Trachypithecus leucocephalus, is endemic to a narrow range in southwest China. This species shows very low genetic diversity in its 2 main relict populations, Fusui and Chongzuo. Whether this has been caused by a short evolutionary history or recent population declines is unknown. Therefore, we investigated the contributions of historical and recent population demographic changes to population genetic diversity by using 15 nuclear microsatellite markers and mitochondrial DNA (mtDNA) control region sequences.Current patterns of population genetic variation may have been shaped by long-term evolutionary history and contemporary demographic processes. Understanding the underlying mechanisms that yield those patterns is crucial for informed conservation of endangered species. The critically endangered white-headed langur, h = 0.486 ± 0.036; π = 0.0028 ± 0.0003). The demographic history inferred from mtDNA and microsatellite markers revealed no evidence for historical population size fluctuations or recent population bottlenecks. Simulations of possible population divergence histories inferred by DIYABC analysis supported a recent divergence of the Chongzuo population from the Fusui population and no population bottlenecks.Using genetic data from 214 individuals we found a total of 9 mtDNA haplotypes in the Fusui population but only 1 haplotype in the Chongzuo population, and we found an overall low genetic diversity  contains supplementary material, which is available to authorized users. Genetic diversity describes the differences in DNA sequences among individuals of a population or of a species and is a key aspect of biological evolution . GeneticMany natural and anthropogenic factors at various temporal and spatial scales, including evolutionary history and recent human-related population declines, can influence current genetic variation in a species, thus figuring prominently in determining population genetic patterns. For example, late speciation or population vicariance events cause lower genetic variance, compared to populations with deep divergence histories , 8, sincAsian colobines are a unique clade of primates including 50–60 species known for their highly diverse morphological characteristics and ecological adaptations , 17. MosTrachypithecus leucocephalus, is a lesser known Asian colobine monkey living in a very narrow range  in southwestern China [The white-headed langur (WHL), rn China . Its suirn China . In the rn China , 23, 24.rn China . Currentrn China  Fig. 1)Trachypitrn China . Previourn China , 28. MitIn this study, we evaluated WHL population genetic diversity using new mtDNA sequence data obtained from most of its range, and then generated the first molecular-based inferences of the historical and recent population demographics of the WHL using a combined dataset of mtDNA and microsatellite genotypes. We aimed to answer 2 questions: (i) Is the current range-wide, low, population genetic diversity the result of a short evolutionary history or of recent population declines; and (ii) Is the extremely low genetic diversity in the CZ population attributable to a recent founder event or to severe population bottlenecks? In light of our findings, we discuss the evolutionary origin and colonization history of the WHL and provide conservation planning suggestions that can help enhance population viability.Between 2012 and 2014 we non-invasively collected fecal samples from wild WHL populations in FS and CZ in the Chongzuo National White-Headed Langur Nature Reserve  in Guangxi Province, China. We collected a total of 403 fecal samples  from 41 wild WHL reproductive groups, with 1–26 (mean = 9.8) samples collected per group. Details of sampling were described previously . The samA previous study found that all the variable sites of a 1213-bp sequence of the mtDNA control region reside within a fragment of 350-bp in the HVRI of the WHL . This stBy using microsatellite and mtDNA sequence data with multiple analytical approaches of population structure, previous studies showed both strong genetic differentiation between the FS and CZ populations and limited within-population divergence , 28. Alts) and haplotypes (n), haplotype diversity (h), and nucleotide diversity (π) [We aligned and edited the mtDNA sequences with CLUSTAL X v 2.1 . Geneticsity (π)  of the ssity (π) . The Hassity (π)  and the sity (π) . Therefosity (π) , we consΦST [FST [To estimate genetic differences among populations, we used mtDNA data in ARLEQUIN v 3.5.1.2 , 49 to cΦST  and FST ΦST [FST  statistiD [FS tests [8 steps for the total population (FS and CZ combined) and 1 × 108 steps for the FS population, sampling every 1000 steps and discarding the first 10% as burn-in. The analysis was repeated 3 times, each with different seed numbers. The log and tree files of different runs were combined using LOGCOMBINER v 1.8.1. Effective sample size, mixing, and convergence were checked using TRACER v 1.5 [Historical changes in the WHL population size were inferred from mtDNA data by using several methods. First, we performed neutrality tests, including Tajima’s D  and Fu’sFS tests , in ARLEFS tests  in DNASPFS tests , implemeFS tests . The anaFS tests . We ran ER v 1.5 .For the microsatellite data, we used BOTTLENECK v 1.2.02  to assest2. Scenarios 2 to 4 all considered FS as the ancestral population but the demographic history of CZ varied. Scenario 2 considered CZ diverging from FS at a recent time t1 (t1 < t2); scenario 3 considered CZ diverging from FS at an earlier  time t2; and scenario 4 had CZ first diverging from FS at a historical time t2, followed by a population bottleneck event in CZ at a recent time t1. N2a and N2 were effective population sizes of the CZ population before and after the proposed population bottleneck, respectively (N2 < N2a)  for each population and FST and the mean classification index between pairs of populations. Posterior probabilities of the modeled scenarios were estimated using a logistic regression approach [We used microsatellite data with the approximate Bayesian computation (ABC) method, implemented in DIYABC v 2.0.1 , 62, to ormation  and usedOf the 403 field-collected fecal samples, 390 (97%) successfully generated sequences of the mtDNA HVRI, corresponding to 213  of the 214 distinct individuals previously identified from microsatellite genotypes. We found 9 haplotypes (Hap A–I) consisting of 10 polymorphic sites among all samples  and low nucleotide diversity (π)   and low nucleotide diversity (π) , the CZ population completely lacked diversity. Within the FS reproductive groups with more than 1 sample (N = 20), 7 groups had 1 haplotype, 10 had 2 haplotypes, and 3 had 3 haplotypes. Pairwise FST values were large and statistically significant between the FS and CZ populations , indicating strong genetic differentiation between the 2 populations.The total population had low haplotype diversity  population  to evaluate population size changes. The overall consistent patterns yielded from these analyses support a constant historical population with no large, long- or short-term size fluctuations. Therefore, the observed low genetic diversity in this species is unlikely to be explained mainly by loss of historical genetic diversity. Two underlying mechanisms may account for the low genetic diversity in the contemporary populations. First, the shallow evolutionary history of the species may have restricted the accumulation of population genetic variations. Mitochondrial sequence analysis showed that the WHL forms a monophyletic clade within Francǫis’s langurs (Trachypithecus francoisi) and that it split from the latter 0.46–0.27 million years ago [2) and on a recent population density census at CZ  or recent demographic bottlenecks  in the FS, CZ, or the population as a whole. However, the population assumptions and detection powers of these methods vary considerably, and potential confounding factors are present in sampling schemes and population genetic characteristics that can lead to false signals . For exaears ago . These 2ears ago . The infears ago . This inurs/km2; ). The toLittle is known about the evolutionary divergence history of the extant WHL populations. The FS population showed considerably greater mtDNA genetic diversity than did the CZ population, as the latter possesses only 1 haplotype that is also the most common haplotype of the FS population, suggesting that the FS population may be the source from which the CZ population has recently descended. Alternatively, the CZ population may have been established early but lost genetic variation during recent, severe demographic bottlenecks caused by human-related habitat destruction and hunting . HoweverAlthough our results suggest that the WHL’s low genetic diversity may be due primarily to its short evolutionary history and its small historical population size, recent anthropogenic range contractions and population declines have possibly caused further reductions in WHL effective population size and genetic variation, thus potentially aggravating the effects of inbreeding and genetic depression. Depletion of genetic variability in wild populations has been linked to reduced reproduction and to lowered resistance to infectious diseases, both of which threaten the fitness and long-term survival of populations . AlthougOur study was the first to apply both mtDNA and nuclear microsatellite markers to a large non-invasive sample of wild WHLs to evaluate the long- and short-term population demographic history of this critically endangered primate. Analysis of mtDNA sequence data revealed extremely low population genetic diversity relative to other endangered primates and a lack of genetic variation in the CZ population. The demographic history, revealed by mtDNA and microsatellites, indicated a small and stable historical effective population size and no recent population bottlenecks. In addition, population demographic history simulations generated by DIYABC analyses supported the hypothesis that the CZ population had recently been established by founders from the FS population. These results suggest that shallow evolutionary history and small historical population size are the primary factors shaping the WHL’s contemporary genetic diversity. Priority must be given to habitat area recovery and the restoration of habitat connectivity in order to combat the potentially detrimental effects of genetic inbreeding in the remaining, small WHL populations.2 [The WHL is endemic to an area bound by rivers and mountains in Guangxi Province, southwest China, and has an estimated historical range of about 360 km2 . It is a2 . Its soc2 . Reprodu2 .This species was first described scientifically in 1955 and named in 1957 , and no Average female reproductive age are 5–6 years at first birth  and abouAdditional file 1:Table S1. Primer information for PCR amplifications of 15 microsatellite markers in the white-headed langur. Table S2. Primer information for PCR amplifications of the white-headed langur mtDNA control region sequences and sex identification marker. Table S3. Prior values of parameters for simulated scenarios in DIYABC analysis. Table S4. Polymorphic sites in the mtDNA HVRI sequences and the number and sampling locations of the haplotypes. Table S5. Parameters of genetic diversity and results of neutrality tests using the mtDNA HVRI sequences in the Fusui (FS) and Chongzuo (CZ) populations. Table S6. Summary of BOTTLENECK analyses based on the microsatellite data from the Fusui (FS) and Chongzuo (CZ) populations. Table S7. Summary of posterior probabilities of 4 demographic history scenarios evaluated in DIYABC analysis using microsatellite data. Table S8. Posterior distributions of population demographic parameters from the scenario with the highest posterior probability (Scenario 2) inferred by DIYABC analysis using microsatellite data. Figure S1. Model checking by applying a PCA on the best-supported scenario (Scenario 2) in DIYABC analysis. Figure S2. Point estimates of effective population sizes (N1 and N2) and the temporal parameter (t1) of the best supported scenario (Scenario 2) from DIYABC analysis. (PDF 374 kb)


