Geng (japonica) rice grown the temperate regions as well as in tropical high lands worldwide. To develop rice varieties with improved cold tolerance (CT) at the reproductive stage, 84 BC2 CT introgression lines (ILs) were developed from five populations through backcross breeding. These CT ILs plus 310 random ILs from the same BC populations were used for dissecting genetic networks underlying CT in rice by detecting QTLs and functional genetic units (FGUs) contributing to CT. Seventeen major QTLs for CT were identified using five selective introgression populations and the method of segregation distortion. Of them, three QTLs were confirmed using the random populations and seven others locate in the regions with previously reported CT QTLs/genes. Using multi-locus probability tests and linkage disequilibrium (LD) analyses, 46 functional genetic units (FGUs) (37 single loci and 9 association groups or AGs) distributed in 37 bins (~20%) across the rice genome for CT were detected. Together, each of the CT loci (bins) was detected in 1.7 populations, including 18 loci detected in two or more populations. Putative genetic networks (multi-locus structures) underlying CT were constructed based on strong non-random associations between or among donor alleles at the unlinked CT loci/FGUs identified in the CT ILs, suggesting the presence of strong epistasis among the detected CT loci. Our results demonstrated the power and usefulness of using selective introgression for simultaneous improvement and genetic dissection of complex traits such as CT in rice.Low temperature stress is one of the major abiotic stresses limiting the productivity of  Oryza sativa L.) grows worldwide. There are two major subspecies of O. sativa, Xian (indica) or Geng (japonica) [Geng rice crops grown primarily in high-latitude or high-altitude regions of China, Japan, Korea, and other parts of the world, low temperature, or cold stress, is one of the most common environmental stresses affecting rice growth and development, and thus grain yields. Cold stress includes two major classes, chilling (0–15°C) and cold deep-water irrigation (CDWI) (18–19°C) stress, both of which are major environmental factors limiting rice productivity [As the staple food for the half world population, rice (aponica) , the foructivity ,3. The fqCT8, qCTB7, qCTB3 and qCT-3-2, have been fine mapped [qCTB10-2 was delimited to a 132.5 kb region containing 17 candidate genes and 4 genes were cold-inducible [Ctb1 and CTB4a, have been cloned and functionally characterized [Ctb1 encodes an F-box protein and functions as part of the E3 ubiquitin ligase complex that is associated with increased spikelet fertility (SF) under cold stress [CTB4a encodes a conserved leucine-rich repeat receptor-like kinase [Geng rice remains slow by the conventional pedigree method. This is because almost all crosses have been made between different varieties of the same Geng subspecies, which is known to have very low genetic diversity [Xian varieties as donors because of the genetic barriers such as hybrid sterility and hybrid breakdown commonly seen in Xian-Geng crosses.CT in rice is a quantitative trait controlled by multiple genes and involving complex biological and genetic mechanisms. To date, QTL mapping using bi-parental or multiple cross populations identified more than 250 QTLs affecting CT at different development stages of rice –10. Mappe mapped , 11–13, nducible , and onlcterized ,16. Ctb1iversity . BreederTo overcome the problem of breeding and genetic research being separate activities in the past study, we have been practicing a strategy of simultaneous improvement and genetic dissection of complex traits by selective introgression, which have been demonstrated to be powerful in several successful applications –27. This2F4 populations derived from crosses between Chaoyou1 , a high yield Geng variety, and 5 donors  (2F4 ILs developed by two rounds of selection for cold tolerance (CT) at the reproductive stage under cold water, as described previously [2 populations without any selection.The materials used in this study included two sets of introgression lines (ILs) from five BCd India) . The fireviously . The sec2F4 bulk populations were sown in the seedling nursery on April 15, 2008, and 450 40-day seedlings of each BC2F4 bulk population were transplanted into a 45-row plot at the Rice Research Institute of Jilin Academy of Agricultural Sciences (JAAS). When the RP (CY1) entered the panicle initiation stage, the cold-water treatment was initiated by constant irrigation of cold water (19± 0.5°C). The cold-water depth in the pond was 20 cm and the treatment was maintained for ~30 days until panicles of almost all plants exerted completely. Then, irrigation with normal temperature water was resumed until the maturity. Under this cold-water treatment, CY1 had spikelet fertility (SF) of 24.8±4.3%, then, 162 BC2F4 plants with SF>50% were selected. The selected 162 BC2F5 introgression lines (ILs) from the five populations were progeny tested under the same condition in 2009. Under this cold-water treatment, CY1 had a mean SF of 35.1±3.5%, then, 132 lines with SF>48% were selected. Further progeny testing of the 132 CT ILs under the cold water treatment in 2010 resulted in final selection of 84 CT ILs (SF>40%) from the five populations were used for genotyping and genetic dissection of CT. Performance of ILs and recipient parent  for SF during the first two rounds of selection under cold water stress were shown in In the summer of 2010, the phenotyping experiments of the two sets of ILs were conducted in two locations, Beijing and Jilin. On 15 April 2010, seeds of the 412 ILs (including 132 CT ILs and 310 random ILs) were sown in the seedling nursery and 15 40-day-old seedlings of each IL were transplanted into a single-row plot the fields in the sheltered pond of JAAS. The field experiment was randomized with two replications and CY1 was inserted in every 50 lines as the check. The LT treatment using cold water was the same as in 2008 and 2009. At maturity, eight representative plants in the middle of each plot were sampled and then eight main panicles of each sampled plant were measured for spikelet number per plant, filled grain number per plant and SF. Under this cold water treatment, the CY1 had a mean SF of 10.5%. In Beijing, seeds of all ILs and CY1 were sown on May 5, and 15 30-day old seedlings of each IL were planted into a one-row plot with a spacing of 25 × 15 cm. The field arrangement was the same as in the LT treatment. Heading date  was visually recorded for all plots when ≥50% of the plants in a plot had headed. At the maturity, five plants of each plot under the cold-water and normal conditions were evaluated for plant height (in cm), panicle length (in cm), spikelet number per panicle (SN), filled grain number per panicle (GN), the spikelet fertility , panicle number per plant (PN), 1000-grain weight , biomass  per plant, grain yield per plant  and harvest index (HI) in the same way as described previously .2F6 ILs with SF>30% and the 310 random BC2F4 ILs (ranging from 59 to 65 per population) were used for genotyping (http://www.gramene.org) were used to survey the polymorphism between CY1 and the donors, from which an average of 113 well-distributed polymorphic SSR markers were used to genotype the selected and random ILs. Marker positions and genetic distances between linked markers were based on the Cornell SSR maps , which covered all 12 rice chromosomes with a total genome size range from 1,125.0 cM in population CY1/Yuanjing7 to 1649.8 cM in population CY1/Chhomrong, with average genetic distances between adjacent markers ranging from 15.2 to 23.7 cM.In the 2010 cold treatment experiment when CY1 had SF of 10.5%, 84 BCnotyping . More tht-tests. In this method, we used the minimum threshold of P<0.05 because of the small population sizes of the random populations. The third methodology was more comprehensive and was used to detect QTL and QTL networks in three steps based on the molecular quantitative genetics theory [r (r≥2) unlinked but perfectly associated loci of equal introgression in CT ILs selected from each BC population. Two types of statistical tests were performed to detect FGUs associated with ST. First, X2 tests were performed to detect whether the allelic and genotypic frequencies at individual loci across the genome in ST ILs from each BC population deviated significantly from the random unselected populations. Second, a multi-locus probability test, PAG)( = (pi)rm • (1−pi)r(n−m), was used to detect individual AGs for CT, where pi is the frequency of the donor introgression in the random ILs from each BC population, n is the number of the selected ILs, m is the number of ILs that have co-introgression of the donor alleles, and (n−m) is the number of ILs having no introgression at the r unlinked loci in the AG. Here (Pi)m is the probability of m ILs having co-introgression of the donor alleles and (1-Pi)n-m is the probability of (n-m) ILs having no introgression at r unlinked loci. The threshold to claim a significant case was P ≤ 0.005 for individual cases. For any detected AG of r (r ≥ 2) perfectly associated loci, there will be r·(r–1)/2 significant pairwise associations between the r loci, which were also confirmed by the linkage disequilibrium (LD) analyses [Three different approaches were used to detect QTL and genetic networks underlying CT that were segregating in the five populations. First, we used the segregation distortion method to detect main-effect QTLs in the selected IL populations. This method takes advantage of several populations together and performs a joint analysis to detect QTL that are respond to directional selection in multiple populations based on a consensus map . We alsos theory , 29. In analyses . Becausep ≤ 0.05), and FGUs in different groups were either independent, or negatively associated; and 2) all associated FGUs within each group were connected, forming multiple layers, according to their progressively reduced FG frequencies and inclusive relationships [To reveal the multi-locus structure of the detected FGUs–the putative genetic network underlying ST in the ILs from each BC population, pairwise gametic LD analyses were performed to characterize the relationships between alleles at all ST FGUs detected in ILs from each BC population , 29. Theionships .Geng donor to CY1) up to 49.1% increased SF for ILs from CY1/Fengaizhan (Xian). The average yield advantage over CY1 ranged from 200% for ILs from populations CY1/Doddi and CY1/X22 to 400% for those from population CY1/Fengaizhan. This improved CT was also associated with an average 36.7% increased biomass, 287% increased number of filled gains/panicle, 35.9% increased harvest index, 11.8% increased height, 9.1% increased grain weight, and 4% delayed heading under the cold-water stress. Under the non-stress conditions, the selected ILs showed same trait values as CY1 except for significantly increased height and reduced HI by 12.7cm and 3.4%, as well as slightly increased SN and delayed heading by 11.1 spikelets/panicle and 3 days. In particular, those selected ILs from population CY1/Fanaizhan had apparently better CT than those from other populations, while ILs from Yunjing7 showed the lowest level of CT.When compared with the normal conditions, the cold-water stress of 2010 caused delayed heading by 14 days and greatly reduced trait values of CY1 for all measured traits . This trGeng subspecies  but