of Mexico and looked for evidence of introgression between sympatric wild and cultivated populations. Traditional varieties have high levels of diversity, even though there is evidence of a severe initial genetic bottleneck followed by a population expansion. Introgression from wild to domesticated populations was detected, which might contribute to the recovery of the genetic variation. Introgression has occurred at different times: constantly in the center of Mexico; recently in the North West; and anciently in the South. Several factors are acting together to increase and maintain genetic diversity in P. coccineus cultivars, such as demographic expansion and introgression. Wild relatives represent a valuable genetic resource and have played a key role in scarlet runner bean evolution via introgression into traditional varieties.The scarlet runner bean ( Phaseolus coccineus L.) is one of the five domesticated Phaseolus species. It is a close relative of common bean (Phaseolus vulgaris) and year bean (Phaseolus dumosus), which were also domesticated in Mesoamerica. In contrast with the common bean, an autogamous annual species, the scarlet runner bean is allogamous and perennial. It inhabits the highlands of Mesoamerica (1000–3000 m.a.s.l.), from northern Mexico (Chihuahua) to Panama , and P. coccineus subsp. striatus, a purple‐flowered type with eight wild varieties.The scarlet runner bean  to Southeast of Mexico (Chiapas) during 2014 and 2015. Wild individuals were sampled in nine locations, ferals in two sites, and traditional varieties at 11 locations. Samples of the breeding line Blanco Tlaxcala and a cultivar from Spain were also included Table . CategorLeaf tissue from wild samples was collected and stored in silica until processed. Seeds from traditional varieties were germinated and DNA was extracted using a DNeasy Plant Mini Kit (Qiagen). Library preparation and sequencing were performed at the Institute for Genomic Diversity at Cornell University. For library construction, a double digestion was performed using PstI and BfaI enzymes, following Genotyping by Sequencing protocol  and then were converted to binary files using samtools 1.5  in at least one wild population were identified with PLINK 1.07 , a second filtering was applied to reduce the difference in the sampling. For this filtering, relatedness was estimated through VCFtools 0.1.15 . The consequence of SNVs within coding regions was predicted with the R package VariantAnnotation  Principal Component Analysis (PCA) performed with SNPrelate  per site were estimated with the Hierfstat package (Goudet FST) among the established populations according to Weir and Cockerham  were plotted along the 11 chromosomes.Three approaches were used to assess gene flow and introgression: TreeMix (Pickrell and Pritchard Results) were evaluated with the ABBA‐BABA test: (1) gene flow from the wild population from Chiapas (Wild‐SUR‐CH) into the branch of all cultivars  and (2) gene flow from the Chiapas wild population (Wild‐SUR‐CH) into the Feral population (Table P. dumosus cultivars into Chiapas wild runner bean populations (Wild‐SUR‐CH) were also evaluated. For all tested scenarios, wild P. vulgaris was used as an outgroup (H4), and the statistical significance (P < .05) was established after applying a Bonferroni correction to the block jackknife P‐value.The ABBA‐BABA approach is based on a resolved phylogeny among four taxa , H3), H4) and determines if the pattern of derived alleles is consistent with the phylogeny  diverged before the split between H1 and H2 (receptors), scenarios where a cultivar was a donor and a wild population was a receptor were excluded. Therefore, the third gene flow scenario suggested by TreeMix . The candidate regions were then compared with the windows with the highest fd values (5% top) per chromosome.To look for evidence of adaptive introgression, candidate regions under selection were identified in the three traditional varieties in which gene flow from the wild relatives was detected  was 0.05 within each population, 25‐Kb windows and 12.5 Kb step size were fixed, the iHS threshold was fixed at 1.5, and P < 0.05. Genes found in candidate regions were annotated using MapMan4  of each population was constructed using the PLINK allele count function. Because the different SNV categories may be under different evolutionary processes, they provide complementary information. Therefore, we constructed the SFSs according to SNV in CDS, nongenic, and intronic regions. The expected SFS was derived by using the Watterson's estimator θ^W=s∑i=1n−11i. To identify signs of demographic bottlenecks, long Runs of Homozygosity (ROH) per individual were estimated with PLINK using a 500 Kb min window size.To find evidence of demographic processes that have affected Based on the SFSs, the ROHs, and the introgression events found with TreeMix and the ABBA‐BABA test, demographic scenarios were constructed and tested using fastsimcoal2 that uses coalescent simulations to model demographic scenarios from the SFS  generations ago. For Cult‐SMOCC and Cult‐SUR‐CH models, we were interested in gene flow from sympatric wild relatives, even though such sympatric taxa are unlikely to be the closest wild populations. We thus expect that the time of divergence (TDIV) in these models will be earlier than the timing of domestication.Results); therefore, we only modeled the ancestral population size (NANC), the time when a bottleneck started (TBOT), population size during the bottleneck (NBOT), demographic expansion time (TEXP), and the current population size , a demographic expansion occurred , at TEXP (time expansion) generations ago. The wild population size (NWILD) was assumed to be constant through time Fig. . The migWe ran 100,000 simulations with 100 independent replicates for each model. For the best‐fit model for those scenarios with gene flow, the likelihoods of the best runs were compared to estimate the AIC weight. Then we performed 50 bootstraps for the best‐fit model to obtain the mean and 95% confidence intervals for each parameter.P. coccineus , 20 of P. vulgaris, and 35 of P. dumosus were kept. The mean missing data were 1.22%, and the mean depth per site was 31.04×. The SNV dataset contained 79,286 SNVs, of which 11,019 variants were found in nongenic regions (13.90%), 35,429 within introns (44.68%), and 32,838 within CDS (41.42%). Regarding the variants within CDS, 13,738 (41.84%) were predicted as synonymous mutations, 18,392 (56.01%) as nonsynonymous, 541 (1.65%) as frameshift, and 248 as nonsense (0.75%).After mapping, SNV calling, and filtering, 237 individuals of P. coccineus and 71,861 SNVs, with a mean missing data 1.21% and mean depth per site of ∼32×.To reduce the difference in sample size among the populations, a second filtering was applied based on relatedness and missingness. This dataset consisted of 183 individuals of striatus (Wild‐striatus); and two from the Sierra Madre Occidental . The other six populations corresponded to cultivars: from Sierra Madre del Sur (Cult‐SUR‐CH); Oaxaca Valley (Cult‐OV); Trans‐Mexican Volcanic Belt (Cult‐TMVB); Sierra Madre Occidental (Cult‐SMOCC); the Spain cultivar, which was grouped within traditional varieties from TMVB in the ancestry analysis  froP. dumosus and P. coccineus occur in the Southern region of Mexico. We tested for introgression between these two species, and the ABBA‐BABA test supported introgression from P. dumosus to Wild‐SUR‐CH. Finally, the test showed evidence of frequent gene flow among traditional varieties, mainly from the Cult‐TMVB into the other traditional varieties  associated with domestication time around 9700 generations ago, followed by a relatively recent expansion TEXP = ∼1500), and a current population size of ∼759,000  of the three populations (Table The best‐fit scenario for Cult‐SMOCC populations suggested a recent introgression (from 3000 generations ago to present), a less severe bottleneck associated with cultivar spreading NAC = ∼18,000), and a current population size of ∼395,000. For the SUR‐CH, the best model predicted an ancient introgression (from 6000 to 3000 generations ago), a bottleneck that led to an ancestral population size of ∼8500, and a current population of ∼742,000 , and its current population size was estimated to be ∼340,000 Fig. . In contFIS) than wild populations  compared to the other domesticated bean species , even though it is higher than that for common bean, in which it started ∼8000 year ago Gepts , but theOur results suggest that the introgression from wild relatives has only taken place during the last 3000 generations in the sympatric SMOCC populations. Meanwhile, the introgression in the SUR‐CH seems to be older  into a wild population (Wild‐TMVB‐CDMX) was inferred using TreeMix , another important legume crop , followed by the breeding line Blanco Tlaxcala . Two different processes that followed the severe bottleneck in these populations (caused by the introduction to Europe or the sampling to create the breeding line) might explain their relatively low ratio: the bottleneck removed low‐frequency variants, which have the highest nonsynonymous/synonymous ratio, decreasing the ratio, and/or the strong artificial selection possibly resulted in the expression and posterior purge of some deleterious alleles. González et al.  reportedThe greatest estimates of genetic diversity were found in wild populations. Nevertheless, there are wild populations with lower genetic diversity than traditional varieties, such as both Wild‐SMOCC and Wild‐striatus. The expectation that high diversity will be maintained close to the center of domestication and decrease with increasing geographic distance is observed in our data Fig. . This suThe proportion of private alleles is lower in traditional varieties compared to their wild relatives, but still significant. In Cult‐SUR and Cult‐TMVB, private variation represents 33% of their segregating sites. The demographic expansions of the traditional varieties that occurred after the domestication bottlenecks have probably favored the emergence of new variants.P. coccineus as a species.Populations with the highest genetic diversity did not have the greatest private variation. This was the case of Wild‐TMVB‐CDMX. In contrast, the proportion of segregating sites that were private to Wild‐SMOCC‐EspDia and Wild‐SUR‐O was remarkably high (46% for both groups). The presence of private alleles might be at least partially explained by population histories. An ancient population, with high population sizes, would show a high proportion of private alleles  at The authors declare no conflict of interest.Table S1. Phaseolus coccineus sampling material used. “Population” column refers to the 15 defined populations that were the clustering factor in the analysis. All samples corresponded to P. coccineus subsp. coccineus, except a wild population from Tres Marías, Morelos. The last column shows the number of individuals that were kept after variant filtering.Fig. S1. Demographic models that were tested using fastsimcoal2. For TMVB, SMOCC and SUR populations recent, constant and ancestral gene flow were tested. The likelihood for each of these scenarios are shown in the right column. For Cult‐OV and the populations from Spain the severity and time of the bottleneck was estimated. NWILD= Ne wilds; NCC= Current Ne cultivars; NAC= Ancestral Ne cultivars; NBOT= Ne during the bottleneck; TBOT= bottleneck time (generations); TEXP= time of demographic expansion; TDOM= domestication time; TDIV= divergence time.Fig. S2. a) Phylogenetic relationship among individuals of P. coccineus from Mexico. The 15 defined populations are indicated and colors in the ancestry plot do not correspond to the colors of the 15 populations. b) PCA plot for the first two principal components including all samples.Fig. S3. Ancestry plots of the wild and traditional varieties of P. coccineus. The ancestry analysis was performed using the complete data set and the data subset (183 samples). The 15 defined populations are indicated in the extreme right of the ancestry plots.Fig. S4. PCA plots for the first two components of a) cultivated and b) wild populations.Fig. S5. Gene flow scenarios inferred by TreeMix. a) Analysis performed using complete data set, and b) with the data subset consisting of 183 samples.Table S2. Gene flow models tested for cultivated, feral and wild populations of P. coccineus using the ABBA‐BABA approach.Table S3. Gene flow models tested with a sample subset for cultivated, feral and wild populations of P. coccineus with the ABBA‐BABA test. Simple size is indicated with (n) in the corresponding columns.Table S4. Gene flow scenarios tested with Dsuite.Fig. S6. Introgressed regions and under positive selection along the 11 chromosomes from the reference genome of P. vulgaris. The left axis shows the log10 for the selection statistic iHS (blue solid line) and the right axis shows the values for the fd statistic (dotted red line) computed in sliding windows of 25,10 SNVs per window and step. a) Introgression from Wild‐SUR‐CH into Cult‐SUR‐CH, b) Introgression from Wild‐SMOCC‐Rego into Cult‐SMOCC, c) Introgression from Wild‐TMVB‐CDMX into Cult‐TMVB, and d) Introgression from Wild‐TMVB‐Tepoz into Cult‐TMVB. The gray line shows the statistical significance threshold for iHS ( log10(0.05) = 1.3).Fig. S7. Venn diagram showing the number of genes found in the introgressed regions (blue circles) and candidate genes under selection (yellow circles) in the three traditional varieties in which gene flow from the wild sympatric populations was detected.Table S5. Genes found in the candidate regions of the three evaluated traditional varieties. The Donor population column shows if the gene was also presented in the introgressed region identified.Fig. S8. Site Frequency Spectrum (SFS) calculated for each population. Colors indicate the SNV category. Points show the expected distribution.Fig. S9. Total length of ROH (Kb) estimated using a 500 Kb min window size. Colors indicate the type of P. coccineus sample.Table S6. Demographic parameters estimated using fastsimcoal2 (95% CI). NWILD= Ne wilds; NCC= Current Ne cultivars; NAC= Ancestral Ne cultivars; NBOT= Ne during the bottleneck; TBOT= bottleneck time (generations); TEXP= time of demographic expansion; TDOM= domestication time; TDIV= divergence time; REXP= expansion rate; MIGWC= migration rate from wild to cult (NMWC/NWILD); MIGCW= migration rate from cult to wild (NMCW/NAC); NMWC= migrants from wild to cult; NMCW= migrants from cult to wild.Fig S10. Heatmaps showing the genetic diversity in terms of H and inbreeding coefficient estimated for the 15 defined populations using the 237 samples.Table S7. Genetic diversity levels in the P. coccineus populations. Expected (EH) and observed (OH) heterozygosity, and inbreeding coefficient (ISF). The confidence intervals were obtained performing 100 bootstraps. Asterisk shows the estimated values using the data subset (183 samples).Fig. S11. Differentiation index among the 15 populations of P. coccineus. a) was estimated using the complete data set, and b) with the data subset (183 samples).Fig. S12. a) Proportion of private and shares alleles for each population. b) Proportion of private and shared alleles within CDS regions, separating into synonymous and nonsynonymous mutations. The numbers inside the columns indicate the proportion of each category. Only segregating sites within populations are included.Table S8. Proportion of segregating sites (SS) and nonsynonymous/synonymous ratio of SS splitted into the shared and private alleles found within each P. coccineus population.Fig. S13. A) Mean allelic richness per site and (B) mean private alleles per site estimated with AZDE for each population.EH) and the distance from the centroid of the Cult‐TMVB locations to traditional variety locations estimated using the data subset  and the distance from the centroid of the Cult‐TMVB locations to traditional variety locations estimated using the data subset (183 samples).Click here for additional data file.