Phoxinus phoxinus ujmonensis  was used as a model to investigate genetic diversity and population structure as well as the influence of environmental factors on population genetics. In this study, we specifically developed 12 polymorphic microsatellite loci. The analysis of microsatellite and mtDNA markers revealed a high and a moderate genetic diversity across seven populations, respectively. Moderate differentiation was also detected among several populations, indicating the impact of habitat fragmentation and divergence. The absence of isolation by distance implied an extensive gene flow, while the presence of isolation by adaptation implied that these populations might be in the process of adapting to divergent habitats. Correlation analysis showed that abiotic factors like dissolved oxygen, pH, total dissolved solids, and conductivity in water as well as biotic factors like plankton diversity and fish species diversity had impact on genetic diversity and divergence in P. phoxinus ujmonensis populations. The results of this study will provide an insight into the effect of environmental factors on genetic diversity and contribute to the study of population genetics of sympatric species.As a widely distributed species along the Irtysh River,  We kept the damage to the fish population to the minimum, especially in the breeding season. All the sampling work was supported by the local institution Fisheries Research Institute of Xinjiang Uygur Autonomous Region, Urumqi 830000, China. From July 2013 to September 2015, we finished the 10 times sampling work and collected a total of 239 individuals from seven locations  using universal M13 primers. Plasmid DNAs extracted from positive colonies were sequenced, and sequencing results were analyzed with the SSRHunter v 1.3.0 . PCR amplifications were performed in a 10 µl of solution containing 50–100 ng genomic DNA, 2 × Es Taq MasterMix , and 5 µM of each primer on a Applied Biosystems (ABI). Thermal cycling parameters were as follows: initial denaturation at 94℃ for 3 min, followed by 35 cycles of denaturation at 94℃ for 30 s, specific annealing at different temperatures (Table Na), heterozygosity, and polymorphic information content (PIC) were computed using Popgene v 1.32 and PIC Calculator v 0.6 , number of effective alleles (Ne), expected heterozygosity (He), and observed heterozygosity (Ho) were calculated for each locus and each population with PopGene v 1.32 , Shannon's information index (iS), and deviations from Hardy–Weinberg equilibrium (HWE) were computed through both Popgene and GenAlEx 6.5 , pairwise genetic differentiation coefficient (Fst_p), and the hierarchical analysis of molecular variance (AMOVA) were performed using Arlequin v 3.5.1.2  dendrogram was constructed with Mega 6.0  was run at iteration of 10. Subsequently, the delta‐K (ΔK) metric of these cluster sets was uploaded to the web interface STRUCTURE HARVESTER  with GenAlEx 6.5 to visualize the patterns of genetic relationships between individuals and populations, basing on pairwise Euclidean distances. Admixture analysis was conducted in STRUCTURE 2.3.4  values and nucleotide diversity (iP) values were calculated, and neutrality test of each population was conducted using DnaSP v 5.1  of these seven groups were calculated with Mega 6.0. To better understand the genetic distance and structure of these populations, we constructed the phylogenetic tree of all the 239 individuals based on the Cox I + Cox II dataset in Mega 6.0. We determined the best‐fitting nucleotide substitution model which produced the lowest Bayesian information criterion (BIC) score using Mega 6.0. , dissolved oxygen (DO), and conductivity (Con) from 2012 to 2017, as well as computed biotic indexes, such as plankton diversity indexes  and Shannon–Wiener's diversity indexes of fish species   were observed in ppu17, ppu47, ppu49, and ppu51 among several populations. Thus, we abandoned the data of the above five loci to ensure the reliability of genetic results  to 0.7957 (TRKT and CHE), while the Fst_p values varied from 0.01319 (BEJ and CHE) to 0.11049 , and only 5.66% of the total variance was attributed to the differences among populations , suggestT Figure b.Gd values revealed by mtDNA haplotypes implied neither significant nor strong genetic structure among these populations. The shared haplotypes in the three networks suggested that some key cytoplasmic genetic information was communicated frequently across these populations, supporting the genetic structure to some extent , which was in accordance with the result of UPGMA dendrogram. Most individuals from KYET, XSK, and KLH fell into one cluster, while most individuals from BEJ and CHE and most individuals from HBH and TRKT were assigned to another two clusters, respectively , DO, and pH were higher than other values with V of −0.635 in the third component, DO of −0.582 in the second component, and pH of −0.510 in the second component, respectively. The highest loading values in the first component were just −0.470 (TDS) followed by −0.453 (T) and −0.452 (Con). Haplotype diversity (HdC) and nucleotide diversity (PiC) were revealed by concatenate sequences of Cox I + Cox II, which presented most information of individual Cox I and Cox II. The correlation analysis showed that iS was positively related to two abiotic factors DO and pH, while Fst was negatively related to iS and these two factors. The HdC and PiC were positively related to Con and TDS, but negatively to V . We also computed the abiotic and biotic indexes in three regions corresponding to three clusters. In addition, we calculated the Shannon–Wiener's diversity indexes of fish species (Sf) in the upper, middle, and lower regions (matched the three clusters). Visualized correlation analysis suggested that Si3 of the three regions was positively correlated to DO3 and pH3, as well as to Multi3 and Sf  or water distance  of the T matrix between Fst_p matrix might imply the potential IBA pattern, though correlations of Fst_p between other five factors were not significant  the mean Fst_p) and genetic distances (Nd) are main indexes of genetic structure. A large number of previous studies have provided the reference for the use of these two indexes  were observed to be higher than others. The iS value in HBH (2.135) was also the highest. Additionally, the mean Fst_p and Nd values in HBH and TRKT were higher than those in the rest five populations with 0.050 and 0.368 in HBH, 0.098, and 0.705 in TRKT, respectively, which could be explained by the unique environment of the Haba River. For example, there existed several dams along the Haba River. The HBH and TRKT populations were located upper stream of the lowest and the second lowest dam ordinally, while streams and irrigation canals were scattered on both sides of the Haba River. Such an environment was favorable for the two populations' adaptation which allowed high genetic diversity and resulted in slight differentiation between these two populations and other five populations. Furthermore, the UPGMA dendrogram exhibited a slight but meaningful convergence; that is, these seven groups showed apparently regional aggregation in upper, middle, and lower reaches, respectively. This might reflect a trend that the genetic relationships among these seven populations developed along the river from upper stream to lower stream, since gene flow between nearby populations could occur more frequently. A similar trend was found in the population genetic structure of California Bocaccio and Atlantic Salmon  was positively related to dissolved oxygen (DO). The previous study reported that dissolved oxygen availability might have great effect on the energy for fish locomotion, growth, and reproduction (1987). Generally speaking, the active locomotion could enhance frequent gene flow among fish populations, and efficient reproduction could ensure better inheritance of genetic information. The evidence supporting above findings could be found in the study of the population differentiation of the African cyprinid Barbus neumayeri. The aquatic dissolved oxygen was reported as a selective force limiting gene flow among populations and therefore played a key role in determining the genetic structure of populations  also showed the importance of water flow velocity  was also highly positively related to both plankton diversity indexes  and Shannon–Wiener's diversity indexes of fish species (Sf). Food availability was reported to be an important factor influencing species richness, in turn, affecting genetic diversity  was positively related to DO but HdC/PiC (cytoplasmic diversity) were positively related to TDS since DO and TDS were always negatively related. We thought that this should also be due to the different inheritance patterns of these two types of markers mentioned above and the unique environment in this watershed. The four tributaries wind down the hillside on the north side of the mainstream. The TDS, to which HdC/PiC were positively related, were undoubtedly higher in the XSK, BEJ, and HBH populations since they were located in the lower reaches of the three tributaries, while the DO seemed to be higher in upper reaches except for the HBH population. Nevertheless, the Pearson correlation coefficient for iS and DO (0.69) was lower than that for iS and Multi (0.72), and they were both lower than that for DO and Multi (0.87). As the Multi represented the abundance of food for P. phoxinus ujmonensis, we might hypothesize that food availability has more influence on the genetic diversity of this species.Nonetheless, one might note the discrepancy that 5P. phoxinus ujmonensis along the Irtysh River, which might be attributed to high genotypic association ensured by the continuous water system. However, there was moderate genetic differentiation among several populations, which could partially be due to the habitat fragmentation. The slight but meaningful differentiation of TRKT population against other populations might imply that human activities are unneglectable factors that influence genetic structure of fish. There was no obvious evidence supporting isolation by distance. However, we found the potential evidence of isolation by adaptation as well as the highly positive correlation between genetic diversity and abiotic or biotic factors. This might also suggest that there exist gene flow along distances, but different populations might be in the process of adapting to divergent environments due to habitat fragmentation. Finally, as P. phoxinus ujmonensis occupies an important niche in the food web, our results will provide reference for the study of population genetics of economic or endangered fish in the same catchment. The relationship of genetic information of various fish species and the detailed interaction between environmental factors and genetic characteristics remain to be further investigated.In this study, we developed and characterized 12 highly polymorphic microsatellite loci. Population analysis revealed high level of genetic diversity in local populations of The authors declare no conflicts of interest.Peng Xie and Xu‐Fa Ma designed the research. Peng Xie, Guang Zhao and Jian‐Gong Niu performed the sampling work related to fish individuals. Jun Wang and Qiong Zhou performed the sampling and data analysis work related to plankton and abiotic factors involved. Peng Xie conducted the statistical analysis of genetic data and environmental data. Peng Xie drafted the manuscript. Xu‐Fa Ma and Yan Guo revised the manuscript. All authors reviewed the manuscript. Click here for additional data file.