higher heterozygosity at 2.9%, when compared to the selected ILs.However, ILs from each of the random populations showed significantly lower SF than CY1 under both the normal and cold-water conditions, except for ILs from populations CY1/Yunjing7 and CY1/Chhomrong derived from parents of the same bspecies . Thus, tqCT1.3, qCT6.7 and qCT9.6 were detected in two of the populations, while qCT6.5 was detected in three populations  were validated in one or two of the random populations  on rice chromosomes 1, 2, 3, 4, 6, 9, 11 and 12 when five populations were combined using a consensus linkage map , Table 3ulations .2 tests (single loci) and multi-locus linkage disequilibrium analyses in 84 cold-tolerant introgression lines (ILs) selected from five populations  (37 single loci and 9 association groups or AGs) for CT were detected by χulations  and distulations . The numulations . These FagCTA1 consisting of three unlinked but perfectly associated loci at RM556 (bin8.4), RM257 (bin9.6) and RM228 (bin10.6) in the downstream. Sub-branch 2 had RM207 (bin2.9) in the upstream and RM154 (bin2.1) in the upstream. agCTC1 (bins 2.8 and 9.7), RM303 (bin4.6) and RM209 (bin11.5) in the downstream. The fourth network (agCTD2 in the downstream. agCTD2 comprises four unlinked but perfectly associated loci near RM315 (bin1.11), RM227 (bin3.12), RM87 (bin5.8) and RM247 (bin12.2) detected with a P value of 7.3−15. Branch D-3 was agCTD3 consisting of two unlinked but perfectly associated loci near RM207 (bin2.9) and RM434 (bin9.5) detected with a P value of 1.8−13. Branch D-4 was agCTD1 consisting of two unlinked but perfectly associated loci near RM7 (bin3.5) and RM282 (bin3.6). The fifth network  and RM216 (bin10.2). Branch E-3 contained 3 unlinked but highly associated loci with RM341 (bin2.5) in the upstream, and RM274 (bin5.8) and RM185 (bin4.4) in the downstream , qCT2.4 (between RM29 and RM341), qCT3.5 (between OSR13 and RM7), qCT3.12 (RM85), qCT4.2 (RM518), qCT4.6 (between RM303 and RM317), qCT6.6 (between RM3 and RM439), qCT9.7 (between RM278 and RM160) and qCT11.5 (between RM457 and RM21). Three of these large-effect CT QTL  were also reported previously [Taking above results together, we found the following nine CT QTL were detected in multiple populations and had high introgression (large effect) in the upstream of genetic networks, suggesting these regions may contain putative regulatory genes for CT. These included eviously , while teviously , we founGeng breeding efforts for improved CT at the reproductive stage, donors for CT are almost exclusively Geng varieties that are known to be more tolerant to cold than Xians. However, we previously demonstrated that Xian varieties could be used as donors for improving CT in Geng varieties [Xian donors in three of the five populations used in this study were from Vietnam, India and south China, and are known to have poor CT. Interestingly, they all appeared to be good donors for CT as far as the number of selected CT progeny concerned. This was true for tolerances to other abiotic stresses such as drought, salinity and submergence in most BC breeding populations involving a diverse set of donors and recipients [In arieties . In factcipients –21, 23. cipients , 31, 32,cipients , 29, theGeng) genetic background as compared with the Mendelian expectation, which was in contrast to the general genomewide over-ingression in ILs of several Xian genetic backgrounds [Geng genomes have smaller pan genome size but larger core genome than Xian genomes [Geng genomes have much higher “genetic load” than the Xian genomes [2F2 plants.The first one was the genomewide under-introgression of the donor genomes in both the random and selected ILs in the CY1  had the introgressed donor alleles at most C-1 loci. Theoretically, pronounced non-random associations between or among unlinked loci in selected progeny of a bi-parental cross suggest the presence of strong epistasis among genes acting in a same positively regulated pathway that leads to the target trait (CT) under strong directional selection . If so, Clearly, the putative genetic networks detected in this study should be verified. While it would be very difficult to clone and verify the molecular functions of the detected CT loci individually using the classical approaches of map-based cloning or of molecular biology, it is not difficult to confirm the putative CT genetic networks genetically. A straightforward strategy is to break, by recombination, each identified network or AG into individual loci and evaluate their effects individually in the progeny derived from the crosses between different ILs or between crosses between the ILs and CY1. Also, various omic tools and bioinformatic analyses can be very powerful to gain useful information for inferring the possible molecular mechanisms of the detected genetic networks by assessing the genomewide responses at different levels of carefully selected progeny from these crosses to cold stress , 34, 40.Geng rice. The Geng rice gene pool is known to have very limited genetic diversity [Xian donors contributed CT enhancing alleles at quite different loci to the Geng recipient, CY1, suggested that there is a rich source of hidden genetic diversity in the Xian gene pool for improving CT of Geng rice. Thus, our strategy of using BC breeding, strong phenotypic selection plus genetic tracking and characterization using DNA markers provides a good solution for broadening the genetic diversity in the Geng gene pool. Thus, genetic complementarity provides an appropriate explanation for the hidden diversity and transgressive segregation of CT observed in this study and other complex traits in rice [Our results have important implications for improving complex traits of iversity , 41. Our in rice –21. FinaqCT3.12, qCT6.7 and qCT9.6) were validated in random BC populations. In addition, 46 functional genetic units (FGUs) including 37 single loci and 9 association groups for CT were detected by χ2 tests and multi-locus linkage disequilibrium analyses. The findings reported herein may be useful for knowledge-based rice improvement of CT. Future research will focus on validating the effects of these putative genetic networks detected in this study. Our results demonstrated that selective introgression is powerful in simultaneous improvement and genetic dissection of complex traits such as CT in rice.Total of 17 QTLs for CT were detected in 84 cold-tolerant ILs selected from five BC2 populations in CY1 genetic background using a consensus linkage map, three of which (S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Salix psammophila (desert willow) is a shrub endemic to the Kubuqi Desert and the Mu Us Desert, China, that plays an important role in maintaining local ecosystems and can be used as a biomass feedstock for biofuels and bioenergy. However, the lack of information on phenotypic traits and molecular markers for this species limits the study of genetic diversity and population structure. In this study, nine phenotypic traits were analyzed to assess the morphological diversity and variation. The mean coefficient of variation of 17 populations ranged from 18.35% (branch angle (BA)) to 38.52% (leaf area (LA)). Unweighted pair-group method with arithmetic mean analysis of nine phenotypic traits of S. psammophila showed the same results, with the 17 populations clustering into five groups. We selected 491 genets of the 17 populations to analyze genetic diversity and population structure based on simple sequence repeat (SSR) markers. Analysis of molecular variance (AMOVA) revealed that most of the genetic variance (95%) was within populations, whereas only a small portion (5%) was among populations. Moreover, using the animal model with SSR-based relatedness estimated of S. psammophila, we found relatively moderate heritability values for phenotypic traits, suggesting that most of trait variation were caused by environmental or developmental variation. Principal coordinate and phylogenetic analyses based on SSR data revealed that populations P1, P2, P9, P16, and P17 were separated from the others. The results showed that the marginal populations located in the northeastern and southwestern had lower genetic diversity, which may be related to the direction of wind. These results provide a theoretical basis for germplasm management and genetic improvement of desert willow. Salix psammophila (desert willow) is a shrub mainly distributed in Northwest China and is endemic to the Kubuqi Desert and the Mu Us Desert. S. psammophila  is dioecious catkin-bearing and perennial shrub. It is about three to four m high. S. psammophila exhibits extraordinary adaptation to drought , and branch size. Plant leaves utilize light energy for carbon fixation; branching pattern is the main factor determining plant architecture, and is also an important trait for the use of plants as raw materials in the above-mentioned applications. Moreover, S. psammophila is a dioecious shrub, females and males may perform differently to ecological stress  phylogenetic analyses, principal coordinate analysis (PCoA) and population structure of S. psammophila. Further, those results would provide a basis for germplasm resource management as well as breeding programs.Therefore, we determined the variance of phenotypic traits and genetic diversity based on SSR, and tried to compare and analyze the genetic diversity and population structure of S. psammophila established in Ordos Dalad, Inner Mongolia in 2008. More than 1,000 genets were collected 21 populations from different areas. S. psammophila genets were randomly sampled within a 100 × 100 m2 plot at each population. The distance between each genets were at least 50 m. About 50 genets were collected from each population by phenotypic traits. We collected genets by phenotypic traits, including PH, ground diameter (GD), tree shape, branch color, and so on. According to phenotypic traits investigation, 3-year-old stem of branches with large and strong shrubs, no diseases and pests genets were selected to collect in the germplasm resource preservation library of S. psammophila.Research materials were collected from the germplasm resource preservation library of A total of 528 genets from 17 populations (P1–P17) were obtained from the germplasm collection to analyze population genetic structure and the association between SSR markers and specific phenotypic traits. There were 15 genets missing and recorded inaccurately in the phenotypic investigation, 22 genets of the same clones were detected by SSR markers; therefore, 491 genets were ultimately analyzed. The origins and locations of the 17 populations are shown in Phenotypic traits of 22–32 genets in each population were evaluated in 2013, including nine quantitative phenotypic traits. Sex was recorded of each genets. PH and GD were measured with meter stick, and BA was measured with a goniometer . Each geFresh and young leaves (0.2 g) were selected from each genet and total genomic DNA was isolated using a Plant Genomic DNA kit . The quality of DNA was verified by 1.2% agarose gel electrophoresis and a NanoDrop2000 spectrophotometer . The DNA was stored at −20 °C until used for PCR.S. psammophila including di- and tri-nucleotide repeats were randomly selected to design based on flanking sequences, and 125 pairs from 168 EST-SSR markers showed successful amplification , 2pmol forward primer, 8pmol reverse primer, 8pmol M13, and double-distilled H2O. The reactions were carried out in a 