Oxytropis diversifolia and O. leptophylla in Nei Mongol, China. For 507 individuals sampled from two populations in the hybrid zone, 12 O. diversifolia populations and five O. leptophylla populations, we measured leaf‐morphological characteristics, quantified genetic structure using 11 nuclear microsatellite loci and five chloroplast DNA intergenic regions, collected micro‐ and macrohabitat data, and conducted geographical cline analysis. We found that the two species differed in leaf morphology, and putative hybrids showed either intermediacy or a bias to O. diversifolia. Parental taxa formed two genetically distinct clusters, while populations in the hybrid zone consisted of both parental forms and various admixed individuals, exhibiting a bimodal pattern. The hybrid zone was coupled to ecological transitions of both microhabitat  and macroclimatic conditions. However, the genetic clines were significantly narrower than the environmental cline. Our results indicate that endogenous selection can be primarily responsible for maintaining the hybrid zone, while local adaptation accounts for the position of the zone. We further suggest the probable outcome of hybridization could be introgression.Hybrid zones have been widely highlighted for their interest in understanding evolutionary processes. It is generally accepted that hybrid zones can be maintained in a balance between dispersal and selection. However, the selective forces can either be endogenous  or exogenous . In this study, we evaluated these alternatives and determined the maintenance of a narrow hybrid zone between parapatric distributed  In this study, we analyzed a narrow hybrid zone generated between two parapatric Oxytropis species in Nei Mongol, China. We found that the genetic clines are abrupt and coincident, while the environmental clines are wide or gradual. Our results indicate that endogenous selection can be primarily responsible for maintaining the hybrid zone, and local adaptation accounts for the position of the zone. Both species belong to sect. Xerobia, which is comprised of 27 species , Mongolia and Russia  against dispersing individuals contribute to shaping and maintaining the hybrid zone.In this study, we analyze a secondary contact between parapatric t Figure , 1 to 3 t Figure , or 3 let Figure . Populatt Figure . The clit Figure . Morphols Figure ; Table 1s Figure  in whichO. diversifolia and O. leptophylla. Specifically, we seek to address the following questions: (1) Considering leaf‐morphological traits, what is the phenotypic difference between parental species and putative hybrids? (2) What is the genetic structure of the hybrid zone? Is it unimodal or bimodal? (3) Do micro−/macrohabitat transition showed either coincidence and/or concordance with the genetic clines?Here, we use an integrative approach by combining morphological measurements, molecular data, and environmental association analyses to evaluate the pattern and maintenance of the hybrid zone between 22.1Oxytropis diversifolia E. Peter  is a diploid perennial herb occurring at an elevation from 1000 to 2200 m  DC. is also a diploid perennial herb occurring at a similar elevation ranging from 800 to 1900 m  , O. leptophylla , or of the hybrid zone with signs of intermediacy (N = 2). In each population, leaf‐phenotype survey was performed by tallying leaf‐phenotype counts , or along three to five transects (3 m wide) spanning the length of the population . This survey was used to estimate population size  and population density . Silica‐gel dried leaf samples were collected, using methods described in Wang et al. . The number of trichomes on a 1 mm × 1 mm surface was counted for five (populations dominated by one phenotype) to 14 individuals  in each parental population. In the hybrid zone, 33 and 28 individuals were quantified at localities G and HUA, respectively. In total, we quantified 211 individuals of all the 19 populations.O. diversifolia, O. leptophylla, and putative hybrids) as a fixed effect and population nested within category as a random factor.Because the six leaf morphological traits were correlated with one other, principal component analysis (PCA) was performed. Data were scaled prior to analyses. The variation in six leaf‐morphological traits, the PCA components, and the trichome density were also analyzed separately by performing linear mixed‐effects models, with category  method , observed heterozygosity (Ho), unbiased expected heterozygosity (He) and FIS, were calculated using GenAlEx version 6.503  steps followed by 50,000 iterations. The best‐fit number of distinct groups (K) was determined based on the maximum ΔK value  was determined by Hartigan's dip test  and hybrids, two additional methods were used. First, the hybrid index for all individuals were calculated using the est.h function from the R package INTROGRESS , they were defined as priori to estimate parental allele frequencies. Individuals with hybrid indexes ≤0.20 were assigned to O. diversifolia, between 0.20 and 0.80 were assigned to hybrids, and ≥0.80 were assigned to O. leptophylla. Second, NEWHYBRIDS Version 1.1 beta . Additional parameter settings included a burn‐in of 10,000 MCMC steps followed by 100,000 iterations. A relatively strict posterior probability of 0.95 was used. Results of those two analyses were quite similar to STRUCTURE results, but discordance still existed  was used to edit, align, and concatenate sequences. Haplotypes were identified using DnaSP v6.12.01 , and multilocus nuclear assignments were used . Moreover, several genetic diversity parameters were calculated for each population using DnaSP, including number of segregating sites (S), number of haplotypes (h), haplotype diversity (Hd) and nucleotide diversity (π)  Nei, . A stati(π) Nei,  was cons(π) Nei, .2.72.7.1The microhabitat axes captured habitat attributes at the local scale of individuals within populations. For each individual sampled, a 40 cm × 40 cm square plot was surveyed Figure , slope . In tota2.7.2http://www.cvh.ac.cn), the National Specimen Information Infrastructure , and the Global Biodiversity Information Facility  . After removing wrong identifications, duplicate records and observations, and records without georeferences, in total we got 35 additional records for O. leptophylla. Nineteen contemporary bioclimatic variables of the time period 1970–2000 at 30‐second resolution were downloaded from WorldClim  diverge in present climate conditions, first, one‐way ANOVA on each bioclimatic variable was performed. Among those significantly differentiated variables, three biologically important variables with pairwise Pearson correlation coefficients below 0.7 were then used to conduct PCA, with the entire environmental space built on variable values associated with 10,000 random points sampled from the whole study area.The macrohabitat axes of bioclimatic data were retrieved based on range‐wide occurrences at the coarse scale. In addition to our field sampling localities . The associated plant species that co‐occurring at the 19 localities we surveyed were also noted.Moreover, soil characteristics can also be important. For example, in the European Alps, hybrids between the acid‐loving species 2.8O. leptophylla cpDNA haplotype and microsatellite QK2 scores) and environmental data . Morphological data (leaf‐shape frequency and leaf morphology PC2 scores) were also used to fit geographical cline to test for any effect of divergent selection or morphological introgression.To explore whether endogenous or exogenous selection is primarily responsible for maintaining the hybrid zone, one‐dimensional (1D) cline analysis was performed based on both genetic data (frequency of O. leptophylla cpDNA haplotype was applied to generate the prediction surface in ARCMAP v. 10.2 (Esri), and the position of the cline center in two dimensions were determined , while the proportions of 1–3 leaflets and 3 leaflets exhibited the reverse trend . We occasionally found two individuals with 3–5 leaflets (one in the population DIANO and one in PE), but they were rare. The five populations of O. leptophylla were predominantly monomorphic with the phenotype of 5–13 leaflets, and exceptions were found in BOPO  and H   Figure . In the ) Figure .3.2O. diversifolia, O. leptophylla, and putative hybrids , while the remaining two variables, mature leaf width and petiole length of mature leaf, were highly correlated with co‐ordinates on PC2 . Populations of the two parental species and putative hybrids were shown to differentiate in the PCA plot  rather than PC1 (p = .59)  Table . Additio3.33.3.1O. diversifolia and O. leptophylla is likely due to the self‐compatibility and/or inbreeding  within‐population genotypic disequilibria were detected prior to correction for multiple testing, but were not specific for any pairwise loci or population. After Benjamini–Hochberg correction, only one pair of linkage disequilibrium in population PSEV remained significant Table . SignifiNa, Ho, He) across loci differed significantly between O. diversifolia, O. leptophylla, and the hybrid zone , or the hybrid zone showed similar estimates as populations of O. diversifolia (He).At the population level, the genetic diversity parameters , 42 hybrids (50.0%) and 27 O. leptophylla (32.1%) , and this remained true when G and HUA analyzed separately .Across the two localities in the hybrid zone, the 84 individuals examined comprised 15 tophylla 2.1% (Tab%) Table . No signtophylla 2.1% , but there was no difference for the floral phenotype of hybrids to be either parental species .Concerning the morphology of individuals in the hybrid zone, most individuals genetically assigned as F1s, F2s, backcrosses to O. diversifolia, and backcrosses to O. leptophylla. No individual detected had greater than a 9.5% chance of being classified as F1s  or the hybrid zone showed similar estimates as populations of O. diversifolia   Table .O. diversifolia haplotypes  or O. leptophylla haplotypes   Table . Of thes%) Table : 17 poss3.53.5.1SD) were intermediate between the two parental species   in the hybrid zone   possessed Calcisols, and five localities to the east  were Kastanozems; the two localities in the hybrid zone were also Kastanozems; For O. leptophylla, the two soil types most common were Kastanozems  and Chernozems  , while those in O. leptophylla were almost haplic Kastanozems (KSh) and luvic Kastanozems (KSl)  Figure . Kastano) Figure . Similar) Figure .3.6R2 = 0.753, p < .0001), and localities of the hybrid zone possessed intermediate values .We plotted the maximum‐likelihood cline for the best‐fit model for each morphological, genetic, and environmental variable Figure . All vars Figure . Populats Figure . Howeverk = 27, AIC = 285.6 vs. InLikelihood = 525.5, k = 29, AIC = −993.0). However, a model in which clines of cpDNA haplotypes and microhabitat constrained to the same center exhibited a better fit . These results indicated that genetic clines and the environmental cline were coincident, but genetic clines were narrower than the environmental cline. Moreover, we found that the leaf‐shape cline showed shifted cline center when compared with genetic clines .We then tested for cline coincidence and concordance based on cpDNA haplotypes, nuclear microsatellite genotypes and microhabitat data. A model in which three clines were constrained to have the same width did not fit the data as well as a model in which the cline centers and widths were allowed to vary independently  and macroclimatic conditions. However, the genetic clines are significantly narrower than the environmental cline. These results indicate that endogenous selection can be primarily responsible for maintaining the hybrid zone, while local adaptation accounts for the position of the zone. Below we discuss the structure of hybrid zone in detail and tentatively propose potential effects of hybridization.Hybrid zones generated through secondary contact between closely related species have been widely highlighted for the interest in understanding the evolutionary processes , the narrow width relative to the dispersal ability . Although the floral structure of both species is quite the same and they can be bee‐pollinated, the difference in flower color is obvious . Because both species are not rewardless , the distinct flower color can somehow prevent interspecies pollen transfer.A pronounced feature of the hybrid zone detected in our study is the bimodal pattern of genotypic distribution. Historical literature review suggests that both bimodal and unimodal hybrid zones have been shown to exhibit postzygotic isolating mechanisms, which may be either intrinsic or extrinsic. However, only bimodal zones are associated strongly with prezygotic barriers .Moreover, the bimodal distribution seems to be asymmetrical. Asymmetric hybrid zones occur when strong asymmetries in gene flow and barrier strength promote hybridization and introgression in one direction, such that gene flow occurs only from one species to another  and/or whole‐transcriptome sequencing (RNA‐Seq) is necessarily important.In summary, we have found evidence of bimodal hybrid zone connecting two parapatric Hui Wang: Conceptualization ; data curation (lead); formal analysis (lead); funding acquisition (lead); resources ; supervision (lead); writing – original draft (lead); writing – review and editing (lead). Xin‐Nuo Li: Data curation (supporting); formal analysis (supporting); investigation . Song‐Hua Mo: Data curation (supporting); formal analysis (supporting); investigation . Min Wang: Investigation . Pei‐Liang Liu: Conceptualization ; formal analysis (supporting); resources . Qin Li: Data curation (supporting); formal analysis (supporting); writing – original draft (supporting); writing – review and editing (supporting). Zhao‐Yang Chang: Conceptualization ; resources ; supervision (supporting).The authors declare no conflict of interest.SupinfoClick here for additional data file.TableS4 ‐ S11Click here for additional data file.


Bulweria bulwerii and provide new insights regarding the drivers of species diversification in the marine environment. We sampled Bulwer’s petrels from the main breeding colonies and used a statistical phylogeography approach based on surveying nuclear and mitochondrial loci (~ 9100 bp) to study its mechanisms of global diversification. We uncovered three highly differentiated groups including the Western Pacific, the Central Pacific and the Atlantic. The older divergence occurred within the Pacific Ocean, ca. 850,000 ya, and since then the W Pacific group has been evolving in isolation. Conversely, divergence between the Central Pacific and Atlantic populations occurred within the last 200,000 years. While the Isthmus of Panama is important in restricting gene flow between oceans in Bulwer’s petrels, the deepest phylogeographic break is within the Pacific Ocean, where oceanographic barriers are key in driving and maintaining the remarkable structure found in this highly mobile seabird. This is in contrast with the Atlantic, where no structure was detected. Further data will provide insights regarding the extent of lineage divergence of Bulwer’s petrels in the Western Pacific.The patterns of population divergence of mid-latitude marine birds are impacted by only a few biogeographic barriers to dispersal and the effect of intrinsic factors, such as fidelity to natal colonies or wintering grounds, may become more conspicuous. Here we describe, for the first time, the phylogeographic patterns and historical demography of Bulwer’s petrel  Mechanisms of population divergence of seabirds with pan-tropical distributions however are not as well understood11. Historical vicariant events, such as the emergence of the Isthmus of Panama in the late Pliocene, can create phylogeographic structure in species that previously occupied a single region, effectively isolating lineages and promoting speciation15. Other oceanic features, such as the Eastern Pacific Barrier, are also emerging as important barriers to gene flow in a variety of taxa16, despite the ability of marine species to disperse long distances. Changes in the oceanic environment driven by historical climatic changes have also been proposed as important drivers of divergence in marine taxa18, although their effect in mid-latitude species is not as clear.Reconstructing the diversification history of species and identifying the main drivers of population differentiation in widely distributed species is key to understand population connectivity and how biodiversity is generated and maintained, particularly in the face of a rapidly changing world. Empirical statistical phylogeographic studies have been unveiling the primary drivers of global diversification of marine species, particularly those of high latitudes21.Cryptic, intrinsic barriers, such as strong philopatry, which is characteristic of most pelagic seabirds, phenological differences driven by variation in non-breeding distributions and adaptation to local environmental conditions, may also play an important role on population divergence, sometimes even in the presence of gene flowBulweria bulwerii is a useful model to study the impact of physical and non-physical barriers in the diversification history of marine species. This small (ca. 100 g), all-dark procellariiform species is distributed in the tropical and sub-tropical oceanic islands of the Pacific and Atlantic oceans. It has also been recently discovered breeding on Round I. off Mauritius in the Indian Ocean, but the population is very small22. The species occurs in deep sea year-round25. It breeds on small oceanic islands for close to five months of the year, a period during which it behaves as a central place forager due to the constraints of incubation of the single egg and chick provisioning25. Individuals perform long migrations between their breeding and non-breeding areas. In the Atlantic, wintering areas include the southern equatorial regions24. In the Pacific, wintering areas are not as well known, but W Pacific populations are thought to migrate SW into the Indian Ocean, while Central Pacific populations are thought to move SE26. Like most procellariiform seabirds, the species is philopatric27. Although the Bulwer’s petrel population in the NW Hawaiian Islands was thought to be the largest with almost 100,000 pairs26, a recent estimate of the population on the Desertas Islands, Madeira, suggests that the population of this species in Madeira might be almost as large28. Elsewhere, populations are smaller22.Bulwer’s petrel 29. In particular, if we consider their tropical regions, the Atlantic Ocean is a much smaller and enclosed basin with a single predominant bio-oceanographic domain. Conversely, there are at least three recognized biogeographical zones in the much larger Pacific basin characterized by very different biological communities29 which might promote isolation and differentiation of seabird populations.Using a gene set of 13 anonymous nuclear loci and one mitochondrial locus and coalescence theory-based methods, we studied for the first time the phylogeography and historical demography of Bulwer’s petrel across its main breeding areas in the Atlantic and Pacific Oceans. We assess the level of genetic differentiation and gene flow and provide temporal estimates for population divergence, testing for a possible effect of biogeographical barriers in the evolutionary history of the species. The Atlantic and Pacific Oceans have very different geographical configurations and oceanographic characteristics which determine the complexity of the different biogeographic domains of the two basinsA total of 9096 bp were surveyed per individual, including 979 bp of the mitochondrial locus and between 511 and 684 bp of each of the thirteen nuclear loci sampled. (Table π = 0.059)  and 0.722 (Bubu 4) and were significant for all loci. Population pairwise tests (based on Φst) for the mitochondrial locus suggests a significant genetic differentiation between ocean basins and also between the western and central population of Bulwer’s petrels in the Pacific Ocean  the alleles sampled in Japan were not shared by any other population  indicating two clearly different genetic clusters, one including all individuals sampled in the Japanese islands, and a second including those of the remaining populations sampled (median posterior probability = 0.99). When STRUCTURE was re-run on the Central Pacific and Atlantic populations alone, it further suggested two genetic clusters corresponding to these two groups, although the Atlantic cluster includes a few individuals with an admixed ancestry  and the largest Ne for the ancestral population of the Central Pacific and Atlantic groups, and for the current Central Pacific group, both estimated at approximately 30,000 individuals (Table t0 peaked at 0.110 and t1 peaked at 0.585 (95% HPD not estimated with confidence), suggesting that the Central Pacific and Atlantic groups diverged ~ 160,000 ya, while their ancestor and the Western Pacific group diverged much earlier   from Vila islet (Azores) that had been previously captured and ringed on the Desertas Islands, which currently hold the largest population of Bulwer´s petrels in the Atlantic28.Other factors may promote population genetic structure in seabirds, including differences in non-breeding areas39, the Bulwer’s petrels from this