Genetic analysis of admixed populations raises special concerns with regard to study design and data processing, particularly to avoid population stratification biases. The point mutation responsible for sickle cell anaemia codes for a variant hemoglobin, sickle hemoglobin or HbS, whose presence drives the pathophysiology of disease. Here we propose to explore ancestry and population structure in a genome-wide study with particular emphasis on chromosome 11 in two SCA admixed cohorts obtained from urban populations of Brazil (Pernambuco and São Paulo) and the United States (Pennsylvania). Ancestry inference showed different proportions of European, African and American backgrounds in the composition of our samples. Brazilians were more admixed, had a lower African background (43% vs. 78% on the genomic level and 44% vs. 76% on chromosome 11) and presented a signature of positive selection and Iberian introgression in the HbS region, driving a high differentiation of this locus between the two cohorts. The genetic structures of the SCA cohorts from Brazil and US differ considerably on the genome-wide, chromosome 11 and HbS mutation locus levels. Five RFLP-assessed haplotypes, named after the locations where they occur more frequently , are classically used to classify the HBB cluster. High fetal haemoglobin (HbF) levels are associated with the Senegal and Arab-Indian haplotypes, compared to the Benin, CAR and Cameroon haplotypes3. Individuals with CAR haplotypes tend to present the lowest HbF levels, while individuals with the Benin haplotype usually have intermediate HbF production levels4. Despite the fact that the protective effect of HbF may vary according to its distribution amongst erythrocytes, as shown by severe SCA cases carrying the Arab-Indian haploytpe5, these findings have motivated abundant characterization of diverse SCA populations worldwide regarding HBB haplotypes.Sickle cell anaemia (SCA) is caused by homozygosity for a point mutation in the beta-globin gene (HBB) on chromosome 11. SCA was the first monogenic disease to be described in humans9. Nonetheless, aspects regarding the effect of European ancestry10 and fine genetic structure on the SCA mutation locus remain elusive. Large association studies have been mostly conducted on SCA patients from the US (SUS). Other studies, such as those conducted in Brazilian SCA patients (SBR), rely frequently on findings from studies of the SUS population. The US and Brazil have the highest prevalence of new-borns with SCA on the American continent, estimated to be 4,351 and 2,978, respectively, in 201011 and are divergent in demographic history regarding migration and admixture.Some effort has been made to describe genetic diversity and structure among SCA patientsHere, we aim to clarify how admixed populations affected by SCA diverge from each other. Also, we aim to further describe the ancestry of SCA patients from Brazil, who have been explored relatively little compared to US patients. To achieve these goals, we propose to compare genetic structures of two populations at the genome-wide and local levels, through the analysis of a North American cohort  and a Brazilian SCA cohort , using high-density genome-wide microarrays .We evaluated the genetic structure at the genome-wide and chromosomal level by comparing SCA cohorts from the United States and Brazil, along with 19 worldwide populations from the African, European, American and Asian continents. Unaffected people (HbAA genotype) sampled from US and Brazil , were also included  as a measure of genetic distance between populations  than to Europeans .Genomic data from all populations were analysed by keeping 155,820 SNPs after quality control for a principal component analysis (PCA), depicted in Supplementary Fig. 9. Moreover, African components are divided into 38.3% Western-Africa Mandé-related and 39.4% Eastern Bantu-related on average for the SUS, while SBR present 15.3% Mandé-related and 28% Bantu-related ancestries  and Eastern African Bantu (LWK) populations to SBR , consistent with a scenario of positive selection. The hypothesis of introgression is also corroborated by the European ancestry local estimates in this location , followed by Europe (approx. 13%), other African regions (approx. 8%) and America (approx. 4%)16. They also described the African Americans to have a 45% Bantu mean ancestry and 22% non-Bantu (Mandinka ethnolinguistic group) mean ancestry, emphasizing that the diaspora encompassed a broad region in Africa, ranging from Senegambia in the west all the way to Angola, in the south16. Our data are consistent with these findings for both the SUS population and non-affected African descendants from the US, which are nearly identical in ancestral composition.By subdividing ancestry origins further to the subcontinental scale, the North American cohort had a pattern of within-Africa ancestry consistent with reports of genetic relatedness to YorubansST = 0.001; Supplementary Table 17.Brazilian affected and unaffected subjects, on the other hand, are somewhat discernible by both PCA and ADMIXTURE plots, although we assert that the non-affected sample was not controlled for skin pigmentation and was rather collected at random. More importantly, Brazilian HbAA were all collected in São Paulo, while the SCA group has also subjects from Pernambuco. Still, this differentiation is markedly small , Brazilian SCA has only been briefly described in terms of genetic structure and ancestry8, and to the best of our knowledge, to date no subcontinental ancestry has ever been evaluated in this population. Kehdy et al. evaluated 6,487 subjects from the general populations of Northeast, Southeast and South Brazil, finding them to display two distinct within-Africa ancestry components: non-Bantu Western and Bantu Eastern and that the former was more prominent in Northeast Brazil, while the latter is more prominent in the South-eastern/Southern areas. Nonetheless, Bantu only accounted for an average of 36% in Southeastern people and 44% in Southern Brazilians, while we found Brazilians, irrespective of disease status, to share 65% of their African heritage traced to Bantu on average. This might be due to the different regional origins of the recruited subjects and/or other methodological and analytical aspects, although both are in agreement with historiographical data, which states that enslaved Yoruban people arrived in large numbers in the Northeast port of Salvador, whereas the Mozambican Bantu slaves disembarked largely in Rio de Janeiro ports, in South-eastern Brazil18. Also, Hudson’s FST on genomic markers confirms that our sample of SCA from Brazil is slightly closer to Bantu than to non-Bantu populations. SCA individuals from the US display a more even sub-continental African composition and greater proximity to the African populations evaluated here, indicating assortative mating may have had great impact on the US cohort. It is noteworthy that the FST values also show that the two affected cohorts are closer to each other than they are to European populations, and that the SBR cohort is closer also to its US counterpart than to any African population surveyed.Unlike the SCA population from the US  could influence LD patterns and generate the aforementioned results. We, then, tested LD between the rs334 mutation region and the region under selection and found that they form independent blocks, not exceptionally linked at any marker 25, while Choi et al. found SHAPEIT switch error to be 2.8% when phasing 85 unrelated individuals from European origin26. We would thus expect our data to fall into the range of approximately 3–6% switch error rate. The local ancestry inference might also be affected by the use of East Asian reference data as proxy of ancestral Americans, since it might inflate the estimates of haplotype contribution of that particular population27.This study was limited by the relatively small sample sizes in SCA cohorts derived from just three sampling localities. These limitations make it difficult to extrapolate the results to larger and more broadly distributed sickle cell individuals from the two countries evaluated and also amplify statistical noise. Although assessed in regard of IBD, individuals might still have cryptic structure/consanguinity that would especially affect the LD patterns observed for Brazilian patients. Differences in gene flow, HbS allele frequency and HBB haplotype composition between sampled subjects from Recife and Campinas may have introduced variance not accounted in the analysis. Although genotyping rate is near 100% for markers included in ancestral analysis (see Methods), technical constraints may apply, as the inference of haplotype phase by population data is known to have greater switch error rates. Lo Here, we quantified divergence between two small cohorts and found this to be a promising way to highlight regions of high divergence that might be of functional importance or to uncover candidate loci based on selection signals. The haplotype structure has important implications on the cis-acting factors leading to variation on HbF production. More generally, these findings underline that the five RFLP-haplotype classifications proposed do not account for population-specific demographic factors and, while still useful, should be analysed carefully.8, and this seems to be the case for SCA patients with regard to HbF production. Admixed mapping, nonetheless, has been applied when the ancestral populations are European and African31. It is still a matter of debate whether HbF levels are influenced by European ancestry8, whereas different ancestries inside the African continent have already been proven to be diverse regarding gamma-globin expression. Moreover, it is still unclear how different levels of admixture will translate to HbF production and other phenotypic traits. Sickle cell disease ancestry studies could lead to novel loci associated with phenotypic variability. Here we demonstrate that SCA samples from different locations may largely vary on the genomic and local ancestry on chromosome 11. Further studies in larger cohorts, sampled from different locations are welcomed to better describe the variation in ancestral background on genomic and HBB cluster levels. Also, more detailed migration history data and the advancement in fine structure inference methods will broaden our understanding of how patterns of gene flow, admixing, selection and linkage disequilibrium act on shaping genomic regions that impact important phenotypic human traits.Genetic studies struggle to deal with admixture and other complex population demographic characteristics in face of association to phenotypic traits. Admixture mapping, a tool to perform this task, has been recently developed and relies on regions of different allele frequency driven by contrasting ancestries. It has been suggested that admixture mapping may only be applicable when ancestral populations differ in the phenotype of interestIn conclusion, we found the two different cohorts of SCA to differ in both genome-wide ancestral composition and locally to the causal locus region. Comparing admixed populations may be a strategy to reveal regions of local adaptation that would