96-well ABI 9902 PCR instrument  under the following conditions: 94 °C for 5 min; 30 cycles of 30 s at 94 °C, 30 s at 57 °C, and 30 s at 72 °C; eight cycles of 30 s at 94 °C, 30 s at 57 °C, and 30 s at 72 °C; The last cycle was 72 °C for 10 min. PCR products was performed by capillary electrophoretic separation using an ABI 3730XL DNA Analyzer , and data were processed using Gene Marker v.2.2.0 software .A total of 168 EST-SSR markers from the de novo transcriptome of fication . A totalfication  and fluofication . The 20 http://darwin.cirad.fr/), and the results were used to construct an UPGMA hierarchical clustering dendrogram of the 491 S. psammophila genets. Moreover, generalized linear mixed models were used to estimate the effect of sex on phenotypic traits (2h) of phenotypic traits were estimated with the multivariate restricted maximum likelihood (REML) animal model , expected heterozygosity (He), and fixation coefficient (F). PIC_CALC v.0.6 (http://bioinformatics.org/populations/). The distance method was using K by setting K from 2 to 14.The microsatellite DNA allele counting-peak ratios method  was usedLC v.0.6  was usedLC v.0.6  between S. psammophila populations is shown in The phenotypic trait distribution of the 491 genets in 17 2h) for phenotypic traits, with the exception of LW and PH. For BA (2h = 0.255), LP (2h = 0.262) and GD (2h = 0.212), the 2h estimates were relatively high. For LL (2h = 0.163), LA (2h = 0.136), LPE (2h = 0.163) and LL/LW (2h = 0.136), the 2h estimates were relatively moderate  for shrubs, which are tufted. The seven phenotypic traits of the 491 genets were standardized and mean Euclidean distances obtained by the UPGMA method were used to construct a dendrogram . The cluStandardized average values of the seven phenotypic traits in different populations were used to generate a heat map of the 17 populations, which formed five clusters . ClusterS. psammophila populations from Northern China. The mean sample size of each population was 27.85. The mean number of alleles per locus (Na) was 7.36 (range: 5.36–8.32). The average number of different alleles per individual and locus was 2.33 (range: 2.14–2.43). The mean genotypic richness  was 14.5 (range: 6.68–18.00). Mean He (0.65) was higher than mean Ho (0.6), and mean PIC was 0.6 . A NJ phylogenetic tree was constructed based on the calculated genetic distances , . The cluK = 2 indicated that the populations could be clearly divided into two distinct subgroups. The highest proportion of genets assigned to the second population were P9 (21.2%), P16 (32.3%), and P17 (87.8%) at K = 2  to 38.52% (LA). The results demonstrated remarkable variation in the leaves of S. psammophila. However, sex did not strongly affect responses in this dioecious species  to LW (2h = 0.031), suggesting that most of the trait variation were caused by environmental or developmental variation. In the present study, the experimental materials were selected from the germplasm resource preservation library with same growth environment. This might be a reason why the heritability values were higher than for S. herbacea     occurred within populations. This is consistent with the analysis of SSR genetic diversity in S. viminalis (94%) (P. nigra (90.8%)  , S. capr = 0.58) , Populus = 0.47) . S. cheiis (94%) , and P.  (90.8%) . The geoulations .S. psammophila populations showed that P1, P2, P9, P16, and P17 were distinct from the other populations. This was confirmed by analysis of population structure and PCoA analysis based on SSR data. Notably, all these separated populations were from the periphery of the distribution area—P1 and P2 from the northeast, P9 from the center, P16 and P17 from the southwest. The 17 populations of S. psammophila basically covered all of them in the Kubuqi Desert and Mu Us Desert, and the populations formed belt distribution from the northwest to the southeast. The Salicaceae species are dioecious, and exhibit a combination of anemophilous and entomophilous pollination  and PIC (0.6) than P1, P2, P16, and P17. The results showed that the marginal populations located in upstream direction of wind had lower genetic diversity, which may be related to the pollination pattern of the wind. P3, P11, and P15 were clustered separately on the basis of distinct phenotypic traits but not on the basis of SSR data. This can be explained by the fact that the expression of plant traits is the result of the interaction between genotypes and the internal and external environments. Moreover, SSR markers are a simple motif that cannot cover the entire genome, whereas phenotypic traits are influenced by specific gene loci . Gene flow was mainly found to be affected by the distance between the donor and acceptor plants .BA, branch angle; GD, ground diameter; LA, leaf area; LL, leaf length; LP, leaf petiole; LPE, leaf perimeter; LW, leaf width; PH, plant height. Means with different letters within a column are significantly different according to Duncan test (Click here for additional data file.10.7717/peerj.6419/supp-5Supplemental Information 5t- and P-values from the mixed model.The data are from general linear mixed models. numDF: numerator degrees of freedom. denDF: denominator degrees of freedom. Estimate, Click here for additional data file.10.7717/peerj.6419/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.6419/supp-7Supplemental Information 7Click here for additional data file.


Pumas are the most widely distributed felid in the Western Hemisphere. Increasingly, however, human persecution and habitat loss are isolating puma populations. To explore the genomic consequences of this isolation, we assemble a draft puma genome and a geographically broad panel of resequenced individuals. We estimate that the lineage leading to present-day North American pumas diverged from South American lineages 300–100 thousand years ago. We find signatures of close inbreeding in geographically isolated North American populations, but also that tracts of homozygosity are rarely shared among these populations, suggesting that assisted gene flow would restore local genetic diversity. The genome of a Florida panther descended from translocated Central American individuals has long tracts of homozygosity despite recent outbreeding. This suggests that while translocations may introduce diversity, sustaining diversity in small and isolated populations will require either repeated translocations or restoration of landscape connectivity. Our approach provides a framework for genome-wide analyses that can be applied to the management of similarly small and isolated populations. Pumas are experiencing increased isolation as human persecution and habitat loss fragment the populations of this once widespread species. Here, the authors estimate the genomic consequences of this isolation by analyzing the genomes of ten pumas from across North and South America. Puma concolor, also known as the mountain lion or cougar, colonized North America approximately 6 million years ago (mya)3. Although their Pliocene fossil record is sparse and felid fossil assignments have been difficult, previous mitochondrial analyses suggested that the ancestral puma lineage diverged from the extinct North American cheetah-like cat Miracinonyx around 3.2 mya4. The geographic origin of P. concolor remains contested, however. At sites across North America, the oldest puma fossils date to the Rancholabrean land mammal age5, ~200 thousand years ago (kya)6. Analyses of mitochondrial and microsatellite data, however, estimated that the common ancestor of North American pumas lived within the last 20,000 years8 and that the genetic diversity of all modern pumas traces to eastern South America7. The combination of genetic and fossil data were interpreted as reflecting a North American origin of the puma lineage followed by local extinction in North America during the late Pleistocene and subsequent recolonization from South America as the climate warmed after the last ice age8. Recently, however, an unequivocal puma fossil was discovered in Argentina that dates to 1.2–0.8 mya9. This discovery pushes back the age of the puma lineage by more than 500,000 years, and suggests that the ancestor of all living pumas may have evolved in South America rather than North America.The ancestors of the puma, 11. During the 19th and 20th centuries, bounty hunting reduced, and in some cases extirpated, puma populations across North America10, restricting them to the North American West and the southern tip of Florida. By the middle of the 20th century, hunting quotas and some outright bans12 allowed puma populations to increase and recolonize parts of their former range. Although some puma populations today are large and well-connected13, others are small and fragmented , and/or critically endangered . Many populations are experiencing increased isolation with the expansion of highways, residential developments, and agriculture15.Today, pumas are among the most widely distributed mammals in the Western hemisphere, ranging from Canada’s Yukon to the southern tip of South America Fig. 10,11. Du17. To rescue the Florida panthers from extinction, eight female pumas from Texas were released in South Florida in 1995, of which five successfully produced offspring. By 2008, the occurrence of phenotypic defects had significantly declined, survival measures had improved, and the population size increased almost threefold18. All Florida panthers genotyped since 2012 show ancestry that includes admixture with the introduced lineages19.The consequences of geographic isolation on puma genetic diversity and fitness have been well documented, particularly in Florida, where they are a federally protected subspecies commonly called the Florida panther. By the 1990s, the canonical Florida panther population in Big Cypress National Preserve was suffering from reproductive failure and phenotypic defects associated with inbreeding20. The admixed ancestry of the Everglades population and potential explanation for the reproductive success of the captive population was later discovered through genotyping21.Florida panthers in Everglades National Park are partially isolated from the core canonical population that persisted in Big Cypress National Preserve by a semipermeable barrier associated with hydrologic fluctuations of the Everglades. Intriguingly, during the 1990s, the Everglades panthers did not show the same high incidence of inbreeding-associated phenotypes as in the Big Cypress population. The absence of observed phenotypic defects in the Everglades population may be attributable to the release during the 1950s and 1960s of captive-bred Florida panthers with mixed Central American ancestry into Everglades National Park. The introduced individuals’ ancestry was unclear at the time of release, although it was known that the captive population had greater reproductive success than wild Florida panthers22. In traditional conservation genetics, researchers sequence a small number of genetic markers across a large sampling of the species of interest. Advances in sequencing technologies have made it possible to sequence whole genomes of non-model organisms, including species of conservation concern. While the cost of sequencing continues to decrease, sequencing whole genomes will undoubtedly remain more costly than sequencing only a handful of genetic loci. This presents a choice: whole genome data sets exchange spatial resolution for finer-scale genomic resolution, allowing researchers