archipelago (at least those from Raso Islet) do not appear to be genetically distinct from their more northern conspecifics. Although our sample size for this population is very small, there is clearly no genetic differentiation for the loci sampled, including the faster evolving NADH2. A larger panel of nuclear SNPs should be used to study in detail current levels of population connectivity between the Atlantic archipelagos. It would be interesting in the future to include individuals from Cima islet in Cabo Verde, where the breeding phenology of the species is very different23.Remarkably, and contrary to the pattern found for most other seabird species breeding in Cabo Verde, which holds several endemic ProcellariiformesB. bulwerii on either side of the Isthmus of Panama. Petrels from the Hawaiian and Atlantic islands share no mitochondrial haplotypes, and even show significant pairwise differentiation at a few nuclear loci, in a pattern common to many other co-distributed seabirds39. Such replicated phylogeographic patterns across co-distributed taxa suggests similar evolutionary processes in response to common barriers, clearly indicating that the Isthmus of Panama plays a fundamental role in the phylogeography of species with historical distributions in the Atlantic and Pacific. However, in many cases, divergence times suggest that the Pacific and Atlantic populations diverged well after the emergence of the Isthmus of Panama, ~ 3 MYA. Even though continental land bridges represent obvious barriers to dispersal for taxa that do not usually fly over land, the historical demography of many species suggests that this barrier is somewhat permeable, and most species are able of rare dispersal over land11.There is a clear phylogeographic break between the populations of 40. The mechanisms by which the glaciation cycles influenced the phylogeographic structure of highly vagile, tropical and sub-tropical seabirds are not well understood. However, the sequential glacial and interglacial periods of the Pleistocene had severe consequences in the global atmospheric conditions and paleo-oceanographic environments41, which likely had profound effects on seabird distribution, dispersal patterns, establishment of new populations and extinction of others42.Interestingly, for many pelagic species including the Bulwer’s petrel, estimates of divergence times suggest that many populations separated during the last 500,000 yearsWe describe patterns of genetic differentiation across the main range of Bulwer’s petrel, identify the main phylogeographic breaks and reconstruct its demographic history. Although the Isthmus of Panama has functioned as a barrier to gene flow, we found that oceanographic barriers may also represent critical drivers of diversification in highly pelagic seabird species, despite their ability to travel long distances. In a rapidly changing world, understanding the mechanisms of divergence and speciation at mid-latitudes can provide insights regarding future biological consequences of global change.39. Further genomic, morphological and behavioural data of Bulwer’s petrels from the Ogasawara Islands should be collected as our data argue for a re-examination of the taxonomic status of the species in the Pacific43.The taxonomy of petrels and shearwaters has often been obscured by the lack of obvious morphological variation even between genetically distinct groups44 solution following standard phenol–chloroform protocols. We screened for polymorphisms in thirteen anonymous nuclear loci isolated from a genomic library made originally from B. bulwerii, as described in30. We also surveyed a fragment of the NADH2 locus using primers L521545 and H106446, as the more frequently used Control Region showed a pattern of variation suggesting significant homoplasy (data not shown). Amplification of this fragment was carried out in 25 µl volume reactions containing the same final reagent concentrations as were used with the nuclear loci30, as well as the same thermocycler profile but with an annealing temperature of 58 °C. All loci were sequenced in both directions.We sampled 105 Bulwer’s petrels from the main breeding localities of the species Fig. . SpecifiSEQUENCHER v.4.2 (Gene Codes Corporation), which was also used to check the validity of the base calls. Seven loci displayed heterozygous length polymorphisms, and all gaps from the data sets were trimmed. The phase of nuclear alleles with multiple heterozygous SNPs were resolved using the Bayesian algorithm implemented in PHASE v.2.1.147. Input files were prepared for PHASE using the software SeqPHASE48.Sequences were assembled into contigs, aligned and edited in DNAsp v5.1049. We used ARLEQUIN v.3.5.2.250 to detect departures from neutrality for each population, by estimating Tajima’s D51 and Fu’s FS52 for each locus. The net pairwise sequence divergence between pairs of populations was calculated using MEGA 553 with the model of sequence evolution selected by the AIC in jModelTest54. Population structure among the three taxa was investigated by estimating global and pairwise Фst for all loci, using the Kimura’s-two parameter model of substitution, as implemented in ARLEQUIN. We tested the significance of results with 10,000 random permutations, considering a significance level of 0.05 after correction for false discovery rate. Median-joining haplotype networks, based on the largest blocks of non-recombining fragments, were estimated for all loci using NETWORK v.4.6.0.055 (https://www.fluxus-engineering.com/sharenet.htm).Polymorphism statistics, such as the number of segregating sites, haplotypic and nucleotide diversities were determined using STRUCTURE 2.356 (https://web.stanford.edu/group/pritchardlab/structure.html). We ran two sets of analyses depending on how the data was formatted: the first set used allelic data for each of the nuclear locus, and the second used the dataset converted to SNPs. There were no differences in results between the two sets and we present results only for the former dataset. In the final analysis, In STRUCTURE, colony of origin was not used as prior information, and analyses assumed an admixture model and correlated allele frequencies among populations. We performed 10 replicate runs for each K value, which varied between 1 (single population) and 6. Each replicate was run for 1 million iterations, with a burn-in of 100,000 iterations. The value of K that best fitted the data was estimated using the method of31 implemented in STRUCTURE Harvester57. Since this method tends to recover the highest hierarchical level of population structure31, we re-ran STRUCTURE to test for further structuring, using the same run parameters as above.To detect population structure without the need to define a priori the populations, we used Bayesian clustering analyses, as implemented in BEAST algorithm based on the multi-species coalescent58 as implemented in the software BEAST 1.759 to estimate a species tree for the sampled taxa, using the mitochondrial and nuclear loci. The recovered topologies of the tree were the same across replicates. As most species trees methods, *BEAST requires a priori assignment of alleles to taxa. We used the well-supported and geographically structured mtDNA network for prior assignment. The input was prepared with BEAUti v1.7 (http://evomics.org/resources/software/molecular-evolution-software/beauti/). We ran *BEAST multiple times to test different priors and settings, and to assess convergence and mixing. The results shown are based on final runs which ran for 109 generations , sampling every 10,000 generations, with a burn-in of 106 generations. We chose the most appropriate model of sequence evolution for each locus (see above), used a strict molecular clock, which could not be rejected for this dataset, and a Yule process for the species tree prior, and other priors were changed according to operator weight suggestions from previous runs. We monitored convergence of the sampling process using TRACER 1.560, ensuring that the ESS values were above 200 and parameter posterior distributions were unimodal. Trees were summarized in TreeAnnotator v.1.761 and visualized in FigTree 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). We assessed nodal support using posterior probability estimates. We used a substitution rate for the NADH2 locus of 0.028 × 10–646, and the nuclear rate was allowed to be estimated relative to the mtDNA rate.We used the *IMa262 using the full data set of twelve anonymous nuclear and NADH2 loci. IMa2 implements a coalescent-based method that uses Markov Chain Monte Carlo sampling of gene genealogies to estimate population parameters scaled by the mutation rate per locus per generation (μ), such as contemporary and ancestral effective population sizes  migration rates  and divergence times  among populations. Implementation of IMa2 with more than two populations requires a known sequence of splitting events, and accordingly we used our *BEAST species tree. It also assumes no recombination within loci, and we used IMgc63 to find the largest block of sequences and individuals with no evidence of recombination and used these data in our IMa2 analysis. We ran IMa2 several times, varying the prior bounds, heating schemes and number of chains, to optimize the priors, improve mixing of the chains and reach convergence of the different analyses. The final runs included 80 independent MCMC chains with a geometric heating scheme . All runs included 15 million generations with 500,000 generations discarded as burn-in. Trend plots, ESS values and comparisons between coupled runs were used to assess mixing and convergence, as suggested in the documentation for IMa2. To translate parameter estimates  to demographic units, we used a substitution rate for NADH2 of 0.028 × 10–646, and a mean generation time of 12 years64.We inferred the demographic history of Bulwer’s Petrel with Ethical statement. The volume of the blood sample collected from each petrel was 50–75 μl (approximately 0.05% vol/weight). Sampling were performed in accordance with the guidelines and regulations of each of the relevant institutions, including the Instituto das Florestas e Conservação da Biodiversidade, Portugal (permit No. 2/2012S), the Direção Regional dos Açores (permits nº4/CN/2002 and nº7/CN/2003), the US Fish and Wildlife Services and the Papahānaumokuākea Marine National Monument (permit No. PMNM-2008-029) and the Ministry of the Environment, Japan (permit No. 080428001). Sampling further followed the requirements of the Directive 2010/63/EU of the European Parliament and of the council for the protection of animals used for scientific purposes.Supplementary Information.