otherwise require a large association study to be unveiled.The present research followed the principles of the Declaration of Helsinki; all patients were presented to the aims and details of the study and signed an informed consent. The institutional review board committee at CHOP and Ethics Committee at the Faculty of Medical Sciences at UNICAMP approved subjects’ enrolment, blood collections and study methods.http://www.internationalgenome.org/data/) representing global populations . The American cohort data is composed of 30 SCA patients with data filtered out from the Epic Care Clinical System , along with 60 auto-declared African Americans not affected by sickle cell diseases, all from CHOP, Pennsylvania. Differently from African-Americans, the Brazilian group of unaffected subjects (HbAA) was not selected regarding skin pigmentation or self-declared African background, since the SBR population is already known to be more heterogeneous in ancestral composition8.The complete dataset comprises a total of 1,994 individuals, 1,822 of which are part of the 1,000 Genomes repository , according to manufacturer’s protocol. Genotype data was analysed along with reference populations from 1000 Genomes Project35. Each individual sample was checked for discordance in relation to the sex register, outlying missing genotype call rate (genotyping rate ≥ 0.90 were kept); we also evaluated relatedness in the collected samples by calculating genome-wide identity-by-descent (IBD), removing one sample from pairs of duplicates or pairs estimated to be second-degree relatives or closer  as the first step. We also excluded regions of known extensive LD across the genome36. For displaying regions of LD we used Haploview v4.237, SNPs with strong LD (D′ ≥0.8) were considered part of a haplotype block using confidence intervals as proposed by Gabriel and colleagues38.For performing PCA, we have controlled the data for regions of high LD through the extraction of local and long-range markers in LD . We identified the optimal value of K (6) by the least error cross-validation method after testing K values ranging from 1 to 18.Genome-wide population structure and admixture were analysed by principal component analysis (PCA), using EIGENSOFT v7.2.141. We then analysed local ancestry in this chromosome using the software SABER+ v1.0, which implements a Markov-Hidden Markov Model for inferring locus-specific ancestry in admixed individuals42. We modelled SCA cohorts as a mixture of chromosomes from three ancestral populations with various global proportions of European, Native American and West African ancestries. Although real admixture histories are more complex than this, we simplified them for the sake of data tractability, since more convoluted admixing models are still poorly addressed by current algorithms43.We phased genotypes of the chromosome 11 on both Brazilian and American SCA patients using the SHAPEIT v2.r790 method34: phased haplotypes from the CEU (117 haplotypes), CHB + JPT (169) and YRI (115) trio-phased panels, as proxy haplotype data from Europeans, Native American and African ancestors, respectively. We also applied a Weir & Cockerham’s makerwise FST estimation44 implemented in VCFLIB package (https://github.com/vcflib/vcflib). Haplotypes blocks images were generated in Haploview37 and VCFLIB.We considered admixed haplotypes as mosaics of segments derived from three of the HapMap phase 3 haplotype panelset al.45. Imputation was performed by IMPUTE2 v2.3.2 method46.For evaluating inference in the classical 5 HBB haplotypes we used phased haplotypes of SCA subjects to impute not-typed markers on chromosome 11, including 4 SNPs  that define these haplotypes as described in Shaikho et al. as a method to describe events of recent selection47. iHS is the amount of extended haplotype homozygosity (EHH) at a given marker along the ancestral allele relative to the derived allele empirically standardized to mean of 0 and variance of 1. Values lower than −2  or higher than 2  are regarded as signals of recent positive selection. A stretch of extended homozygosity for haplotypes on a high frequency allele relative to the other is a signature of a sweep resulting from positive selection. We computed iHS values for SCA from Brazil and the US using the VCFLIB package. By linearly interpolating between SNPs, EHH was integrated with respect to genetic distance for markers that reached EHH of 0.05 in both directions from the core SNP, otherwise, that SNP was skipped. Normalization is then performed to account for regional differences in allele frequencies.The integrated haplotype Score (iHS) was proposed by Voight 48. We used the selscan v1.1.0 software to perform XP-EHH calculation49.Cross Population Extended Haplotype Homozygosity detects sweeps resulting from selected alleles that have trend towards fixation in one population but not the otherST values shown in Fig. ST values are high between Brazilian and American cohorts but drop between Brazilian and Iberian populations. We modelled HbF as response variable on a linear regression, performing a single test for each of the 31 SNPs as predictor variables, along with age, sex and hydroxyurea treatment as covariates. All variants had MAF >0.05 and association with HbF was tested using a standard linear regression of phenotype on allele dosage implemented in PLINK v1.935. The gvlma R package50 was used to test fitness of data to the linear regression assumptions. We provide plots showing normality of residuals for significant SNPs in Supplementary Fig. 51 tool (see Supplementary Fig. We selected the GRCh37 chr11:5.54–5.59 Mb region on account of the FSupplementary Information


Panthera uncia syn. Uncia uncia) but the ecological studies on this animal are scarce since it is human shy by nature and lives in difficult mountainous tracts. The pilot study is conducted to exploit the genetic diversity and population structure of the snow leopard in this selected natural habitat of the member of the wildcat family in Pakistan.The Hindu Kush and Karakoram mountain ranges in Pakistan’s northern areas are a natural habitat of the snow leopard  along with a control sample of zoo maintained male snow leopard were collected for comparison. Significant quality and quantity of genomic DNA was extracted from scat samples using combined Zhang–phenol–chloroform method and successful amplification of cytochrome c oxidase I gene (190 bp) using mini-barcode primers, seven simple sequence repeats (SSR) markers and Y-linked AMELY gene (200 bp) was done.Cytochrome c oxidase I gene sequencing suggested that 33/50 (66%) scat samples were of snow leopard. AMELY primer suggested that out of 33 amplified samples, 21 (63.63%) scats were from male and 12 (36.36%) from female leopards. Through successful amplification of DNA of 25 out of 33 (75.75%) scat samples using SSR markers, a total of 68 alleles on seven SSR loci were identified, showing low heterozygosity, while high gene flow between population.The low gene flow rate among the population results in low genetic diversity causing decreased diversification. This affects the adaptability to climatic changes, thus ultimately resulting in decreased population size of the species. Panthera uncia) has evolved in the mountainous ranges of Asia  markers are considered excellent, being co-dominant, highly polymorphic and easily transferable between populations . These mAccording to a recent estimate, the number of snow leopard individuals is 7,463–7,980 worldwide . Retribun = 50) were collected between December, 2016 and February, 2017 from five localities of GB (Pakistan), viz., Shagarthang, Basho, Thally, Kharkoo and Astore  Zhang Method: Sample (1.5 g) was washed with ethanol, purified by phenol and gDNA was isolated by binding buffer and column filter . (2) PheMini-barcode primers pair having sequence TCCACTAATCACAARGATATTGGTAC and GAAAATCATAATGAAGGCATGAGC  was usedConfirmed snow leopard samples were genotyped by using seven microsatellites (SSR) markers, that is, PUN82, PUN100, PUN124, PUN132, PUN225, PUN229 and PUN327 . AmplifiCombined Zhang–Phenol–Chloroform method was efficient, giving significant extraction success rate, average quantity and quality of gDNA ; Fig. 2.p > 0.05).Polymerase chain reaction amplification of Y-linked AMELY marker (200 bp) was achieved in control male sample and in 21 field collected samples, suggesting a sex ratio of 1.75:1 male to female  of alleles were recorded in Shagarthang valley, followed by Thally (33), Astore (27), Basho (26) and the lowest (25) in Kharkoo valley with Shannon indices in different populations ranged between 1.17 and 1.58. Allele frequencies of different SSR loci ranged between 0.02 and 0.75 in different populations .Observed heterozygosity  was lower compared to expected heterozygosity . Gene flDendrogram constructed using UPGMA, showed low genetic differences and high genetic similarities among three populations . Shagarthang population exhibited one degree separation from the above three populations, while Basho population appeared as a separate clade . AMOVA sIn current study DNA was extracted from 50 putative snow leopard scat samples following three protocols . ExtractThe sample from a control male was amplified by AMELY primer, suggesting the validity of the technique in determination of sex using scat samples of snow leopard. AMELY gene (200 bp) is located on Y-chromosome and hence amplified in males only . Out of We successfully amplified 75.75% of the field collected scat samples using SSR markers. The lack of amplification in other samples could be due to inhibitors  in the extracted DNA templates . The effIn current study, low genetic variation/heterozygosity (0.33–0.5) was observed in snow leopard population of GB compared to the expected value (0.62–0.75). Low heterozygosity has been reported previously for many populations of snow leopards in Central Asia including Southern Mongolia, Central China, Nepal, north-western India, Pakistan, Tajikistan and Kyrgyzstan , 2017.The low genetic diversity in these areas caused by smaller isolated size of the population where higher inbreeding results in increased homozygosity and genetic fixation or loss of unexploited genetic potentials . It can The findings of genetic diversity, population structure and function played a vital role in formulation of conservational strategies. The low genetic diversity in snow leopard populations of the study leads to the conclusion that the gene flow among the populations is too low and the genetic diversification of the animal is not enough to aptly adapt to the environmental changes which would not result in the efficient promotion of this species. Therefore, proper planning and management in the protected and non-protected areas is required to get the output. The illegal hunting, poaching and human-animal conflicts have to be stopped by arranging proper protective and safety measures to the natural habitat of the inhabitant species in order to maintain the effective population size of the threatened wild fauna.10.7717/peerj.7672/supp-1Supplemental Information 1Allele frequency on different SSR markers at different loci analyzed by POP Gen for snow leopard populations.Click here for additional data file.