to test different hypotheses. Each whole genome contains a multitude of largely-independent genealogies, which provides increased power to infer past events24. In particular, the dense haplotype information provided by whole genomes is necessary to examine the very short timescales26 relevant to conservation efforts.Genetics has a long history as a tool in wildlife conservationHere, we reconstruct the last two million years of puma demographic history by generating and analyzing a draft genome from an individual sampled in the Santa Cruz Mountains , along with nine resequenced genomes from pumas from North and South America. We confirm the recent maternal ancestry of North American pumas and describe genomic diversity in the sampled populations. We use shared tracts of homozygosity to predict the effectiveness of assisted gene flow in restoring lost genetic diversity. Finally, we analyze the genome of a Florida panther with admixed ancestry that was collected 30 years after the first release of Central American admixed pumas into the Everglades. This genome allows us to assess the long-term efficacy of inter-population admixture as a means to rescue small and isolated populations from the deleterious effects of inbreeding.27 data (see Methods section). Our PumCon1.0 assembly has a BUSCO28 score of 93.04%, a scaffold N50 of 100 Mb, and 87.6% of the genome represented on 26 autosomal scaffolds, each larger than 20 Mb  from the Santa Cruz Mountains using a combination of shotgun Illumina (47× coverage), long-range linking Illumina, and Oxford Nanopore Technology (ONT) (1.2× coverage)We obtained 27×−55× coverage whole-genome resequencing data from nine additional pumas from locations in North and South America Fig.  and 4, a16.We reconstructed puma demographic history using both mitochondrial and nuclear genomes. Analyses of mitochondrial DNA estimate the most recent common maternal ancestor of all sampled pumas ~300 kya Fig. . North A29 of the nuclear genomic data suggested that two puma lineages, one represented by the two Brazilian individuals and the other represented by all individuals sampled in North America, diverged by 300–100 kya  modelingkya Fig. , similar30. To test whether this observed peak was the result of population structure, we modeled pseudo-diploid individuals using the X chromosomes of our male pumas (see Methods section). We found evidence of a cessation of gene flow between all Brazil–North America pseudo-diploid male puma pairs by at least 100 kya, as shown by the sharp increase in the inferred effective population size (Ne), signifying no coalescent events occurred more recently than the estimated divergence time  American lineage into the Everglades lineage Fig.  and 9. F= 3 Fig. . Notably32. This artifact is particularly likely to occur when geographically widespread samples are taken from well connected species where limited dispersal results in the accumulation of local genetic variants, resulting in genetic isolation by distance33. However, the observed geographic structure could also be the result of discrete genetic structure due to population isolation. Some puma populations have experienced persecution and degradation of their habitat, resulting in limited gene flow between populations36. These isolated populations would show increased divergence over time, resulting in geographic structure.We note that the discrete populations identified in these analyses could simply reflect the spatial sampling of our data set. Spatially structured sampling can cause analyses to report distinct populations even when no discrete population structure exists38. The distribution of ROH across the genome varied among scaffolds and individuals  across the 26 largest autosomal scaffolds , where g is the time in generations, r is the recombination rate, and L is the length of the ROH tract in Mb40  between individuals Fig. . Long ROWe present a draft assembly of a puma genome, which we use to reconstruct the demographic history of the species and measure genome-wide heterozygosity and ROH, the latter of which is less practical with lower-quality or reference-guided genome assemblies. Our assembly strategy combined short-read Illumina data with long-read data from ONT to generate a scaffold N50 of 100 Mb, making this one of the most contiguous wild felid genomes assembled to date.8. Our results using complete nuclear and mitochondrial genomes are consistent with previous genetic analyses in that we show that North American pumas represent a subset of puma genetic diversity. However, the nuclear genomic data suggest that the lineage leading to North American pumas diverged from South American pumas ~300–100 kya, considerably older than the 20 kya inferred previously. While we are unable to exclude the possibility of a local late Pleistocene extinction in North America followed by a recolonization from an unsampled lineage elsewhere in South or Central America, we argue that this nuclear genomic data in combination with a recently identified puma fossil in South America that dates to 1.2–0.8 mya9 supports a simpler demographic hypothesis in which the puma lineage originates in South America, disperses into North America by 300–100 kya and persists there to the present day. We note that new fossils or genomic data from late Pleistocene aged pumas or pumas from other locations in South and Central America will be necessary to test this demographic hypothesis.Our analyses of ten complete puma genomes revealed the dynamic history of a once widespread species whose population size is now reduced across much of its range. We showed that extant North American pumas are descended from a population that dispersed northward from South America by at least 200 kya, consistent with the age of the oldest puma fossils in North America. Previously, the incomplete fossil record paired with divergence estimates based on rapidly evolving microsatellites and partial mitochondrial genomes led to the hypothesis of a North American origin of the species, followed by a late Pleistocene local extinction in North America and then a recolonization from South America within the last 20,000 years10. Differences in habitat selection between the two continents probably reflect a long history of competition with a diverse carnivore guild on both continents. For example, jaguars are better adapted than pumas to living in habitats that flood periodically41, and predation by wolves in North America probably precludes pumas living in open habitat without escape terrain42.If true, the new model for puma demographic history means that pumas would have been present in North America for at least one complete glacial/interglacial cycle, indicating that pumas were capable of surviving in a broad range of habitats and environments. This hypothesis is supported by data from living pumas, which, despite a preference in North America for mountainous habitats, are also known to occupy grassland habitats in South America, such as Patagonia43. Forests would have been reduced significantly at that time, as would available habitat for the smaller prey preferred by pumas, providing a potential mechanism for a reduction in puma population size around that time.Intriguingly, North American pumas share a common maternal ancestor around the peak cold period of the last ice age, ~20 kya. This period is associated with a reduction of available habitat across the continental United States, as the coalesced Laurentide and Cordilleran glaciers covered much of present-day Canada and the Upper Midwestern United States13. The result may be small, isolated populations in South America similar to those currently seen in North America. Thus conservation efforts and findings taken from isolated populations in North America may need be applied in the future to other parts of the puma range.The recent history of pumas is marked by human persecution and encroachment on their habitat, resulting in small and isolated populations that are susceptible to loss of genetic diversity and predisposed to inbreeding. Over many generations, without the input of novel variation from migrants, isolated populations can accumulate local genetic variation while losing overall genetic diversity. Loss of genetic diversity may be a common situation for top predators, as their population densities are usually low and successful migrants are infrequent. Consequently, even moderate levels of fragmentation will affect their genomic diversity. While pumas in South America currently experience less habitat degradation than pumas in North America, pumas in South America will likely face further habitat loss and fragmentation as rapid human population growth and land development continues on the continent10. Hunting was so severe until regulations were put in place during the mid 20th century that pumas likely experienced a population bottleneck. All North American pumas sampled in this study exhibit short ROH that date to approximately the early 20th century, indicative of small effective population sizes during the time when hunting was severe.In North America, pumas were hunted extensively, resulting in low population densities in many areas of their range45. Our sampling focused on populations in North America that are known to be isolated and, as such, our results highlight the genomic consequences of this isolation—reduced diversity and signatures of close inbreeding. Pumas in the isolated populations of Big Cypress (CYP), Santa Monica Mountains (SMM), and Santa Cruz Mountains (SC) all have many ROH of all length categories, indicating ongoing inbreeding as a result of continued small population sizes. In contrast, the Yellowstone individual had a similar number of short ROH to these more isolated populations, but fewer long ROH. This pattern is consistent with the known history of hunting and habitat availability in the Yellowstone area. Pumas in the Yellowstone area were hunted to low densities into the mid 20th century10, but today Yellowstone National Park is a large protected area surrounded by wildlands. This connectivity between the Park and wildlands facilitated the recovery and maintenance of genetic diversity in the local puma population once hunting pressures were reduced.In many areas of North America, including California and Florida, large-scale hunting was followed by shrinking habitat availability, resulting in small, isolated populations20. One Florida panther that we sequenced, EVG21, is admixed, having both Floridian and Central American ancestry. Her genome is a combination of regions with comparatively high heterozygosity, similar to that observed in the Brazilian pumas, and long ROH, similar to the highly inbred Florida panthers. The distribution of the lengths of ROH suggest that her maternal and paternal lineages shared a common ancestor that lived shortly after the release of the admixed pumas into the Everglades population. This suggests that the genomic consequences of inbreeding happen quickly, with much of the gains from the genetic rescue being quickly erased. EVG21’s genome provides evidence that when the population is small, it is likely that an individual’s parental lineages will share a very recent common ancestor, even after genetic rescue through admixture  within 50 years after survival rates first began to decrease due to inbreeding, unless connectivity was increased48. Our genomic analyses of the samples from the Santa Monica Mountains also support the effectiveness of population connectivity. The