Populus alba  and Populus davidiana (P. davidiana) are important plant species for answering a variety of issues on species evolution due to their wide distribution and ability to adapt to a variety of environments and climates. Even though P. alba and P. davidiana belong to ecologically and economically important forest trees in the Northern Hemisphere, little is known about their genomic landscape and genome divergence during speciation. We re-sequenced 20 and 19 members of P. davidiana and P. alba, respectively, and found that the Dxy value between P. alba and P. davidiana was 0.2658, whereas the FST values were 0.2988, indicating that the genetic divergence was fairly clear. Populus davidiana and P. alba diverged from the ancestor in the middle Pleistocene, c. 0.80 Ma (95% HPD: 0.79–0.81 Ma). The population sizes of P. davidiana increased ~20,000 years ago after a considerable long-term decline following divergence. However, after differentiation, the effective population size of P. alba expanded slightly before experiencing a long-term bottleneck effect. According to the expectation of allopatric speciation, we found a significant number of genomic differentiation sites in both species' speciation events, and the majority of these genomic differentiation regions can be attributed to neutral evolutionary processes. Nevertheless, the regions with extreme divergence exist in abundance, indicating that natural selection has had an impact. Positive selection can be found in highly differentiated regions, while long-term balancing selection traits can be easily observed in low differentiated regions. According to these findings, climate differences over the Quaternary, as well as variance in linked selection and recombination, all contributed significantly to genomic divergence during allopatric speciation of the two aspens. One of the fundamental goals of evolutionary genomics is to understand the relative contributions of diverse evolutionary factors in the generation and shaping of genetic diversity within and among species  to evaluate the evolutionary forces affecting patterns of genome-wide variation. The two species are all from the section Leuce Duby of the genus Populus. Populus alba and P. davidiana are the most ecologically important forest trees in China, as well as ecologically important members of mesic forest ecosystems that were isolated by Pleistocene glaciations   explore their population structure; (ii) estimate the historical demographic processes and species divergence time points; (iii) infer the overall patterns of heterogeneous genomic differentiation and fine-scale genomic landscapes of diversity; (iv) identify signatures of long-term balancing selection and positive selection over the entire genome. Specifically, the main purpose of this study is to understand and disentangle how the patterns of genetic variation are shaped by the multitude of evolutionary forces within and among species during the process of speciation.Both  Tuskan, , the twoP. alba and 20 P. davidiana individuals covering their geographical distributions in China were collected for DNA extraction  to remove reads with identical external coordinates and insert lengths. For downstream analyses, we only retained the reads with the highest summed base quality. We further filtered the reads that could potentially cause mapping bias according to three additional standards: (1) those with extreme read coverage (<4 or higher than twice of the mean coverage); (2) RepeatMasker detecting overlapping known repeated elements   was used to perform the Principal component analysis (PCA) with a Tracy–Widom test to ascertain the significance level of eigenvectors. The neighbor-joining (NJ) model with TreeBest software (http://treesoft.sourceforge.net/treebest.shtml) was used for the construction of the phylogenetic tree.NGSadmix was implemented to deduce the genetic structure of the −8 per base per generation, and we adopted a generation time of 15 years. We carried out 100 bootstrapping simulations to estimate the variance fluctuation of population size  from each population and applied the Pairwise Sequentially Markovian Coalescent (PSMC) model to reconstruct demographic history , which was estimated with 100,000 coalescent simulations in each model. Alternative models of historical events were fitted to the joint site frequency spectra data  No Migration and no population expansion; (2) Asymmetric Migration but no population expansion; (3) Asymmetric Migration along with population expansion; (4) complicated model, comprising a bottleneck in P. alba; (5) complicated model, containing a bottleneck in P. davidiana , nucleotide diversity (π), and Tajima's D for individual species. We then took an average of (within each 10 Kbp non-overlapping window) FST, π, and Tajima's D values of all sites  to those expected under different demographic models (see Results). All simulations were performed using the program msms v3.2rc based on demographic parameters derived from the best-fitting model inferred by fastsimcoal2.5.1. Population-scaled recombination rates ρ were assumed to be between 1 and 5 Kbp−1 given the large variation. We simulated genotypes corresponding to a 10 Kbp region with the same sample size as the real data for 100,000 replications, from where we simulate genotype likelihoods using the program msToGlf in ANGSD  of observing more extreme inter-specific FST values among simulated data sets than among the observed data. The determination of significance was based on running 500,000 coalescent simulations of the most acceptable demographic null model. We then corrected for multiple testing by using the False Discovery Rate (FDR) adjustment, and only windows with FDR lower than 1% were considered as candidate regions affected by selection.In order to examine thresholds for detection of outlier windows that may have been targets of natural selection, we conducted coalescent simulations to compare observed patterns of genetic differentiation  for evaluating the levels of LD within each 10 Kbp window, we used VCFtools v0.1.12b  dividing the dxy of the P. alba and P. davidiana species by the dxy between the P. alba population and P. tremula to estimate the relative node depth (RND); (3) the posterior probability of the sample allele frequency at each locus and averaged over each 10 Kbp window was used to calculate dxy. Wilcoxon ranked-sum tests were used to analyze the genome-wide mean for all population genetic data presented, as well as the significance of differences among outlier windows.In both u and Li  and Fay u and Li . Furtherhttp://bioiSo.cau.edu.cn/agriGO/index.php)  Muled 31.86 . FollowiK = 3, P. alba showed additional sub-structuring of the population, but the bulk of P. alba members were deduced to be a product of two genetic components mixing, demonstrating very minor clinal variation with increasing latitude. When K = 4, hybridization and introgression can be found between P. alba and P. davidiana  for the associated variables alongside. Around 0.80 million years ago (Mya), the progenitors of the two aspens underwent divergence into P. alba and P. davidiana populations (bootstrap range [BR]: 0.79–0.81 Mya). Currently, the functional population sizes (Ne) of P. davidiana (Ne−P.davidiana) and P. alba  are 1,882,692  and 2,663,695  respectively. However, both species have a significantly low effective population sizes in comparison to their common ancestor . The rate of migration (m) also distinguishes between the two species, the least generation migration rate (m) between P. alba and P. davidiana (3.97 × 10−8 and 9.16 × 10−7), which is not accidental as the two populations are separated by a significant geographical distance.The demographic histories and timings of divergence of the two species were further estimated by employing a coalescent simulation-based technique carried out in Fastsimcoal 2.6.1 , genetic differentiation was found to be evident among the two populations. Total sequence differentiation among the populations which stands as a universal criterion for evaluating interspecific differentiation and is referred to as dxy was also calculated. The two populations were found to have evident sequence differentiation among them, with the mean value of dxy between P. davidiana and P. alba being 0.2658 (Table 5 and We have previously shown that linkage disequilibrium (LD) decays within 10 kilobases (Kbp) in both FST with that obtained from simulations based on the best-fitting demographic model, we identified 379 and 1,156 outlier windows exhibiting significantly  high and low interspecific FST compared to the expected null distribution obtained from the coalescent simulations , whereas the highest HE was harbored by individuals of P. alba. In light of the results of HE, relatively lower genetic diversity (π = 0.0089) is exhibited by P. davidiana in comparison to P. alba . P. alba possesses had a slower LD decay and a slightly higher recombination rate (Table 5) compared to P. davidiana (P. davidiana (Tajima's D = 0.13), P. alba (Tajima's D = −0.06) has a lower Tajima's D-value. The physical distance after the average LD coefficient r2 decays to half of the maximal value is generally referred to as the LD decay distance. Different LD decay curves are manifested by