Psidium guajava), considered one of the greatest threats to the local biodiversity due to its effective spread in the archipelago and its ability to outcompete endemic species. To better understand its history and genetics, we analyzed individuals from three inhabited islands in the Galapagos archipelago with 11 SSR markers. Our results reveal similar genetic diversity between islands, and the populations appear to be distinct: the islands of San Cristobal and Isabela are genetically different while the population of Santa Cruz is a mixture from both. Additional evidence for genetic bottlenecks and the inference of introduction events suggests an original introduction of the species in San Cristobal, from where it was later introduced to Isabela, and finally into Santa Cruz. Alternatively, a second introduction in Isabela might have occurred. These results are contrasted with the historical record, providing a first overview of the history of P. guajava in the Galapagos islands and its current population dynamics.The threat of invasive plant species in island populations prompts the need to better understand their population genetics and dynamics. In the Galapagos islands, this is exemplified by the introduced guava ( Invasive plant species are a threat to natural ecosystems and biological diversity , 2. StudPsidium guajava), a species cultivated in South Asia, Central and South America, North America and Australia for its edible fruit [P. guajava originated in Central and South America [th century [P. guajava is widely distributed in the Galapagos, growing in both disturbed areas and natural forests in the islands that host human populations: Isabela, Santa Cruz, San Cristobal and Floreana [The Galapagos Islands are an example of such an isolated ecosystem vulnerable to invasive species due to their unique biodiversity and high biological endemism , 9. Thisle fruit . It is b America  and was  century , 14. It  century , 13. CurFloreana .Rubus niveus on the species richness of the Scalesia forests [Understanding invasive processes can also benefit conservation efforts in island ecosystems. Invasive species have been formally recognized as a serious threat to the Galapagos Islands’ endemic flora and fauna, which can rapidly become extinct when alien species are introduced , 13. A l forests ), highli forests .P. guajava populations on the Isabela, Santa Cruz and San Cristobal islands in the Galapagos (Ecuador) to understand the history and current status of this species in the archipelago. The combination of the well documented introduction history of the common guava in the Galapagos and the relation to its genetic diversity and population structure can provide a model on how a plant species invades and spreads on an island ecosystem.Here we analyze the genetic diversity and population structure of P. guajava individuals were sampled from selected locations of three islands in the Galapagos archipelago: 94 samples were obtained from San Cristobal (shortened SCY), 80 from Santa Cruz (shortened SCZ) and 95 from Isabela (shortened ISA). The taxonomic identity of the samples was confirmed through morphologic examination and comparison with voucher specimens from the Charles Darwin Foundation CDS Collection  and the Universidad San Francisco de Quito Herbarium . Due to the historical link between P. guajava and human settlements, the sampled sites encompass each island’s farming zone , expected heterozygosity (HE) and F-statistics were estimated on the hierfstat package [poppr package [diveRsity R package [adegenet R package [E between islands. Finally, Hardy-Weinberg Equilibrium for each marker was tested with the pegas package [Maps of the georeferenced sampling regions on each island were drawn using ARCGIS Desktop 10.2 . For each SSR locus analyzed, the total number of alleles (A), observed heterozygosity  was plotted with the ggord package [ST) among the sampled regions within all the three assessed islands. STRUCTURE 2.3.4 [K) was evaluated between 1 and 10, with 10 independent runs performed for each K value. 1,000,000 Markov Chain Monte Carlo (MCMC) steps were used, with a 100,000-step burn-in period. The optimum value of K was evaluated using the Evanno method [pophelper package [Pairwise Fistances  were cal package  to quantRE 2.3.4  was usedo method  as impleo method , and indo method . The fin package  implemenP. guajava in the Galapagos Islands. A total of 14 different colonization scenarios , post-bottleneck population sizes , times of divergence (t1 and t2) of populations and the length of bottleneck events (db) (both in generations) were all drawn from uniform prior distributions (range: 10.0–10000.0). For scenarios 13 and 14, admixture rates (ra) were drawn from a uniform distribution (range: 0.001–0.999). Posterior probabilities of each scenario were computed using the logistic regression approach in DIYABC 2.0. The introduction times (in generations) for each island  according to the best supported scenarios were also estimated. To better understand these patterns and confirm possible introduction events, the potential of population bottlenecks in every island was inferred through the BOTTLENECK software, following both, the SMM and TPM models [Approximate Bayesian Computation (ABC) analyses were run on DIYABC 2.0  to infercenarios  were comcenarios . The simM models . HeterozP. guajava individuals from three islands in the archipelago was successfully obtained for 11 of the 13 nuclear SSR markers tested (Genetic information for s tested . Two of s tested . The nums tested . The meas tested .E) estimates show a greater genetic diversity in Santa Cruz  and San Cristobal , with the lowest value found in Isabela  (E between islands were not significant (given a Bonferroni correction for the test) between Isabela and San Cristobal (p = 0.037), but were significant between Santa Cruz and both Isabela (p = 0.001) and San Cristobal (p = 0.005). Linkage disequilibrium between markers was not found for Santa Cruz  but appears to be relatively common in Isabela (The expected heterozygosity (H= 0.133) . These d Isabela . This lo Isabela . Null al Isabela , while a Isabela .IS statistic, revealed values ranging from 0.341 to 0.621  that best explain the population structure in our data set suggested an optimal number of two lineages which cluster San Cristobal and Isabela under different groups  than to Santa Cruz (FST = 0.120). This pattern coincides with the geographic distances between the three islands. The genetic distance between Santa Cruz and San Cristobal is considerably lower (FST = 0.074), suggesting the more widespread gene flow between these two islands throughout the history of P. guajava in the archipelago. When corrected for null alleles, the FST distances did not change significantly. The Santa Cruz-San Cristobal FST distance remained almost the same (FST = 0.076); while, the FST values between Isabela and the other two populations, experienced a slight decrease .To further explore the degree of genetic structure in these populations, different approaches were considered. A PCoA based on genetic distances shows minor differentiation between islands, even when the top two principal components explain over 60% of the total variance . This apt groups . A similpulation –S4 Figs.p < 0.001, r2 = 0.343) and to FST genetic distances, with a lower linearity observed for the latter . This suggests that some isolation by distance among different populations of P. guajava found on different islands is observed.The geographic distances between sampling regions of the three islands were consistently correlated to Nei’s  approach [P. guajava generations ago (mean = 124 generations), depending on the model. Meanwhile, the split of the Santa Cruz population (event t1) could date from 5 to 113 generations ago (mean = 35.3 generations) (t2(h), P. guajava in the archipelago.The population history of approach  in orderrations) . Given tP. guajava in all three islands (HE = 0.356) is low when compared to the species diversity within its native range, an expected pattern for an insular invasive species [P. guajava accessions from germplasm collections, the genetic diversity found in those countries (HE = 0.678 and HE = 0.740 respectively) is considerably higher than that found in the Galapagos P. guajava populations. This low genetic diversity in island populations is related to a founder effect, where the first plants that arrive on an island are low in number and are sampled at random from the source population. Because of this, much of the ancestral population’s diversity is left behind, lowering the possibility of obtaining heterozygote individuals [The overall genetic diversity of  species . Accordi species  and Vene species  with P. ividuals ,43,44. ICortaderia jubata and C. selloana in New Zealand [Miconia calvescens in several Pacific islands [E = 0.061, HE = 0.095 and HE = 0.110 respectively). However, other invasive species in island settings, like Senesio madagascarensis [Paraserianthes lophantha [E = 0.790 and HE = 0.600 respectively), argued to be a product of gene flow or multiple introductions. By contrast, the P. guajava populations in Galapagos show an expected heterozygosity which is intermediate to these scenarios. This suggests that factors such as multiple introductions, the magnitude of the founder effect, the reproductive system of the species and the geography of the invaded ecosystem [P. guajava populations.Other plants that are known invaders in island ecosystems, such as  Zealand  and Mico islands  exhibit carensis  and Paraophantha  in Hawaicosystem ,49,50 coP. guajava populations in different islands from the Galapagos archipelago share some features that provide an insight into the forces that have shaped their current status. The equivalent allelic diversity in all three islands, particularly if rare alleles are excluded, suggests that similar introduction dynamics have shaped these populations, or that a certain degree of homogenization through gene flow between islands has occurred. Upon closer inspection, the significantly higher expected heterozygosity index in Santa Cruz (0.365), when compared to Isabela (0.284) and San Cristobal (0.326), opens the possibility of multiple introductory events that lead to a higher genetic diversity [P. guajava introduced to these islands, a common occurrence during the colonization of new island ecosystems [Furthermore, the iversity . The lowosystems , and a lThe degree of differentiation among different islands is noteworthy, as it suggests that gene flow is considerably higher than might be expected for plant populations in different islands. Only 13% of the total differences occur between islands , suggestP. guajava has proven to be an aggressive invader in the Galapagos [P. guajava. A proportion of the analyzed individuals showed higher values of the F inbreeding index  [Psidium socorrense and its congener P. sp. aff. Sartorianum which is resulting in the local extinction of the native species at the southern slope of the island [P. guajava and P. galapageium.Due to pageium) . In the pageium) . Extinctpageium) ,72. ThisP. guajava with the well documented historic events that describe the first human settlements on the archipelago. Most of our historical sources are based predominantly on the memories and journals kept by the first settlers and resident sailors of the Galapagos, as well as their descendants’. The information from these sources matches the historical documents obtained from the National Archives, archaeological evidence  and publications from academic historians [The relatively recent colonization of the Galapagos Islands allows us to link the population genetics of storians ,74.P. guajava was present in the highlands of San Cristobal at least since the 1930’s [P. guajava was documented in historical sources several decades prior, where early Galapagos settlers wrote about their concern of the invasive species and saw it as a potential plague [P. guajava dates from the late XIX century (ca 1889–1890), where it is said that the very first three guava trees on the archipelago were planted by Manuel J. Cobos in his personal garden on San Cristobal Island for ornamental purposes. Cobos was an eminent settler of the Galapagos and was well-known throughout the archipelago. In 1870 he established a massive and profitable sugar cane plantation over the highlands of San Cristobal [P. guajava monotypic forests [P. guajava, to no success. By the 1950’s the plague had spread to Santa Cruz [P. guajava is poorly documented in the Isabela records, but according to Lundh (2006) [Palynological research confirmed that e 1930’s . Howeverl plague ,73,76,77ristobal . Unfortu forests ,73. In t forests ,77. Havinta Cruz ,73. P. gh (2006)  the plagP. guajava population is the origin of the plague that spread over the four inhabited islands of the Galapagos. All but two rare alleles from the San Cristobal population are also present in the Isabela and Santa Cruz populations  planted in Tahiti Island, started an aggressive invasion that spread over several islands of the Pacific [The San Cristobal population shows evidence of having gone through a genetic bottleneck which created a transient excess of heterozygosity , an even Pacific .P. guajava occupies a larger area on Isabela than on any other island within the Galapagos [P. guajava invasion persists in Isabela and implies that the arrival of several new settlers after the 1950’s triggered the invasion on this island. Nevertheless, both historical events and our results suggest that P. guajava could have been present in Isabela several decades before the 1950’s. This island was the third to be permanently colonized by humans, following Floreana and San Cristobal [P. guajava was introduced from San Cristobal to Floreana during this period. From here, we could speculate that Antonio Gil, who worked with cattle, might have carried P. guajava seeds or fruit to Isabela. This statement is supported by both of the scenarios obtained from our ABC analysis which show that the Isabela population derived from the original population of San Cristobal, before the Santa Cruz population appeared . During the 1960’s, the penal colony was removed and more people  began to move to Santa Cruz, where tourism was being developed [P. guajava in Santa Cruz occurred at this time. The important gene flow detected between the populations of San Cristobal and Santa Cruz  may be dated around this time as well. Meanwhile, the Isabela lineage is almost absent in San Cristobal . It is interesting to note that the population from Santa Cruz, which is located in the center of the archipelago, has gene flow with both the San Cristobal and the Isabela populations. On the other hand, these two populations, located on opposite extremes of the archipelago, show very little gene flow between them. Thus, gene flow between P. guajava populations from different islands seems to be correlated with their geographic locations, potentially reflecting the heightened human mobilization between islands that are closer together. It must be noted that all of the gene flow between P. guajava populations located on different islands would be presumably human-mediated, since this invasive species’ natural dispersers cannot travel across the ocean [P. guajava seedlings, fruit or seeds between islands and between the mainland and the islands .The time when the Isabela lineage arrived in Santa Cruz is unclear. During the first half of the XX century, Isabela remained isolated due the presence of a penal colony and the lack of a freighter ship serving this island regularly . This fa Isabela . Howevereveloped ,76,82. Tristobal , a fact he ocean ,67,68. NP. guajava populations from the Galapagos Islands, and provide a valuable insight into the history of the invasive process of this species. However, the addition of new information to the presented framework could aid in better understanding the full extent of the risks that P. guajava represents to the local ecosystems.Genetic information has increasingly become a powerful tool for the reconstruction of a population’s history and the characterization of its diversity and the forces that shape it . Our curP. guajava populations. Historical sources pinpoint different provinces in mainland Ecuador as the possible source [P. guajava in the archipelago, and it remains the only inhabited island with unsampled guava populations. Further studies will address some of these questions, to continue to elucidate the history of the P. guajava invasion.Our reconstruction of the history of the invasion is backed by historical records but was insufficient to explore the source population for the Galapagos e source ,74,75,82P. guajava is particularly noteworthy due to the occurrence of a native member of the same genus in the archipelago: Psidium galapageium. The tools that have been used to explore the invasion of P. guajava could also be used to explore the effects of the co-occurrence of both species in the same spatial context. Firstly, a deeper exploration of the adaptive processes occurring on both species and their signature at the molecular level could allow us to quantify the degree to which both species compete directly with each other, and to account for the toll that P. guajava places on its endemic counterpart. Secondly, a complete analysis of the potential for hybridization between these two species can account for the potential noxious effects that P. guajava could place on the genetic diversity of the P. galapageium populations in the archipelago.The purpose of describing an invasive process such as this is to ultimately recognize the extent of the risk it poses to the local flora and fauna, and to assist in the development of effective control and mitigation strategies. The case of S1 FileP. guajava population. Pop2/N2: Present Santa Cruz P. guajava population. Pop3/N3: Present San Cristobal P. guajava population. N1b: Isabela first P. guajava colonizers.N2b: Santa Cruz first P. guajava colonizers.N3b: San Cristobal first P. guajava colonizers.Time is not shown to scale and is measured as number of generations, considering t2> = t1. Pop1/N1: Present Isabela (PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 FigF values were obtained by computing the likelihood function.(TIF)Click here for additional data file.S2 FigThe results are indicated for K = 2, this being the optimum K value (ΔK = 19.62). The values of K correspond to the number of clusters (represented by different colors) in which are grouped the sampled individuals. White dotted lines separate different regions.(TIF)Click here for additional data file.S3 FigThe results are indicated for K = 4, this being the optimum K value (ΔK = 39.57). The values of K correspond to the number of clusters (represented by different colors) in which are grouped the sampled individuals. White dotted lines separate different regions (GR = Granillo Rojo).(TIF)Click here for additional data file.S4 FigThe results are indicated for K = 2, this being the optimum K value (ΔK = 1.83). The values of K correspond to the number of clusters (represented by different colors) in which are grouped the sampled individuals. White dotted lines separate different regions.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableThe names of the linked loci appear in rows and columns, whereas population names appear as entries in the table. ISA = Isabela population; SCY = San Cristobal population. No LD was found in Santa Cruz.(DOCX)Click here for additional data file.S3 TableResults shown correspond to those after Bonferroni correction.(DOCX)Click here for additional data file.S4 TableThe mean frequencies over the three populations are shown as well.(DOCX)Click here for additional data file.S5 TableMissing data was ignored for this analysis.(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


The frequency of r2 values greater than 0.75 was lower in the biparental population (0.2–0.5%) and higher in the other populations (0.2–1.6%). The average distance for SNPs with r2 values greater than 0.75 was much higher in the biparental population (approximately 80 to 126 kb). In the other populations, the ranges were approximately 6 to 19 and 6 to 35 kb. The heatmaps for the regions covered by the first 100 SNPs in each chromosome, in each population , provided evidence that the comparatively few high r2 values (close to 1.0) occurred only for SNPs in close proximity, especially in the synthetic and breeding populations. Due to the reduced number of SNPs in the haplotype blocks (2 to 3) in the populations, it is not expected advantage of a haplotype-based association study as well as genomic selection along generations. The results concerning LD decay (rapid decay after 5–10 kb) and LD decay extent  are in the range observed with maize inbred line panels. The LD maps indicate that maize chromosomes had a pattern of regions of extensive LD interspaced with regions of low LD. However, our simulated LD map provides evidence that this pattern can reflect regions with differences in allele frequencies and LD levels (expressed by |D'|) and not regions with high and low rates of recombination.Linkage disequilibrium (LD) analysis provides information on the evolutionary aspects of populations. Recently, haplotype blocks have been used to increase the power of quantitative trait loci detection in genome-wide association studies and the prediction accuracy of genomic selection. Our objectives were as follows: to compare the degree of LD, LD decay, and LD decay extent in popcorn populations; to characterize the number and length of haplotype blocks in the populations; and to determine whether maize chromosomes also have a pattern of interspaced regions of high and low rates of recombination. We used a biparental population, a synthetic, and a breeding population, genotyped for approximately 75,000 single nucleotide polymorphisms (SNPs). The sample size ranged from 190 to 192 plants. For the whole-genome LD and haplotype block analyses, we assumed a window of 500 kb. To characterize the block and step patterns of LD in the populations, we constructed LD maps by chromosome, defining a cold spot as a chromosome segment including SNPs with the same LDU position. The LD and haplotype block analyses were also performed at the intragenic level, selecting 12 genes related to zein, starch, cellulose, and fatty acid biosynthesis. The populations with the higher and lower frequencies of |D'| values greater than 0.75 were the biparental (65–74%) and the breeding population (26–58%), respectively. There were slight differences between the populations regarding the average distance for SNPs with |D'| values greater than 0.75 (in the range of approximately 207 to 229 kb). The level of LD expressed by the r The statistic D' is the ratio between D .P(ab)–P(Ab).P(aB)) and the deviation of the actual gametic frequency from linkage equilibrium [2 is the square of the correlation between the values of alleles at two loci in the same gamete, where D is the covariance [Linkage disequilibrium (LD) analysis is important to humans, other animal species, and plant geneticists because the results can be used for positional cloning, provide information on the rate of recombination, gene conversion, and evolutionary aspects of populations, including recombination history, mutation, selection, genetic drift, and admixture, and allow for the selection of populations and single nucleotide polymorphisms (SNPs) for association studies . The mosvariance .et al. [et al. [et al. [Additional information on historical recombination is provided by analysis of the haplotype block pattern in populations. A haplotype block is a chromosome region in which there are few haplotypes  (2–4 per block), and for which the LD analysis provides evidence of a low rate of recombination . Recentl [et al.  observed [et al. . Their cet al. [et al. [2 between 0.23 and 0.61, LD decay after 5–10 kb, and LD extent along 200–300 kb. Faster LD decay and shorter LD extent (less than 4 kb) were observed by Maldonado et al. [Characterization of the LD and haplotype block patterns in human, domesticated animal, and plant populations has provided variable results concerning the degree of LD, LD decay, LD decay extent, and number and length of the haplotype blocks. Most maize LD studies have been done with inbred line panels. Thirunavukkarasu et al.  and Trun [et al.  observedo et al. . The numo et al. .In several investigations in human populations, the structure of LD was described based on LD maps. In an LD map, each SNP has an LD position in LD units (LDUs). One LDU is the distance in kilobases at which disequilibrium  declines to approximately 0.37 of its starting value. Assuming unrelated individuals, ρ equates to the absolute value of D'. The difference between the LD positions of two SNPs divided by the distance in kilobases (d) is the exponential decline of disequilibrium (ε). LDUs share an inverse relationship with the recombination rate. Thus, regions with extensive LD have few LDUs (plateaus or blocks), and regions with many LDUs have high levels of recombination rate (steps). Holes in the LD maps are regions where greater marker density is required to provide a full characterization of the block and step patterns of