two pumas sequenced from the region (SMM12 and SMM22) both currently reside in the small subpopulation south of US 101 freeway. However SMM12 migrated into the subpopulation from north of US 10115, a larger area that shows greater connectivity to surrounding regions. Migrations between these two areas are now rare, but the two subpopulations were probably part of a larger panmictic population prior to the existence of US 101. The genomic analysis of ROH highlighted that SMM22 had an increased number of large ROH relative to SMM12, consistent with SMM22 originating in a population that is smaller and more isolated. The examination of IBD ROH between SMM12 and SMM22 showed that only 4% of their genomes are in ROH that are IBD between the two individuals. In contrast, individuals that originated from the same population have a much larger proportion of their genomes in IBD ROH . This indicates that while inbreeding has reduced diversity in a considerable proportion of the genomes of individuals within small populations, these low diversity regions are generally not shared between populations. Thus, reconnecting the populations on either side of US 101, as currently proposed via a wildlife crossing over the freeway, would help restore the lost genetic diversity.In many areas of the current puma range, human land use has reduced the connectivity that is critical to recovery and maintenance of healthy populations. Despite these barriers, gene flow among neighboring populations can be facilitated by enhancing landscape connectivity through coordinated land use planning and by adding bridges or underpasses across freeways49. While in some species average genome-wide heterozygosity is highly correlated with the level of inbreeding estimated using ROH26, in systems with admixture, average heterozygosity estimates can be deceptive, as demonstrated with our admixed Everglades puma (EVG21). The heterozygosity of EVG21 is almost as high as the Brazilian pumas, but EVG21 has a large portion of her genome in ROH. We would infer two very different genetic conditions when considering each metric separately, and thus both heterozygosity and proportion of the genome in ROH should be considered in assessing genomic health. Finally, knowledge of shared ROH, an analysis which can only be done with very high density markers across the genome, is critical when designing mitigation plans, as this analysis predicts whether enhancing connectivity would restore lost genetic diversity and helps identify potential candidates for translocation. In this context, this study can serve as a template for future conservation genomic research targeting species living in small, isolated populations.Genome-scale data sets have the potential to inform conservation planning. Our results highlight how whole genome data can provide new insights when compared to traditional conservation genetic techniques. For instance, measures of average heterozygosity are the most commonly used metrics to characterize the genetic health of a species, as estimates are relatively simple to generate and are easily comparable among organisms. However, average heterozygosity provides only a narrow insight into the health and genetic potential of a species50  who lived in the Santa Cruz Mountains in California, USA in accordance with guidelines and regulations of local governing bodies . We extr28 (version 2.0.1) to evaluate genome completeness based on a set of conserved single-copy orthologous genes . In the PumCon1.0 genome, 93.0% of these genes are complete and present in a single copy only  by alignment to the domestic cat genome (GCA_000181335.4) .We generated and sequenced a cDNA library from whole blood collected from a wild puma (SC85) from the Santa Cruz Mountains . The Pum16 (EVG21), two pumas from the Santa Monica Mountains in Southern California , and two pumas from eastern Brazil . Capture, handling, and sampling of all pumas involved in this study were approved by the appropriate governing bodies (52 (SRR2737512-SRR2737518) to use as the outgroup for our analyses.We generated genomic data for a total of 11 pumas , and a third included the ten pumas plus the cheetah (LD filtered). For all variant files, we masked or removed sites that were not biallelic SNPs, and did not pass our filtering criteria  . We then56  modelulations . We perfulations . We alsoulations . In addiulations .We ran principal component analysis on the LD-filtered variant file for the ten pumas, which consisted of 166,037 SNPs . We consWe calculated genome-wide heterozygosity using three different methods: two reference-based and one non reference-based (Supplementary Methods and Supplementary Table https://github.com/russcd/Heterozygosity_HMM). We estimated HMM model parameters from the data and used the filtered fasta files with IUPAC codes as input (We used a hidden Markov model (HMM) to identify ROH by identifying transitions between inbred and outbred regions of the genome (as input . We convas input .58 (version 1.90b4.4) to identify ROH for comparison with our ROH HMM. Even with relaxed parameters, PLINK still tended to break up long tracts  relative to the other Florida panthers. We hypothesized that, because an individual cannot have a shared maternal and paternal ancestor that dates to before the admixture event, admixture in previous generations may have prevented the formation of short ROH. To test our hypothesis, we used an HMM to classify tracts of ancestry in the IUPAC coded fasta file of EVG21 into three types: pure Central/South American ancestry, pure Floridian ancestry, and mixed Central/South American and Floridian ancestry. The genome of EVG21 was composed of 21.98% Central/South American ancestry, 28.24% Floridian ancestry, and 49.58% mixed ancestry based on the HMM (Supplementary Fig. We estimated the proportion of the ROH that are shared between pairs of pumas by finding genomic regions where ROH overlap between pairs of samples . For eacFurther information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Catla catla (Hamilton) fertilised spawn was collected from the Halda, Jamuna and Padma rivers in Bangladesh from which approximately 900 individuals were retained as ‘candidate founders’ of a breeding population. These fish were fin-clipped and genotyped using the DArTseq platform to obtain, 3048 single nucleotide polymorphisms (SNPs) and 4726 silicoDArT markers. Using SNP data, individuals that shared no putative parents were identified using the program COLONY, i.e. 140, 47 and 23 from the Halda, Jamuna and Padma rivers, respectively. Allele frequencies from these individuals were considered as representative of those of the river populations, and genomic relationship matrices were generated. Then, half-sibling and full-sibling relationships between individuals were assigned manually based on the genomic relationship matrices. Many putative half-sibling and full-sibling relationships were found between individuals from the Halda and Jamuna rivers, which suggests that catla sampled from rivers as spawn are not necessarily representative of river populations. This has implications for the interpretation of past population genetics studies, the sampling strategies to be adopted in future studies and the management of broodstock sourced as river spawn in commercial hatcheries. Using data from individuals that shared no putative parents, overall multi-locus pairwise estimates of Wright’s fixation index (FST) were low (≤ 0.013) and the optimum number of clusters using unsupervised K-means clustering was equal to 1, which indicates little genetic divergence among the SNPs included in our study within and among river populations.The online version of this article (10.1186/s12711-019-0454-x) contains supplementary material, which is available to authorized users. Catla catla (Hamilton) is the sixth most important finfish aquaculture species, with approximately 2.8 × 106 tons produced globally in 2015 [In terms of quantity produced,  in 2015 . In spitFertilised spawn were collected in 2012 from the Halda, Jamuna and Padma rivers as part of a program to replace inbred broodstock in Bangladeshi hatcheries . Fish frFor the purpose of the current study, in 2016, archived fin-clip samples were genotyped. Genotyping was conducted using the DArTseq platform  accordin2) of genotypic allele counts were computed, and then a random SNP from the pair with the highest r2 was excluded iteratively until all pairwise r2 values were lower than 0.2. Finally, SNPs that deviated from Hardy–Weinberg equilibrium (HWE) were filtered out. To achieve this, a preliminary analysis (method outlined below) was undertaken to identify and remove close relatives, and thus reduce the risk of false identification of SNPs with genotyping problems .Quality control procedures were implemented to ensure that only high-quality and informative SNPs, in approximate linkage equilibrium, were retained for analysis. First, SNPs with an observed minor allele frequency (MAF) lower than 0.05 or a rate of missing observations higher than 0.05 were excluded. Second, only one randomly-selected SNP was retained from each unique DNA fragment. Third, as a measure of linkage disequilibrium (LD), pairwise squared Pearson’s correlations (rems (see ). Then, ems (see ) and theems (see ). All thG), the method of VanRaden [G matrix was constructed separately for individuals from each river. Clustering of genomic relationships using the ‘Ward2’ algorithm was implemented using the ‘hclust’ function [To construct genomic relationship matrices (VanRaden  was implVanRaden  that wasfunction . Individfunction ). For thfunction ), using function . Errors G matrices with the pedigree-based additive (i.e. numerator) relationship matrices (A) [G matrices were assigned as half-siblings by COLONY, particularly in the case of the Padma river fish. This disparity was attributed to COLONY falsely splitting large full-sibship groups into multiple full-sibship groups [A matrix  to other individuals in A and then removing these individuals from A; (3) appending to the list that was generated in step (2) the individual remaining in A with the lowest average relationship with the other individuals and then removing this individual and its relatives (aij > 0) from A; and (4) iteratively repeating step (3) until no individuals remained in A. Then, allele frequencies using data from the listed individuals only were taken as estimates of allele frequencies in the sampled river populations. These allele frequencies were provided as inputs to regenerate the G for each river. These G matrices were then used to manually assign sibship and dummy parents. This was achieved by (1) visually identifying groups of individuals with genomic relationships of approximately 0.5 and assigning these to full-sibling groups, and (2) visually identifying genomic relationships between these full-sibling groups of approximately 0.25 and defining these as half-siblings.Comparison of the ices (A) , derivedp groups , 21, 22.2.16.0.0 ); (2) liobs) and expected (Hexp) heterozygosities were estimated by SNP and river of origin (‘summary’ function of ‘adegenet’). The significance of pairwise river of origin differences in mean Hexp were estimated  as was the difference between Hobs and Hexp within rivers (paired t-tests). Allelic richness and private allelic richness among rivers were compared visually using the rarefaction method implemented in ADZE [Data from putatively unrelated individuals only were used for the