the P. alba and P. davidiana populations . For both populations, the highly differentiated regions manifested distinct positive selection characteristics  . The more negative Fay & Wu's H values reflected frequently appearing derived alleles , whereas the more negative Tajima's D values reflected frequently appearing rare alleles . A more visible characteristic is the regions with high differentiation having powerful signals of linkage disequilibrium (LD)  . Inter-specific shared polymorphisms among the two populations as well as the alleles fixed in the P. alba and P. davidiana populations were also compared. The results indicated that in the regions with high differentiation, the ratio of inter-specific shared polymorphisms was very low   . Frequenhism (π) , with LDhism (π) , possiblhism (π) . In the hism (π)  whereas ulations .Nec) over a 10,000 bp window size. Since ρ = 4Nec, a decline in Ne in an area linked to selection decreases the local estimate of ρ. In an attempt to get rid of this impact, the influence of recombination rate upon genomic differentiation was evaluated by estimating ρ/θπ in regions with high or low differentiation. Particularly, a notable negative correlation was found between the rate of recombination and FST.Highly differentiated genomic regions exhibited a very low rate of recombination, whereas a low differentiated genomic region had a much higher rate of recombination , and 34 significantly overrepresented GO terms were found for genes located in zones with high genetic differentiation. The majority of these GO categories were associated with stimulus-response, catalytic activity, and positive regulation of biological processes  and the FST values corresponding to P. davidiana and P. alba were around 0.2988 , thus driving the divergence of species. Gene flow had been impeded among populations due to geographical isolation  that took place around 20,000 years ago . A varieFST values  that rely on within-species diversity are expected to be higher in regions with restricted recombination   . The powhism (π) , indicathism (π) . SelectiFST and increased genetic diversity   . Balancisity (π) . The fresity (π) , with lesity (π) . The prosity (π)  in both gligible . While sgligible . Our resgligible  or “trengligible  of co-evgligible .P. davidiana and P. alba. The genetic differentiation between the two species was found to be obvious, with an FST value of 0.2988 for P. davidiana and P. alba. Coalescent simulations suggest that the divergence of the two species occurred in the middle Pleistocene, with only minimal gene flow after the divergence. Following divergence, the two species encountered a significant long-term bottleneck, with population increase beginning roughly 20,000 years ago, at the end of the last glacial maximum. Though neutral processes explain the vast majority of regions of genomic differentiation between the two species, a few outlier regions that are noticeably influenced by natural selection have also been investigated. The highly differentiated regions of both species showed substantial positive selection characteristics, and long-term balancing selection was also observed in the regions with limited differentiation in both species. Our results point out that Natural selection and Quaternary climate changes both played a role in the divergence of the P. davidiana and P. alba populations.Here we provide insights into the speciation and recent evolutionary histories of two closely related forest tree species, http://bigd.big.ac.cn/gsa), accession number CRA003302 and CRA001674.The data presented in the study are deposited in the Genome Sequence Archive repository , China West Normal University (Grant No. 20B007 and 19E044), the Open Fund of Ecological Security and Protection Key Laboratory of Sichuan Province, Mianyang Normal University (Grant No. ESP2004), and the Open Fund of MOE Key Laboratory of Biodiversity and Ecology Engineering, Beijing Normal University (Grant No. K202001).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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S. foetida and S. waldsteiniana in the eastern European Alps, was investigated to study the genomic structure of populations within and outside their contact zone and to analyze congruence of morphological phenotypes with genetic data. Eleven populations of the two species were sampled across the Alps and examined using phylogenetic network and population genetic structure analyses of RAD Seq data and morphometric analyses of leaves. The results showed that a homoploid hybrid zone between the two species was established within their sympatric area. Patterns of genetic admixture in homoploid hybrids indicated introgression with asymmetric backcrossing to not only one of the parental species but also one hybrid population forming a separate lineage. The lack of F1 hybrids indicated a long‐term persistence of the hybrid populations. Insignificant isolation by distance suggests that gene flow can act over large geographical scales. Morphometric characteristics of hybrids supported the molecular data and clearly separated populations of the parental species, but showed intermediacy in the hybrid zone populations with a bias toward S. waldsteiniana. The homoploid hybrid zone might have been established via secondary contact hybridization, and its establishment was fostered by the low genetic divergence of parental species and a lack of strong intrinsic crossing barriers. Incomplete ecological separation and the ability of long‐distance dispersal of willows could have contributed to the spatial expansion of the hybrid zone.Natural hybridization of plants can result in many outcomes with several evolutionary consequences, such as hybrid speciation and introgression. Natural hybrid zones can arise in mountain systems as a result of fluctuating climate during the exchange of glacial and interglacial periods, where species retract and expand their territories, resulting in secondary contacts. Willows are a large genus of woody plants with an immense capability of interspecific crossing. In this study, the sympatric area of two diploid sister species,  We detected a hybrid zone of willows in the European Alps by using population genomics and morphometrics. Introgression and broad geographical scale characterize this hybrid zone. HybridiAreas where hybridization naturally occurs are described as hybrid zones , between 30 and 18 thousand years ago   Argus, . Willows) Argus, . In EuroSalix species of section Arbuscella   Does hybridization occur between the two sister species? (ii) Is hybridization only local or is the hybrid zone larger than previously anticipated? (iii) What is the genetic structure of those hybrid zone populations and what are the evolutionary processes involved ? (iv) Can analyses of morphological traits unravel hybrid phenotypes, distinguish them from parental ones, and are they concordant with genetic structure among individuals/genetic ancestry of individuals?The scope of this study was to investigate the putative hybrid zone of 22.1S. foetida, S. waldsteiniana, and putative hybrids were sampled over the species' distribution area in the European Alps. In total, 102 individuals were included in the study representing five populations of S. foetida, five populations of S. waldsteiniana, and two intermediate hybrid populations. For details, see Figure In the years between 2014 and 2020, leaf samples of 2.2PstI restriction enzyme followed by sequencing on an Illumina HiSeq 2500 platform .Between 10 and 20 mg of silica‐dried leaf material per individual was extracted using the DNeasy Plant Mini kit  following a modified manufacturer's protocol. The volume of AP1 buffer was adjusted between 400 and 450 μl, depending on the quantity of leaf material used in the extraction, the volume of RNase A was decreased to 2.5 μl, and incubation at 65°C was 30 min long. Incubation on ice was increased to a minimum of 30 min. The final elution was done using two times 50 μl of AE with an incubation time of 30 min at room temperature, each resulting in 100 μl DNA extract. The DNA quantity was measured with a Qubit 3.0 Fluorometer . DNA extracts were sent to Floragenex Inc. , for single‐end RAD‐seq library preparation using the 2.3FastQC (v.0.11.9)   required to create a stack was set to 10, the maximum number of nucleotide mismatches (M) allowed between stacks within individuals to merge the stacks into one locus was set to 3, and finally, the maximum number of nucleotide mismatches (n) allowed between stacks between individuals to merge the stacks was set to 3. Further filters were applied to remove problematic aspects of the full RAD‐loci assembly, such as missing data, non‐informative loci, and linkage disequilibrium. In ipyrad, the parameter for minimum samples per locus was set to 81, which roughly corresponded to 80% of all individuals. An additional ipyrad assembly, with an inclusion of two outgroup S. viminalis samples . The amount of missing data was minimized by the use of min‐maf flag set to 0.05, where loci appearing less frequently than 5% were discarded. Potentially paralogous loci, putatively consequential to a recent salicoid duplication event  . Less informative parental loci  that were out of Hardy–Weinberg equilibrium (HWE) at p‐value < 0.05 were extracted to a blacklist in the same manner  are not to be expected. Because several analysis tools require unlinked markers, the write‐single‐snp flag was used where only the first SNP per locus was kept. To ensure the reproducibility of the results, the option of keeping the first SNP per locus was made. Next, the appropriate outputs (generated by the plink flag) were used in PLINK (v.1.90)   . Phylogenetic networks extend phylogenetic trees and can more explicitly represent the reticulate evolutionary history of taxa in processes like hybridization   hybridization events, and values between 0.2–0.4 and 0.6–0.8 hint to introgression or ancient hybridization events. The data can be tested for populations or individuals as specified in a population map. For this analysis, the complete RAD‐loci alignment of the ipyrad assembly with outgroups was used. The population map defined all parental individuals from populations outside the contact zone as either S. waldsteiniana or S. foetida population, and each hybrid individual was assigned its own group. A file of 55 triplets was created to test each individual within the contact zone as a hybrid of both parental species, where S. foetida population was assigned as the first parent and S. waldsteiniana as the second parent. Triplets with significant hybridization  were tested with 100 bootstrap replicates, using bootstrap_hyde.py script. The average γ‐values of the replicates were visualized with ggplot2 (v.3.3.5) package   building approach with a GTR + Γ model of nucleotide substitution was used in the software Wickham,  in R  , and has probably undergone a recent drastic size reduction and genetic bottleneck. To avoid bias due to strong departure from HWE       image analysis software . The ratios between length and width, as well as length and widest point, were calculated to quantitatively represent the shape of each leaf. A higher length‐to‐widest point ratio describes an ovate or lanceolate (in combination with a high length‐to‐width ratio) leaf shape and a lower ratio describes an obovate leaf shape. In total, it was possible to obtain 781 leaves of sufficient quality for morphometrics, adding up to approximately eight leaves per individual. A multivariate analysis approach was used to investigate several morphometric variables in a single analysis. Based on the correlation inspection performed in R with the cor function, four independent metrics  were selected for the multivariate principal component analysis (PCA), which was performed with the function prcomp and visualized with ggbiplot (v.0.55)  , as assigned in the sNMF analysis for K = 2.Morphometric measurements were applied to the 95 herbarium vouchers of the collected specimens. To represent an individual's average leaf, up to 10 mature leaves per herbarium specimen were selected. Each individual leaf was positioned on a blank paper sheet with a 10 × 10 mm black square as a scale, and scanned with a Canon CanoScan LiDE 220  scanner, producing a high‐quality image. The number of teeth on the perimeter of each leaf was counted manually, and parameters length, width, widest point (distance from the base of the leaf to the widest part), perimeter, and area were measured on the scanned images with .55) Vu,  in R. Di33.1ipyrad run on 102 samples generated 47,297 RAD‐loci with 315,576 SNPs with 6.3% of missing data. The final ipyrad assembly including outgroup consisted of 47,899 RAD‐loci with 336,649 SNPs and 6.6% missing SNP data. The final STACKS assembly generated 50,924 RAD‐loci with 329,518 SNPs, comparable to the outcome of the ipyrad assembly. The final STACKS assembly with unlinked SNPs used for most population genetic analyses consisted of 6173 SNPs with 4.4% missing SNP data.On average, 6.94 million reads were assembled per individual. No population significantly deviated from the mean. The 3.2S. waldsteiniana), formed well‐supported clusters. The populations of S. foetida and S. waldsteiniana sampled outside the contact zone were positioned farthest apart, indicating the two most diverging monophyletic clades, with the remaining contact zone populations forming paraphyletic groups positioned intermediately to both parental clades. Some populations seemingly consisted of several highly related individuals, with this pattern being the most prevalent in the population IT5, where all individuals but one showed high genetic similarity. The topology of the ML tree . Population IT8, identified as S. waldsteiniana, also showed high proportions of the S. foetida genetic patterns , however, several individuals showed a nearly equal admixture of both parental patterns. Population IT7, identified as S. waldsteiniana, displayed the most equally admixed patterns of both parents . Populations IT6 and IT9, both identified as hybrids, showed the highest variance , with admixture patterns slightly shifted toward S. waldsteiniana.In the e Figure . Populat3.3K = 6 and therefore K = 2, 4, and 6 were used . The three individuals collapsed into the AU10 population showed even higher admixture, with posterior probabilities for S. foetida genetic cluster ranging between 34.5% and 43.6% (mean = 37.8%). Comparably, several S. foetida individuals from outside the contact zone showed no S. waldsteiniana genetic patterns, and the highest observed posterior probability was 25.1% (mean = 6.2%). With higher values of K, internal structures of local populations were recognized as genetic patterns. Decreased variation in posterior probabilities of different genetic groups could be observed in S. waldsteiniana populations outside the contact zone when K was 4 and 6 (the exception was population AU10). On the contrary, the posterior probabilities of other genetic groups increased in S. foetida individuals outside the contact zone with an increase in K.The plot of calculated entropy suggested that the plateau was reached around Appendix  and thosAppendix . HoweverRADpainter . The only exceptions were two S. waldsteiniana individuals from population AU10 that were nested in one of the paraphyletic groups of the contact zone populations. For some populations, clear internal structuring with high coancestry coefficients  or to the second parent  with high posterior probabilities and could identify several individuals from the contact zone as hybrid progeny . The highest FST‐values were observed between population IT5, identified as S. foetida (0.31–0.43), and all other populations. Of that, the lowest (0.31) was the FST‐value with the parental S. foetida population and the highest (0.43) with population IT7, identified as S. waldsteiniana. Of all identified S. waldsteiniana populations, population IT7 was the most diverged to parental S. foetida, showing the highest FST‐value (0.22), followed by the parental S. waldsteiniana population (0.16). Populations IT8, identified as S. waldsteiniana, showed similar FST‐values with both parental populations . Populations IT6, IT9, and IT7 had lower FST‐values with parental S. waldsteiniana  than with the parental S. foetida population . The IBD analysis showed insignificant spatial correlation of the genetic and geographic distances, hinting at a stronger prevalence of genetic structuring that is not influenced by spatial segregation.The 43 Table . Salix f3.4S. foetida and S. waldsteiniana individuals outside the contact zone showed separation on the axis of the first principal component  and PC2 (25.3%) were only slightly different  may result in the same patterns and therefore cannot be excluded from the interpretation . The genetic structure of populations showed that parental S. waldsteiniana individuals contain higher portions of S. foetida genetic patterns, which could be due to limited population sampling of the former in the northeastern range outside the contact zone. However, it could be also hypothesized that gene flow between these two species has historically been more directed toward S. waldsteiniana. The process might have been mitigated by a positive selection of ecologically favorable alleles that were incorporated into the genome through admixture, as was observed in other plant genera  and the majority of the population IT7 were seemingly classified as parental species' clusters with no (or only little) admixture with other populations in the 2 Figure  and S3. Appendix . It is pm et al. . More poSalix in the Alps ; investigation (lead); methodology (lead); visualization (lead); writing – original draft (lead); writing – review and editing (supporting). Loic Pittet: Formal analysis (supporting); investigation (supporting); writing – review and editing (supporting). Natascha Dorothea Wagner: Conceptualization (supporting); formal analysis (supporting); investigation (supporting); resources (supporting); supervision (supporting); writing – original draft (supporting); writing – review and editing (supporting). Elvira Hörandl: Conceptualization (lead); funding acquisition (lead); project administration (lead); resources (lead); writing – review and editing (lead).Open Access funding enabled and organized by Projekt DEAL.None declared.Appendix S1.Click here for additional data file.Appendix S2.Click here for additional data file.Appendix S3.Click here for additional data file.Appendix S4.Click here for additional data file.