Plasmodium falciparum malaria cases in Africa represent over 90% of the global burden with Mali being amongst the 11 highest burden countries that account for 70% of this annual incidence. The persistence of P. falciparum despite massive global interventions is because of its genetic diversity that drives its ability to adapt to environmental changes, develop resistance to drugs, and evade the host immune system. Knowledge on P. falciparum genetic diversity across populations and intervention landscape is thus critical for the implementation of new strategies to eliminate malaria. This study assessed genetic variation with 12,177 high-quality SNPs from 830 Malian P. falciparum isolates collected between 2007 and 2017 from seven locations. The complexity of infections remained high, varied between sites, and showed a trend toward overall decreasing complexity over the decade. Though there was no significant substructure, allele frequencies varied geographically, partly driven by temporal variance in sampling, particularly for drug resistance and antigen loci. Thirty-two mutations in known drug resistance markers  attained a frequency of at least 2% in the populations. SNPs within and around the major markers of resistance to quinolines (pfmdr1 and pfcrt) and antifolates (pfdhfr and pfdhps) varied temporally and geographically, with strong linkage disequilibrium and signatures of directional selection in the genome. These geo-temporal populations also differentiated at alleles in immune-related loci, including, protein E140, pfsurfin8, pfclag8, and pfceltos, as well as pftrap, which showed signatures of haplotype differentiation between populations. Several regions across the genomes, including five known drug resistance loci, showed signatures of differential positive selection. These results suggest that drugs and immune pressure are dominant selective forces against P. falciparum in Mali, but their effect on the parasite genome varies temporally and spatially. Interventions interacting with these genomic variants need to be routinely evaluated as malaria elimination strategies are implemented. Plasmodium falciparum is one of the most deadly parasitic diseases, particularly in Africa, where fatalities have been exacerbated by interruptions in interventions due to the COVID-19 pandemic  and multidrug resistance 1 (pfmdr1) genes, which spread through Africa from Southeast Asia, leaving strong signatures of selective sweeps in the genome of P. falciparum (pfdhfr) and dihydropteroate synthetase genes (pfdhps), which are associated with SP resistance, combination drugs that were introduced to contain chloroquine resistance  and a long-lasting partner drug such as lumefantrine, piperaquine, mefloquine, or amodiaquine, to limit resistance development  gene on chromosome 13  is a village in the suburban area of Sikasso, which is located 375 km southeast of Bamako. Malaria transmission is seasonal and mainly occurs from May to September . The samDangassa is in the south savannah grassland of eastern Mali, along the Niger River and has year-round access to fresh water. Malaria transmission in the area is both seasonal and perennial with parasite prevalence ranging from 40 to 50% of the population in the dry season (October–May) and 70–85% in the rainy season (June–September) . Most ofFaladje village is located 80 km northwest of Bamako with malaria transmission being both seasonal and perennial. Prevalence of parasitemia ranges from 40 to 50% of the population in the dry season (October–April) and 70–85% in the rainy season (May–September) . The samKenieroba is a village of about 2,000 people and is located approximately 75 km southwest of Bamako . The 89 Kolle is a rural village of 2,500 people located 55 km southeast of Bamako. Prevalence of parasitemia also ranges from 40 to 50% of the population in the dry season (October–May) and 70–85% in the rainy season (June–September) . This siFinally, Nioro-du-Sahel (Nioro) is in the Sahelian region of Mali near the Islamic Republic of Mauritania with desert climate, limited rainfall, and the lowest malaria transmission . The samwww.malariagen.net/projects/SPOTMalaria), a project led by the Malaria Genetic Epidemiology Network  to harness genomic technologies in monitoring the evolution of malaria parasites. The filter papers were subjected to DNA extraction using the Qiagen DNA extraction protocol and the genome of P. falciparum in each sample was sequenced at the Wellcome Sanger Institute as previously described . For downstream analysis, vcf files of variants were filtered with vcftools (vcftools.github.io) to retain only biallelic coding SNPs supported by at least five reads and with a mapping quality greater than 30  sample was collected by spotting whole blood obtained by finger prick onto filter paper cards. The collection was carried out in accordance with the SPOTMalaria protocol (escribed . The sho than 30 . Using R than 30 .FWS index) as described previously  between alleles at physically separated loci was calculated using PLINK version 1.92.0 for each chromosome per population. The geom_smooth function from ggplot2 was used to fit the distribution of r2 for SNPs within a physical distance of 50 Kb.For each sample, within-host genetic diversity was assessed using the within-sample inbreeding coefficient (eviously . FWS wated in R . Pairwisr2) of not greater than 0.02 between any pair of SNPs. The samples were clustered by principal component analysis (PCA) and multidimensional scaling analysis (MDS). PCA was performed using the findclusters and dapc within the R adegenet package. PCA clustering was also evaluated with snpRelate snpgdsPCA function, which calculates the genetic covariance matrix from genotypes and computes the correlation coefficients between sample loadings and genotypes for each SNP. For MDS, pairwise identity by state (IBS) distance was generated in PLINK, followed by MDS in R using the cmdscale function.To determine the