analysis of the genetic architecture of the river populations , 24. Obs in ADZE .ST values between river populations [After data conversion , pairwise overall Wright’s  FST valuulations , and booulations ). For thulations ). AnalysTo investigate the possibility that a population structure other than that due to river of origin might fit the data better, unsupervised (K-means) clustering  was implemented using output from principal component analyses . In the implementation of the ‘find.clusters’ function, the maximum number of clusters was constrained to 20 and the number of randomly chosen starting centroids to be used in each run of the K-means algorithm was set at 1000. Then, the optimum number of clusters was identified as the level of K with the minimum Bayesian information criterion (BIC) .2 ≤ 0.2, and ultimately 978 SNPs remained after removal of those that were not in putative HWE.In total, 3048 SNPs and 4726 silicoDArT markers were identified from 2630 and 4720 DArTseq-generated sequences (i.e. fragments), respectively  and 23 (8.0%) individuals with no putative parents in common were identified from the Jamuna and Padma fish, respectively, in contrast with 140 (46.8%) from the Halda. The 8.4% and exp and Hobs was very small for all rivers . Furthermore, differences between river populations in allelic richness and private allelic richness were not substantive  indicated that variation among populations represented a very small < 0.02) proportion of the total molecular marker variance, albeit significantly different from zero P < 0.001). In addition, overall multi-locus pairwise estimates of Wright’s FST  were low. In addi proportiThe degree of sibship among Jamuna and Padma fish was higher than anticipated, and indicated that catla sampled from rivers as fertilised spawn are not necessarily representative of river populations. This has implications for the interpretation of past population genetics studies, the sampling strategies to be adopted in future studies, the management of broodstock sourced as river spawn in commercial hatcheries, and for pedigree-based genetic analyses and the management of inbreeding in the Bangladeshi catla breeding population. The high level of sibship observed among the Padma river individuals Fig.  was partPoor performance of hatchery-produced seed in Bangladesh has been attributed, in part, to inbreeding caused by uncontrolled mating among a limited number of parents over multiple generations in closed hatchery populations , 5. Our Sibship assignment and the generation of dummy parents were undertaken to improve pedigree-based analyses of breeding population data. These assignments were particularly problematic for the Jamuna river Fig. , possiblST estimates (< 0.013), using data from putatively unrelated individuals only, were generally lower than those previously published: Halda-Jamuna 0.014 [ST estimates [ST estimates than other markers [Pairwise inter-river Fna 0.014 , 0.017-0na 0.014  and 0.08na 0.014 ; Halda-Pna 0.014  and 0.05na 0.014 ; and Jamna 0.014  and 0.05na 0.014 . In thesstimates . However markers .The low molecular marker differentiation among rivers observed in our set of SNPs indicates a lack of genetic differentiation due to drift or adaptive selection and implies that there has been ongoing gene flow among the river systems. This was not unexpected in the case of the Padma and Jamuna rivers, since the Jamuna is a tributary of the Padma, but the Halda river is geographically and hydrologically isolated—although it is possible that natural gene flow between the Halda and other rivers has been exacerbated in recent history by translocation through restocking and aquaculture activities.In this study, (1) we have successfully developed and described a panel of catla DArTseq-based silicoDArT and single nucleotide polymorphism (SNP) markers for future genomic studies ; (2) we Additional file 1: Table S1. Summary statistics for all genomic markers identified by DArTseq. Standard errors are in parentheses. Table S2: Overall multi-locus pairwise estimates of Wright’s FST.Additional file 2:Figure S1. Minor allele frequency across all river populations and within populations using (a) all SNPs and founders prior to quality control and (b) SNPs and founders used in population genetic analyses. The minor allele for each loci was identified in the dataset containing all rivers for (a) and (b) separately. White filled bars before zero represent SNPs for which the minor allele was absent (i.e. MAF is exactly 0). Figure S2. Mean number of (a) distinct alleles per locus and (b) private alleles per locus, as functions of standardized sample size for three rivers (excluding known relatives). Figure S3. Bayesian information criterion (BIC) against the number of clusters (K) from unsupervised K-means clustering.


Anopheles bellator is a primary malaria vector in the Atlantic Forest. Partial sequences of timeless and Clock genes were used to assess the genetic differentiation of five Brazilian populations, which showed strong population structure  in all pairwise comparisons between Bahia sample and the others from Paraná, São Paulo and Rio de Janeiro states. Also, the resulting phylogenetic trees clearly grouped the sequences from Bahia in a different cluster with high bootstrap values. Among southern and southeastern populations low levels of genetic differentiation were found suggesting a general stability of the genetic structure. Anopheles (Kerteszia) bellator, a mosquito species whose immature stages develop in water accumulated in bromeliads, occurs in Trinidad and Tobago and coastal areas spanning eastern Venezuela to southern Brazil.,,An. bellator isoenzymes in populations from three locations in Brazil , and one from Trinidad Island (Trinidad and Tobago). They concluded that the Trinidad Island population was genetically distant from the Brazilian populations, mostly from BA. Also, regarding the three Brazilian samples, SC and SP were genetically close to each other, and quite distant from BA. Recently, a handful of studies have tried to understand the evolutionary relationship among different species from Kerteszia subgenus ,,An. bellator populations. Therefore, to date, there have been no further investigations regarding the relationship between An. bellator populations from Brazil.An. bellator, An. homunculus and An. cruzii are sympatric in the Atlantic Forest.,An. homunculus populations from these regions showed no clear genetic separation ― they were in fact considered a single species ― An. cruzii was shown to be a complex of at least two siblings: one major group with broad range dispersion from south and south-east Brazil and a distinct sibling species in BA.,,,An. bellator populations highly structured like those from An. cruzii; or, like An homunculus, is it a species without significant genetic differentiation in its wide range distribution? To answer this question and to better understand the genetic variability and divergence among An. bellator populations, partial fragments of timeless and Clock genes were evaluated in five Brazilian locations: Ilha do Mel (PR), Cananéia (SP), Abraão and Sítio Forte (RJ) and Camacan (BA). Clock and timeless loci are involved in the circadian rhythms of insects,An. cruzii complex.,,,From the south of Brazil to the north-east, Sampling sites - The mosquitoes used in this study were captured along the Brazilian Atlantic Forest at the following localities: Ilha do Mel (PR), Cananéia (SP), Abraão and Sítio Forte (RJ), and Camacan (BA)  . The mapAn. bellator adults and immatures were obtained, treated and preserved as described in de Rezende Dias et al.Supplementary data (Table I).Molecular data - The timeless and Clock partial gene sequences from the five An. bellator populations were obtained by polymerase chain reaction (PCR), cloning and sequencing as described in de Rezende Dias et al.,An. bellator sequences for each gene is presented in Supplementary data .timeless gene fragment from Anopheles (Kerteszia) laneanus and Anopheles (Kerteszia) cruzii s.s. were used in order to verify the relationship of An. bellator samples analysed in this study with other closely related Kerteszia species. The sequences from An. laneanus were obtained by PCR, cloning and sequencing as described above and submitted to GenBank under the Accession Numbers: KT803975 - KT803978. The sequences from An. cruzii s.s. were those previously published by Rona et al. (10) .Sequences from the DNA sequence analysis - Anopheles bellator DNA sequences were aligned with MAFFTape and phangorn packages,Clock) and TIM3e+G+I (timeless) were selected following the AIC criterion using the modelTest function from phangorn.timeless phylogenetic tree using the coding regions from An. bellator, An. laneanus and An. cruzii s.s. were also constructed (K80+G model) as described above.ROSEQ v 2.91FST , Dxy and Da).DnaSP v5 softwarePopulation structure analysis - A total of 94 sequences  were analysed for the two loci [Supplementary data (Tabela II)]. Clock and timeless fragment sequences were 164 bp and 368 bp long, respectively [Supplementary data ]. Polymorphism data from a variety of measures - e.g. nucleotide diversity  - showed that in general the timeless gene has lower genetic diversity compared with Clock [Supplementary data (Tabela II)], although the first has a bigger fragment size. None of the Tajima’s D results were significant, which means that there is no evidence of natural selection acting in any of the populations [Supplementary data (Tabela II)].FST values (ranging from ~ 0.62 to 0.67) were found, there were few shared polymorphisms and a large number of fixed differences ]. The average number of nucleotide substitutions per site (Dxy) and the number of net nucleotide substitutions per site between populations (Da) was also higher in the BA pairwise comparisons, confirming the FST measurements. The southern and southeastern populations exhibited limited differentiation, characterized by shared polymorphisms and no fixed differences.In all pairwise comparisons with the BA population, very high and significant ferences , some ofHaplotype genealogies analysis - The genealogical relationship of haplotypes was inferred by Maximum Likelihood for the two genes (An. bellator A), however they clearly grouped the sequences from BA  in a different cluster with high bootstrap values.wo genes . The resAn. bellator A and B with other closely related Kerteszia species (An. cruzii s.s. and An. laneanus), a Maximum Likelihood tree was constructed using the coding regions of the timeless gene fragment, since the introns showed a substantial number of indels in the alignment [Supplementary data ]. The resulting phylogenetic tree showed that An. bellator A and B are a monophyletic group, clustering them in a separated clade with high bootstrap value. The final topology was very similar to those from Foster et al.An. cruzii s.s. and An. laneanus are genetically closer when compared with An. bellator. An attempt was also made to compare An. bellator Clock sequences with those from different Kerteszia species available in the GenBank. Although, the same problem found in the timeless non-coding regions (indels) was also disclosed in the Clock alignment, but in a more challenging way, since more than 70% of the fragment is composed by an intron.In order to verify the genealogical relationship between Anopheles bellator A and B - Two highly An. bellator structured groups (An. bellator A and B) were disclosed