the LD. Holes are identified by an LD map interval of 3, which is an arbitrary value because disequilibrium is indeterminate for εd > 3 and of doubtful reliability for εd > 2 , 11.Because there is no information on LD and the structure of haplotype blocks in popcorn populations and no LD maps for maize, the objectives of this study were: (1) to compare the degree of LD, the LD decay, and the LD decay extent in popcorn populations; (2) to characterize the number and length of haplotype blocks in the populations; and (3) to elaborate the first LD map for maize, for elucidating whether maize chromosomes also have a pattern of interspaced regions of high and low rates of recombination.2 generation) temperate population, a tropical synthetic (Synthetic UFV), and a tropical breeding population (Beija-Flor cycle 4). A biparental population is the most used maize population for deriving doubled haploids and inbred lines in hybrid breeding. Maize synthetic varieties are used as germplasm sources in breeding programs or as improved populations in developing countries. Theoretically, a biparental population shows LD only for linked genes and molecular markers. In a synthetic there is LD for genes and molecular markers with independent assortment. Because selection can change the LD degree, we also included a breeding population. The biparental population was derived from the single cross AP4502, developed by the Agricultural Alumni Seed Improvement Association, Romney, IN, USA. Synthetic UFV and Beija-Flor cycle 4 (BFc4) were developed by the Federal University of Viçosa (UFV), Minas Gerais, Brazil. The synthetic was derived by random crossings involving 20 elite inbred lines from the tropical population Viçosa and 20 elite inbred lines from the tropical population Beija-Flor. The inbred lines were selected based on expansion volume . Beija-Flor cycle 4 was developed after four cycles of half-sib selection based on expansion volume.We used a biparental (FLeaf samples of young plants were collected for DNA extraction. The DNA extraction was performed using the CTAB (cetyl trimethylammonium bromide) protocol with modifications. After quantification, the DNA samples of 574 plants (190 or 192 from each population) were sent to the Institute of Biotechnology at Cornell University  and Institut de Recherche en Immunologie et en Cancérologie/IRIC at University of Montreal  for GBS services based on HiSeq 2500 (paired-end reads of 125 bp) and NextSeq500 (single-end reads of 85 bp), respectively. The SNP variant call services were provided by the Institute of Biotechnology and Omega Bioservices, Norcross, GA, respectively, using B73 version 4 (current version) as the reference genome , we filtREALbreeding software (available by request). This software has been recently used in studies on population structure [For Hardy-Weinberg equilibrium analysis by population and chromosome, the Bonferroni criterion was adopted to keep a global level of significance of 1%. To characterize the block and step patterns of LD in the populations, we constructed LD maps by chromosome using the interval method . We defitructure , QTL maptructure , genomictructure , and gentructure . We simu2 values, defining intervals of 50 kb (0–50 to 451–500). To define a haplotype block, we adopted the criterion proposed by Gabriel et al. [We then evaluated the degree of LD by chromosome in the populations concerning SNPs separated by up to 500 kb, using a two marker expectation-maximization (EM) algorithm . For thel et al. . The hapl et al.  to generl et al. .2 values defining intervals of 1 kb (0–1 to 10.1–11 kb). All analyses were performed using LDMAP [Haplotype blocks summary) developed in REALbasic 2009 by Prof. José Marcelo Soriano Viana.The LD and haplotype block analyses were also performed at the intragenic level. We choose 12 genes related to zein (one), starch (four), cellulose (five), and fatty acid biosynthesis (two) . With twng LDMAP  and Haplng LDMAP . HeatmapWith the exception of chromosome 10 in the breeding population, the number of SNPs was generally in proportion to the chromosome length, providing an SNP density in the range of 23.5 to 44.3 kb (one SNP per 30.0 kb on average) . The aveThe LD map from the simulated data provided evidence that the LD units were lower for the generation with lower LD (generation 10) . Thus, t2 value in the biparental population was approximately half of the corresponding average values observed in the other populations . Regardless of the chromosome, the populations with the higher and lower frequencies of |D'| values greater than 0.75 were the biparental population (65–74%) and the breeding population (26–58%), respectively. However, the frequency of r2 values greater than 0.75 was lower in the biparental population (0.2–0.5%) and higher in the other populations (0.2–1.6%) . In the other populations, the ranges were approximately 6 to 19 and 6 to 35 kb. There were slight differences between the populations regarding the average distance for SNPs with |D'| values greater than 0.75 (in the range of approximately 207 to 229 kb).Concerning SNPs separated by up to 500 kb, the biparental population and the synthetic had similar average |D'| values (0.77 and 0.75). The values were approximately 10–14% greater than the average value in the breeding population . Interes.2–1.6%) . Further2 values (close to 1.0) occurred only for SNPs in close proximity, especially in the synthetic and breeding populations , provided evidence that the comparatively few high rulations . Althougulations  there ar2, on average) and then a gradual decrease to the minimum LD value for SNPs separated by 451–500 kb. Because there were no significant differences between chromosomes, we can state that following an initial higher decrease after 50 kb, the |D'| and r2 in the biparental population extended with similar magnitude for an interval of 450 kb   were only observed for the closest SNPs . The biparental population presented an average rnd 0.14) . Regardlest SNPs  Figs. Thest SNPs . With reynthetic . Concernynthetic . Genes Zet al. [2 statistic (0.004 versus 0.038).It is difficult to characterize the LD and haplotype block patterns in two or more unrelated random cross populations based on an LD map and two measures of linkage disequilibrium. Based on studies of the LD pattern in human populations, LD maps demonstrated that the human chromosomes have a pattern of regions of extensive LD (plateaus or cold spots), interspaced with regions of high recombination rate (steps or hot spots) , 26. Botet al. . To bett2 should provide a comparable characterization of the LD pattern if there are similar allele frequencies. If the populations have distinct distributions of allelic frequencies, D' can be used for analyzing the recombination history, and r2 should be the choice if recombination and mutation are important factors affecting the LD [2 have been used [2 is the most relevant for association mapping because it has a simple inverse relationship with the sample size required to detect association [When comparing populations that share a common origin, have a similar effective population size, and did not face an extreme reduction in size (population bottleneck), the statistics D, D', and rg the LD . Howeverg the LD , 27. In een used , and beceen used . The staociation . The use2 values and the reduced frequency of SNPs with r2 values greater than 0.25 (defined as useful LD in some studies) in the popcorn populations. In the study of Yan et al. [2 was only 0.009. However, for SNPs separated by up to 100 kb, the average was 0.2 . Even higher LD values were reported in the maize NAM (nested association mapping) population [et al. [2 values observed in the popcorn populations are also lower than the values observed in cattle and chicken populations (0.1 to 0.8 for SNPs separated by up to 100 kb) [et al. [2 averaged across all chromosomes of 0.015 and 0.016 for the Dutch and HapMap-CEU populations, respectively.Because of the differences regarding molecular marker type and density, sample size, and genome coverage, comparison of LD values of human, domesticated animal, and plant populations should be made with caution, even when the studies involve the same species. We were surprised by the low average rn et al. , involvipulation  and in t 100 kb) –34. The  [et al.  observedet al. [et al. [2 dropped below 0.8, and LD extent as the distance over which the average r2 fell below 0.2. Concerning LD decay, our results showed differences between LD measures and populations. There were slight differences between chromosomes, but the higher r2 decay occurred after 5–10 kb (36 to 73%). Yan et al. [2 value of 0.01 in the interval of 1–5 Mb (LD extent of 5 Mb). A similar LD extent (5 Mb) was observed in eight breeds of cattle, but a comparable LD decay (62%) occurred along 100 kb [et al. [2 value of 0.05).The absence of a uniform criterion for defining the LD decay and the LD extent also makes comparison of the results with human, domesticated animal, and plant populations difficult. Angius et al.  used LD  [et al.  used LD n et al.  observedg 100 kb . From th 5–10 kb  to 73%. 2. The criterion of Gabriel et al. [et al. [et al. [If there is a higher LD between QTLs and haplotypes than with individual SNPs, haplotype blocks can provide substantial statistical power in association studies  and incrl et al.  appears  [et al.  observed [et al. , 33. Low [et al. , 28. How [et al. , from le2 values observed in the synthetic and breeding populations (for 11 of the 12 genes) are due to selection for quality. Characterization of the LD and haplotype block patterns regarding specific chromosomal regions has only been made by human geneticists, generally aimed at SNP tagging. From the analysis of SNPs within the HLA region on chromosome 6, Evseeva et al. [et al. [et al. [et al. [2 value for the five genes in the region ranged from 0.03 to 0.89.Concerning the low intragenic LD and the minimum size of the haplotype blocks observed in the three populations, we believe that the lower LD for the biparental population is due to crossing two genetically similar high-quality inbred lines. Because there is no information on the LD and haplotype block patterns in the base populations Viçosa and Beija-Flor, we cannot infer that the higher average intragenic ra et al.  observed [et al.  observed [et al.  identifi2 values in the three popcorn populations with different genetic structures—a biparental population, a synthetic, and a breeding population—is low but comparable to some non-isolated human populations. This finding does not imply that these populations cannot be used for GWAS because there is a fraction of high r2 values for SNPs separated by less than 5 kb. The populations are also not excluded for genomic selection because the most important factor affecting this selection process is the relatedness between individuals in the training and validation sets. However, we do not expect a significant advantage from haplotype-based GWAS and genomic selection along generations due to the reduced number of SNPs in the haplotype blocks (2 to 3). The results on LD decay (rapid decay after 5–10 kb) and LD decay extent  are in the range observed with maize inbred line panels. Our most important result is that, similar to human chromosomes, maize  chromosomes also have a pattern of regions with extensive LD (plateaus or cold spots), interspaced with regions of low LD (steps or hot spots). It should be highlighted, however, that our simulated LD map provides evidence that this pattern can reflect regions with differences in allele frequencies and LD level (expressed by D') and not regions with high and low rates of recombination as evidenced by Jeffreys et al. [In conclusion, the level of LD expressed by the rs et al. , since tS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 FigMAF distribution in the biparental population (a), in the synthetic (b), and in the breeding population (c).(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 FigAverage |D'| values by chromosome and by distance interval (kb) in the biparental population (a), in the synthetic (b), and in the breeding population (c).(PDF)Click here for additional data file.S6 FigAverage r2 values by chromosome and by distance interval (kb) in the biparental population (a), in the synthetic (b), and in the breeding population (c).(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 FigOverall intragenic |D'|  and r2  by distance interval (bp) in the biparental population (a and d), in the synthetic (b and e), and in the breeding population (c and f).(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.