in this study. The population divergence between them is well supported by (i) fixed differences   and the other more southern populations from Florianópolis (SC) and Cananéia (SP).An. bellator from Camacan belong to the same group as An. bellator from Itaparica Island? Also, are there additional structured An. bellator groups in the Atlantic Forest? A more extensive sampling across the geographic distribution of An. bellator, mainly focusing in Espírito Santo (ES) and BA, and the use of a larger number of molecular markers are desirable in order to better understand the evolutionary forces that establish and maintain the population structure of An. bellator. Accordingly, a number of other studies have shown the same separation pattern between northern and southern Atlantic Forest segments in many fauna (including An. cruzii s.s.) and flora groups, which converge around northern ES and southern BA, suggesting a common vicariant event.,,ferences , (ii) hiof 0.65) , and (iiof 0.65) . The resFST values in the pairwise comparisons between PR and SP, with the two populations from RJ (average FST of only 0.07), which do not have a negligible distance apart (~ 500 km). Even so, among the southern and southeastern populations, we found a widespread lack of significant differentiation.The high genetic differentiation observed between BA and the more southern populations might also be explained by the geographic distance between them (at least 980 Km away) . HoweverAn. bellator groups in the Brazilian Atlantic Forest: An. bellator A is widespread in Southern and Southeastern regions, and An. bellator B is found in the north-east Brazil. Interestingly, in spite of the fact that malaria cases are accounted for yearly in BA, An. bellator has never been viewed as a malaria vector in this state,An. bellator groups regarding malaria susceptibility.This study uncovers two structured


Population comparison analysis manifested that the Hui and Tibetan populations had genetic affinities with the Han, Yi and Korean populations, while the Uygur group had a close relationship with the Kazakh population. The aforementioned results suggested that the Huaxia Platinum System is a polymorphic and effective tool that is appropriate for personal identification and population genetics.Short tandem repeats (STRs) with features of high polymorphism and abundant evolution information play a significant role in genetic applications such as human forensics, anthropology and population genetics. The Huaxia Platinum System was specifically exploited to allow coamplification of all markers in the expanded Combined DNA Index System and the Chinese National Database. Herein, in continuation of our previous studies, 493 unrelated individuals were firstly genotyped to investigate the efficacy of this novel system in three minority ethnicities of China . Additionally, genetic relationships among our three investigated populations and other previously published populations were analyzed using pairwise genetic distances, multidimensional scaling (MDS), principal component analysis (PCA), cladogram and STRUCTURE. The combined match probabilities (CMP) for the Hui, Tibetan and Uygur groups were 1.6894 × 10 In 1998, the Federal Bureau of Investigation (FBI) Laboratory selected 13 STR loci to constitute the Combined DNA Index System (CODIS), which has recently blossomed into the expanded CODIS containing an additional 7 STRs to increase the power of discrimination (PD)4. There are several commercial STR kits, such as the GlobalFiler PCR Amplification Kit 5 and PowerPlex Fusion Systems 6, that contain 20 expanded CODIS loci  was specially developed to allow multiplex amplification and fluorescent detection of the 23 unique STRs as well as Amelogenin and Y-InDel (rs2032678) for sex determination. In light of our previous validation study, this assay was proven to be a highly polymorphic, informative, reliable and efficient tool for forensic investigation9.Short tandem repeats (STRs), also known as microsatellites, are DNA regions scattered throughout the genome with tandemly repeated short sequence motifs varying from 2–6 bases, and they have been the most commonly used genetic marker for human forensics11. The Tibetan population is one of the oldest ethnic groups in China, and their ancestor is the Diqiang, who are mainly distributed in the Tibet Autonomous Region, Qinghai and Western Sichuan. Tibetan belongs to the subbranch of the Tibeto-Burman of the Sino-Tibetan language, which is classified into Sinitic and Tibeto-Burman12. A previous study suggested that there are relatively small genetic diversities among Sino-Tibetan populations13. The Uygur minority is the fifth largest ethnic group in China and chiefly inhabits the Xinjiang Uygur Autonomous Region of Northwest China. Their language pertains to the Turkic subbranch of the Altaic language family. The evidence from a physical anthropology study showed that the Uygur people have an admixture between Europeans and East Asians, which makes it a research hotspot14.China has 56 officially recognized ethnic groups and is the most populous country in the world. It is highly diverse, and its substructure is extremely complicated. The Hui, Tibetan and Uygur minority ethnicities are the most representative populations of West China. Among them, the Hui group is the largest and most widespread minority in the country, and most of them live in the Ningxia Hui Autonomous Region, Xinjiang Uygur Autonomous Region, and Qinghai and Gansu provinces. Their language belongs to Sinitic. The demographic history and previous population genetic studies indicated that the Hui minority originates from West and Central Asian-migrating Muslim groups and has a substantial admixture of the Han, Uygur and Mongolian groups16, herein, we characterized the genetic diversity of the Huaxia Platinum System in the three aforementioned minority ethnicities . Then, we evaluated population comparisons between the three studied populations and previously published populations based on 20 expanded CODIS loci. Furthermore, we conducted genetic structure analysis based on the genotype data of 23 STRs included in the Huaxia Platinum System from 11 populations. The details of relevant populations and their abbreviations are displayed in Supplementary Table In a continuation of our previous studiesThis study was implemented to obtain batches of genotype data  were 0.999999999646627, 0.999999999304935 and 0.999999999433994.The allele frequencies of 23 autosomal STR loci for the three populations are given in Supplementary Tables st) and calculated Nei’s standard genetic distances (Rst) between our three studied populations and 47 previously published worldwide populations  based on allele frequencies of 20 expanded CODIS loci to infer interpopulation similarity and differentiation , 0.2240 (SCT) and 0.2140 (XKU). The smallest Rst values were 0.0072 , 0.0181 (between the SCT and Sichuan Liangshan Yi populations) and 0.0324 (between the XKU and Xinjiang Uygur-3 populations). The NWH and SCT populations had a relatively small Rst with the Han, Yi, Tibetan and Korean populations, and the XKU ethnic group had a relatively small Rst with the other three Uygur and Kazakh populations.We performed locus-by-locus pairwise comparisons , Nei’s standard genetic distances were calculated  plot using R43. The first principal component accounted for 30.943% of the variation and the second accounted for 16.316%. Four conglomerate groups were observed: the first group mainly consisted of our three studied populations and most Asian populations and was located at the middle-right part of the y-axis. The NWH and SCT ethnic groups densely aggregated with the Han, Yi, Tibetan and Korean populations, and the XKU population intensively grouped with other Uygur, Kazakh and Bangladeshi populations; the second group included most European and Oceanian populations, as well as some South American and South African populations, and was located at the middle-left part of the y-axis; the third one contained the North American and four South American populations and was positioned at the lower-left part of the graph; and the last group contained the Southern Portugal Angolan and two South African populations and was clustered at the upper-left part. In addition, a PCA diagram of 17 Chinese populations  software44. As shown in Fig. To further analyze the population genetic background of our three studied populations, a phylogenetic tree was reconstructed via the neighbor-joining method based on RFurthermore, as shown in Fig. 15. The probable admixture levels and cluster membership patterns of each population are presented in Fig. To illustrate the genetic structure of our three studied populations, a STRUCTURE plot was implemented based on the genotype data of 23 STRs by combining eight previously published populations46. However, the limited genetic research of the three populations in China is far from sufficient. In this study, we focused on the genetic makeup and phylogeny of the three ethnic groups using 23 autosomal STRs included in the Huaxia Platinum System. First, we confirmed that these STR loci conformed to HWE and that there was no LD existing among them. Thus the 23 STRs could be used as independent genetic markers for forensic investigation and population genetics in subsequent research. The assay performed well in the three populations and it could be a robust and effective tool for forensic applications, such as for individual identification and paternity tests.Three minority ethnicities, the Hui, Tibetan and Uygur, have always been a hot topic in research by linguists, anthropologists, archaeologists and population geneticists45. The SCT population also had genetic affinities with the Han and Yi populations, and our results indicated that the high altitude Tibetan (Tibet Tibetan) and the low altitude Tibetan (SCT) populations had strong genetic similarity, this feature is maintained in accordance with previous findings and is indicated by high-throughput genotyping and sequencing data49. Although the XKU had close genetic relationships with other Uygur and Kazakh ethnic groups, it was not as close as that between other Uygur groups. This may be caused by the special geographical position and history of Kumul. Kumul is a multiethnic region  and is located at the border of Gansu Province in the East and Mongolia in the North, which is the throat of the Silk Road and the gateway to inland China. It was founded by a people known in Han Chinese source during the 1st millennium BCE, and the Uygur people and other minority ethnicities gradually settled in this area subsequently51. Furthermore, structure analysis based on the 23 autosomal STRs manifested that the populations of the same language family had a similar genetic makeup, and this was consistent with the results of population comparison analysis, namely the genetic structure is strongly correlated with linguistic affiliations13.Population substructure dissection is pivotal in population genetic studies, genotype-phenotype association research and forensic genetics, especially in East Asia with its high cultural, ethnical and geographical diversity. We performed population comparisons based on 20 autosomal STR loci (more loci can provide a higher refinement in the construction of a phylogeny than other similar studies) and found that the results of the interpopulation genetic distances, MDS, PCA and phylogenetic tree were consistent. For population