Eranthis have not been previously studied. We investigated the speciation and establishment histories of four Eranthis species: Eranthis byunsanensis, E. pungdoensis, E. stellata, and E. pinnatifida. The sampling localities were Korea, Japan, Jilin in China, and the area near Vladivostok in Primorskiy, Russia. We used 12 chloroplast microsatellite loci  and two chloroplast noncoding regions . The genetic diversity, genetic structure, phylogenetic relationships of the four species were analyzed, and their ancestral areas were reconstructed. The high genetic diversity of the Jeju island population of E. byunsanensis and Russian populations of E. stellata indicated these species’ northward and southward dispersal, respectively. The genetic structure analyses suggest that the populations in these four species have limited geographical structure, except for the Chinese E. stellata population (SCP). The phylogenetic analyses suggest that E. byunsanensis and E. pinnatifida are sister species and that Chinese SCP may not belong to E. stellata. The ancestral area reconstruction revealed that the most recent common ancestor of the four species existed in the current Chinese habitat of E. stellata. This study shows that E. byunsanensis and E. pinnatifida originated from a southern Eranthis species and speciated into their current forms near Jeju island and near western regions of Japan, respectively, during the Miocene. E. stellata may have dispersed southward on and near the Korean peninsula, though its specific origin remains unclear. Interestingly, the Chinese E. stellata population SCP suggests that the Chinese population might be most ancient among all the four Eranthis species. E. pungdoensis may have allopatrically speciated from E. byunsanensis during the Holocene. The Korean peninsula and the surrounding areas can be considered interesting regions which provide the opportunity to observe both northern‐ and southern‐sourced Eranthis species.The temporal and spatial origins and evolution of the genus  This has led to diverse distributional patterns for a wide range of species in these regions. For example, Korea, which is surrounded on three sides by sea and contains large mountainous regions, exhibits high geographical complexity and provides the combinations of diverse geography and ecology for many taxa on this area. Also, interestingly, these regions can be appropriate for observing many closely related species exhibiting various distributions.Eranthis (Ranunculaceae) as an intriguing model for evolutionary study. The genus Eranthis is an early flowering herbaceous plant which has a tuberous rhizome, petaloid sepals, and palmately divided leaves. This genus is made up of 8–9 species that range from Europe to East Asia, and exhibits interesting distributions. This genus generally exhibits high levels of endemism and is distributed in both mainlands and islands. Its constituent species seldom co‐occur, and the sizes of these species’ distributional ranges usually vary much.To aid in our understanding of various distributional patterns of the taxa in these regions and investigate their origins and speciation histories based on these patterns, we propose the genus Eranthis. For example, previous research has investigated the genetic variation of the populations of the Korean endemic Eranthis byunsanensis  Eranthis stellata, found all across the Korean peninsula, in Jilin and Liaoning in China, in the western land near the Sea of Okhotsk and in Primorskiy Krai, in eastern‐most Russia; (b) Eranthis pinnatifida, endemic to Japan and found in the central and southern areas of Honshu; (c) E. byunsanensis, endemic to Korea and found across the Korean peninsula, with its northern limit possibly in North Korea; and (d) E. pungdoensis, endemic to Korea and found only on the small island of Pungdo in the Yellow Sea . The voucher specimens were deposited in the herbarium of Chungbuk National University.2.2Eranthis species were genotyped at 12 chloroplast microsatellite loci  which were randomly selected from the 24 cpSSR loci isolated by Oh and Oh  and five outgroup species, were sequenced at two chloroplast noncoding regions, rpl16 intron and petL‐psbE , with each run composed of 100,000 burn‐in steps, followed by 100,000 MCMC steps. The admixture model and the correlated allele frequencies model were applied. The optimal number of populations (K) was determined using Evanno, Regnaut, and Goudet  was performed using the cpSSR data to define groups of populations that are geographically homogeneous and maximally differentiated with each other , A. asiatica (ND), E. longistipitata (LC), E. albiflora (HR) used as outgroups. E. hyemalis (HYE), which is European Eranthis species, was excluded from this analysis, because the sequencing for rpl16 intron region in this species was not successful.Bayesian inference and maximum likelihood were applied in the reconstructions of the phylogenetic trees. The two chloroplast noncoding sequences, E Edgar,  using thE Edgar, , and manE Edgar, . One seqIn Bayesian inferences, in which tree topology and divergence times were sought, nucleotide substitution model of HKY + G + I and uncorrelated lognormal relaxed clock were applied, and the Yule process was chosen as a tree prior. The length of the MCMC chain was set to 800,000 generations, and parameters were logged every 200 generations. In TreeAnnotator 1.8.0  for E. stellata, A. asiatica, and C. simplex was determined to be 57.55 Ma with 95% highest posterior density of 56.41–59.21.Since we did not have any fossil record which can be used to estimate the divergence times, we obtained two calibration points from a previous phylogeographical study which included the genus rpl16 intron region and petL‐psbE region were analyzed separately; for the petL‐psbE region, E. hyemalis was included in the outgroup, because the sequencing was successful.Maximum likelihood phylogenetic trees were constructed in RAxML 7.0.3  South Korea , including a small island Pungdo in the Yellow Sea (E. pungdoensis); (B) Jilin in China (E. stellata); (C) the area around Vladivostok in Primorskiy, Russia (E. stellata); and (D) Japan (E. pinnatifida).We performed a statistical dispersal vicariance analysis (S‐DIVA) in the software RASP 4.0  than the populations on the Korean peninsula (H_e = 0.439–0.876) and the Jilin region of China (H_e = 0.286–0.821). For E. byunsanensis, the southernmost Jeju island population (BJ) was more genetically diverse (H_e = 0.837) than the populations on the Korean peninsula (H_e = 0.353–0.739). For E. pinnatifida, in general, the Hiroshima populations tended to exhibit higher genetic diversities (H_e = 0.572–0.674) than other populations (H_e = 0.254–0.709). For E. pungdoensis, its only population (H_e = 0.582) seems to need other approaches to be clearly described in the perspective of genetic diversity.The genetic variation of the 33 populations from 3.2E. byunsanensis, E. pungdoensis, and E. pinnatifida were mostly assigned to cluster 1, and E. stellata to cluster 2. This shows that E. byunsanensis, E. pungdoensis, and E. pinnatifida are genetically closely related, while E. stellata does not have a significant relationship with the other three species  were assigned to cluster 1, and two E. byunsanensis populations  and a population of E. pungdoensis (P) were mostly assigned to cluster 2  and the two Chinese populations  were assigned to cluster 1, and the others were assigned to cluster 2  was chosen for its best grouping. In the case of E. byunsanensis and E.  pungdoensis, two groups were identified—(a) BJ and (b) BA, BG, BM, BS, BU, P—with the highest FCT value of 0.98465. This grouping was inconsistent with the results of STRUCTURE analysis in which BJ was clustered with three other populations. Interestingly, the population P, which exists on Pungdo, a small Korean island in the Yellow Sea, was grouped with other five E. byunsanensis populations, even though it's classified as separate species. For E. pinnatifida, the highest FCT value (0.88036) was for five groups—(a) PS5; (b) PS2; (c) PH5; (d) PH8, PH9; (e) PY. For E. stellata, the optimal number of groups with the highest FCT (0.84619) was two—(a) SCP and (b) all the other 19 populations. It appears that this grouping does not reflect any distinct geographical patterns, meaning closer investigation is required to determine the factors which differentiated SCP population from other E. stellata populations, especially Chinese populations.In the SAMOVA for each species, the number of population groups with the highest E. byunsanensis and E. pungdoensis, 98.47% of all the variation was partitioned among population groups, 1.52% among populations within groups, and 0.02% within populations , and E. pinnatifida were distinctly grouped by species  and the MRCA of E. pinnatifida, E. byunsanensis, and the population SCP, appears to have taken place during the early Oligocene (27.15 mya). The divergence between the population SCP and the clade including E. byunsanensis, E. pinnatifida is likely to have occurred during the late Oligocene (24.60 mya). The divergence of E. pinnatifida and E. byunsanensis dates to the early Miocene (15.38 mya), showing reciprocal monophyly. In each species of Eranthis, many splits between the populations took place during the Pleistocene. In the case of E. stellata, interestingly, clustering patterns of some populations from China, Korea, and Russia were quite inconsistent with their geographical distributions —E. byunsanensis, E. pungdoensis ‐ and Japanese endemic E. pinnatifida (D) was a vicariant event, and that in E. stellata, some divergences between Chinese (B), Korean (A), and Russian (C) populations were also vicariances  was determined as an ancestral range. Overall, seven dispersal events and ten vicariant events are thought to have occurred.S‐DIVA analysis revealed that the divergence between Korean endemic s Figure . For the44.1E. byunsanensis and E. pungdoensis populations, is the oldest of these populations. This means that the populations of these two species have primarily dispersed northward, even though it is fundamentally possible for them to disperse in any number of directions over a sufficiently long timescale. In contrast, according to our phylogeny reconstructions, the Jeju population diverged during Pleistocene  can be refuted by the Bayesian phylogeny, in which these two species are sister taxa, and the crown group of E. pinnatifida (7.03 mya) is slightly younger than that of E. byunsanensis (9.47 mya) , in the western sea of the Korean peninsula, and in East China Sea, since at some points in the past, these regions were all connected by land bridge  Figure . In contdge Ota, . HoweverEranthis species a long time ago, and may have acted as a dispersal route for this species and many other plants. In particular, E. byunsanensis inhabits many southern Korean islands between the Korean peninsula and the Jeju island, suggesting that in the past when Jeju and the Korean peninsula were connected, many populations of E. byunsanensis were located in the southern regions near the Jeju island. When assuming the speciation from an Eranthis species in eastern China, western sea of Korea, or East China Sea, even though we cannot conjecture the exact location of the speciation event, we can at least roughly infer that most probable site of speciation would have been near Jeju island. In the previous study, which analyzed genetic variation in E. byunsanensis, E. pungdoensis, and E. pinnatifida,  were the last to diverge of the six E. pinnatifida populations , this divergence time not being very far from the divergence between other E. stellata populations and the clade of E. byunsanensis, E. pinnatifida, and SCP population  and southern‐sourced  species of Eranthis, and these regions are phylogeographically valuable in terms of determining complex origin patterns for closely related species. Also, the observation of unique Chinese SCP population provides the opportunity to infer the ancestral location for these four focal species, thereby adding an important description to their evolutionary history.When combining the possible origins of these None declared.Byoung‐Un Oh contributed to project design, sample collection and writing manuscript. Ami Oh contributed to project design, sample collection, lab work, data analysis and writing manuscript. Click here for additional data file.