comparisons worldwide, only Asian populations can be distinguished from other continental populations, which proved that the Huaxia Platinum System is useful for the identification of Asian populations. For population comparisons within China, the NWH group showed significant genetic homogeneity with the Han, Yi and Tibetan populations, which supports the origin theory of Hui via simple cultural diffusionTo further understand the genetic background and differentiation of Sino-Tibetan language  populations, we are looking for and are validating a large number of new genetic markers with ethnic distinguishing potency. Large-scale population genetic studies using different high-density genetic markers and even whole-genome sequencing (WGS) will be conducted in the future.Blood samples were collected with the approval of the Ethics Committee at the Institute of Forensic Medicine, Sichuan University. Written informed consent was obtained from all participants. All methods were conducted according to the guidelines approved by the Institute of Forensic Medicine, Sichuan University, and this study was ratified by the Ethics Committee of Sichuan University .Blood samples were collected from 493 unrelated individuals belonging to three Chinese ethnic minorities. These individuals included 183 Hui individuals living in Wuzhong City in Ningxia Hui Autonomous Administrative Subdivision, 200 Tibetan individuals residing in Chengdu City in Sichuan Province and 110 Uygur individuals located in Kumul City in the Xinjiang Uyghur Autonomous Region of Northwest China. During the sample collection process, we ensured that each individual’s family has been living in the respective region for at least three generations and did not intermarry with other ethnic groups and that any two individuals had no kinship within a minimum of three generations.® Genomic DNA Mini Kit  according to the manufacturer’s instructions. The 7500 Real-time PCR System  was used to quantify the DNA concentration with the Quantifiler Human DNA quantification kit . Then, the DNA samples were normalized to 1 ng/μL and stored at −20 °C until amplification.We isolated the genomic DNA with the PureLinkTM PCR system  following the manufacturer’s protocol. The 25 μL PCR volume contained 10 μL of master mix, 10 μL of primer set, 4 μL of deionized water and 1 μL of template DNA. Thermal cycler conditions were as described below: predenaturation at 95 °C for 1 min, followed by 26 cycles of 94 °C for 3 s, 59 °C for 16 s, and 65 °C for 29 s, and a final extension at 60 °C for 5 min. Separation and analysis of PCR amplified products was performed on the Applied Biosystems 3500 Genetic Analyzer  using POP-4 polymer , and injections were conducted at 1.2 kV for 16 s. Allele identification was conducted using the Huaxia Platinum panels, bin sets, stutter files and a 175 relative fluorescence units (RFU) threshold, unless otherwise stated, and were compared with the allele ladder provided by the corresponding kit via Applied Biosystems GeneMapper ID-X version 1.2 software.A total of 25 forensically related genetic makers  were coamplified using the Huaxia Platinum PCR amplification system. Multiplex amplification was conducted on a ProFlex52. Interpopulation differentiation was performed in Arlequin v3.5 software to compute pairwise Fst using locus-by-locus pairwise population comparisons. Nei’s genetic standard genetic distance (Rst) was calculated using the Phylip3.695 package. Multidimensional scaling analysis (MDS) was conducted by SPSS software , and principal component analysis (PCA) was carried out based on allele frequencies using MVSP v3.22 software43. The phylogenetic tree based on the neighbor-joining method was delineated in the Molecular Evolutionary Genetics Analysis v7.0 (MEGA v7.0) software44. The genetic distribution of 11 populations was conducted using STRUCTURE v.2.3.4 software46, for which the parameters were a100,000 lenth of burnin period after 100,000 steps for the Markov Chain Monte Carlo (MCMC) using the standard admixture model, five independent runs for each K value and K values from 2 to 7.Allele frequencies, forensic statistical parameters (containing the match probability (MP), power of exclusion (PE), polymorphism information content (PIC) and typical paternity index (TPI)) and p values of the Hardy-Weinberg equilibrium test (HWE-p) of 23 autosomal STR loci were calculated in the modified PowerStat spreadsheet . Subsequently, the observed heterozygosity (Ho), expected heterozygosity (He) and p values of the linkage disequilibrium test (LD-p) were assessed in Arlequin v3.5 software2O in each batch of genotyping was conducted.Our laboratory is accredited by ISO 17025 and the China National Accreditation Service for Conformity Assessment (CNAS). The recommendations published by the DNA Commission of the International Society for Forensic Genetics (ISFG) were followed in the overall experimental procedure. The positive control of Control DNA 007 and negative control of ddHSupplementary materials


Fst pairwise genetic distances emerged for some markers among the studied populations and also between our data and those from other countries and continents. Of particular interest, Alu insertions demonstrated the most differences, and could be of use in ancestry studies for these populations, while INDELs and SNPs variation were informative for differentiation within the country.Alu insertions, INDELs, and SNPs in the X chromosome can be useful not only for revealing relationships among populations but also for identification purposes. We present data of 10 Alu insertions, 5 INDELs, and 15 SNPs of X-chromosome from three Argentinian north-east cities in order to gain insight into the genetic diversity of the X chromosome within this region of the country. Data from 198 unrelated individuals belonging to Posadas, Corrientes, and Eldorado cities were genotyped for Ya5DP62, Yb8DP49, Ya5DP3, Ya5NBC37, Ya5DP77, Ya5NBC491, Ya5DP4, Ya5DP13, Yb8NBC634, and Yb8NBC102 Alu insertions, for MID193, MID1705, MID3754, MID3756 and MID1540 Indels and for rs6639398, rs5986751, rs5964206, rs9781645, rs2209420, rs1299087, rs318173, rs933315, rs1991961, rs4825889, rs1781116, rs1937193, rs1781104, rs149910, and rs652 SNPs. No deviations from Hardy-Weinberg equilibrium were observed for Posadas and Corrientes. However, Eldorado showed significant values, and it was found to have an internal substructuring with two groups of different origin, one showing higher similarity with European countries, and the other with more similarities to Posadas and Corrientes.  The human X chromosome contains multiple types of non coding markers distributed along its sequence, including Alu insertions, insertion-deletions (INDEL), and single nucleotide polymorphisms (SNP) markers. Alu insertions are short interspersed nuclear elements classified into 12 subfamilies that appeared at different times during primate evolution  it travels between both sexes in each generation, telling a story which differs from uniparental genomes; (ii) it has an overall genetic diversity within populations higher than autosomes due to its reduced effective population size, which makes it more sensitive to the effects of genetic drift, population substructuring and selective sweeps; (iii) the lower rate of recombination leads to an increase in levels of linkage disequilibrium (LD) in comparison to autosomes; and (iv) the hemizygous state in males provides a direct access to haplotypes  the Guaraní people, who came from Amazonia, settled in the current Argentinian territory between the end of the fifteenth century and the beginning of the sixteenth century Heguy, . They enOn the other hand, European immigration in Misiones was characterized by two types of settlements: one official (1883–1927) and one private (1920–1945). Private colonization took place in Eldorado, Puerto Rico and Montecarlo, in the northern region of Misiones, where companies of European origin promoted the arrival of foreigners, preferably Germans  that changed with the arrival and expansion of the Guaraní people. As a consequence of this expansion, the language and customs of extant communities was replaced by those of the Guaraní. In particular, the city of San Juan de Vera de la Siete Corrientes was founded by Spaniards and their descendants born in America (dubbed “criollos”), who employed these lands as farms for the breeding and exploitation of cows and horses. The initial settlers of the city of Corrientes gradually joined the local Amerindian population Vara, . In contIn this work, we studied 10 Alu insertions, 5 INDELs, and 15 SNPs to gain insight into the genetic composition of these particular populations. First, we focused in discerning the different contribution of European and Native American genetic components on the studied populations to understand the diversity that they represent. Second, we analyzed the different information that each kind of marker can offer. This constitutes the first study in South American populations that uses this set of 30 markers.n = 92; 32 females, 60 males), the capital city of Misiones , and another important city of Misiones ; the geographical location of these cities is shown in Figure n = 27; 13 females, 14 males) the donors who knew that their four grandparents were German and/or Swiss, and we labeled donors who did not know the origin of all four grandparents as Eldorado B . All samples were obtained with informed consent and were analyzed anonymously; the project was approved by the Ethics Commitee at IMBICE. DNA was isolated from buccal cells and peripheral blood samples as in Gemmell and Akiyama , PE (power of exclution) and PD (power of discrimination) were calculated with an online software ChrX-STR.org 2.0 Calculator (http://www.chrx-str.org/) , rs1781104 for Posadas , Yb8NBC634 for EldoradoA , and Yb8NBC102, rs9781645, 1781104 for Corrientes , respectively. Allele frequencies of all populations are shown in Table Fst-values of the Alu insertions were not significantly different for the populations analyzed. On the other hand, INDELs showed significant Fst-values for most of the comparisons. In the case of SNPs, they showed significant Fst-values for the comparisons between Posadas-Corrientes and EldoradoA-EldoradoB  (data not shown) probably as a result of the Bolivian differentiation from one side, and the presence of Siwa Oasis in the African group, and Crete Island in the European one. The outlier position of these two samples may be an artifact due to the relatively low number of loci analyzed. The proximity of Basque data to our populations can be explained given that Argentina is the country with highest rate of Basque immigration in the world  was graphed employing x Figure . The firFst values obtained . Figure The genetic diversity found in this work between the four populations  was similar to those referenced in the bibliography  resulted in its separation from our four populations. This feature might be indicative of a low rate of slave traffic in the past in this region, unlike the more significant flow of slaves occurred across the central and north-west regions of Argentina (Morales and Alfaro, A notable differentiation was found between the geographically closer Native Americans from Bolivia and our four populations Figures , 5. The GD and CC: study design; CA and IA: data collection; GD and SdP: data processing; GD, CC, and ME: data analysis; GD, SdP, and CC: manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.