potential structure between the geo-temporal populations, dimension reduction methods were used on a pruned set of 5541 SNPs with pairwise LD  as applied in vclib. Values of indices for SNPs were visualized using Manhattan plots. SNPs with values beyond the 99.5 percentile were further analyzed as outliers.Overall genetic differentiation between geo-temporal population pairs was evaluated by age in R , followiage in R . FinepopPfcrt), dihydrofolate reductase (Pfdhfr), dihydropteroate synthase (Pfdhps), kelch 13 (Pfk13), multidrug-resistance protein 1 (Pfmdr1), multidrug-resistance 2 (Pfmdr2), and plasmepsin II (Pfpm2). The frequencies of SNPs in these antimalarial drug resistance markers were calculated for each temporal or geo-temporal population using vcftools. Linkage disequilibrium between the SNPs was calculated and visualized using the LDheatmap R package (https://sfustatgen.github.io/LDheatmap/).SNPs in the following drug resistance genes were extracted; chloroquine resistance transporter ), where Φ (- |iHS|)) represents the Gaussian cumulative distribution function. The generated p values were adjusted for multiple testing following the method of Benjamini, Hochberg, and Yekutieli to control for the false discovery rate (fdr), as implemented by the p.adjust function in base R. SNP loci with fdr adjusted -log10 p-values  over the 99.5 percentile were considered as outliers and marking selective sweep regions.To test if variation within genes deviated from neutrality, we calculated Tajima’s D for each gene with at least three SNPs per geo-temporal population using the age in R . Genes w R , ranged from 0.32 (most polygenomic infections) to 1.0 (monogenomic infection). The distribution of FWS was different across geo-temporal populations , , except between Nioro (0.59) and Kolle (0.39), where the difference was significant , (FWS increasing from 0.82 in 2007 to 0.88 in 2017 (FWS fluctuated but the highest median (0.92) was for samples collected in 2017. The overall LD (r2) between SNPs per population did not align with differences in complexity, as LD for all geo-temporal populations decayed rapidly below 0.05 within 5000 base pairs (Within-host complexity of infection determined by the  1e-06), . The low.03012), . Classif.03012), , though  in 2017 . Considese pairs .P. falciparum from all seven geo-temporal populations clustering into a single genetic cluster with a few outliers  and between all temporal samples or from Faladje only; 2007 against 2013 and 2007 against combined samples from 2015, 2016, and 2017 using differentiation indices (FST and Jost’s D). Genome-wide pairwise FST and Jost’s D were generally low between geo-temporal and temporal population pairs (pfcrt), and Pf3D7_13_v3.400162 that codes for L264V in PF3D7_1308600 conserved protein of unknown function on chromosome 13  in 2013 samples from Bamako (65% of infections), while the alternative 220A variant was most frequent in 2016 samples from Kenieroba . The antigenic loci differentiating geo-temporal populations were mostly those coding for members of dominant gene families in blood stages, including erythrocyte/Duffy binding proteins (EBA/DBL), merozoite surface proteins (MSP), cytoadherence proteins (CLAG), surface-associated proteins (SURFINS), hypervariable exported proteins (HYPs), early-transcribed membrane protein (ETRAMP), a reticulocyte-binding protein (pfrh1), and protein E140 (PF3D7_0104100). The highest number of differentiating SNPs was in pfhyp15 (five non-synonymous SNPs). Differentiating SNPs were also identified in gametocyte-associated genes, pfgig and pfgexp05, as well as pfcap93 which play a role in sporozoite development.Considering the top 0.5% of per locus Jost’s D distribution between each geo-temporal or temporal population pair, 185 SNPs (126 coding for non-synonymous variants) were identified as relatively stronger drivers of differences in allele frequencies . These wpfcrt, pfdhps, pfdhfr, pfmdr1, pfmdr2, and pfk13) attained a frequency of 2% or above in at least one of the geo-temporal populations , A220S (p = 0.000009416), Q271E (p = 0.001798), I356T (p = 0.0002915), R371I (p < 2.2*10−16), and PFMDR1 N86Y (p = 0.01123). For antifolate resistance markers, high frequencies (mean 70%) of PFDHFR mutants 51I, 59R, and 108N were observed across all sampling sites. None of the common artemisinin-resistance-associated variants in pfk13 were detected, though four nonsynonymous SNPs coding for PFK13 R255K, K189N, K189T, and G112E in the non-propeller domain of the Kelch 13 protein were detected. PFK13 K189T was detected in over 50% of isolates from all geo-temporal populations.Thirty-two SNPs in selected drug resistance genes . Selective sweeps were also evident around pfdhfr and pfdhps, markers of SP resistance. Several antigens were also identified with sweep signatures, with the highest number of sweep SNPs within the pftrap (PF3D7_1335900) coding region. Considering only the temporal populations from Faladje, regions of selective sweeps were similar to those described for geo-temporal populations  on chromosome 12, thrombospondin-related anonymous protein (TRAP) on chromosome 13, and a conserved Plasmodium protein (PF3D7_1475800) on chromosome 14, , 143 out of 9,359 common SNPs in 19 genomic regions and 53 genes, marked candidate signatures of selective sweeps across the seven geo-temporal populations . Consideulations . These rsome 14, . Togethepfphist, pfhyp, pfsurfins, and pfmsp), candidate vaccine targets , and housekeeping genes as well as known drug resistance loci. For the latter, the widest span was the region on chromosome 7, around the chloroquine resistance transporter (pfcrt). The discriminating genomic loci around pfcrt were mostly observed when the largest number of isolates collected from Bamako in 2013 was compared to those from other sites. Additional positive selection regions differentiating populations were within the vicinity of other known drug resistance genes, pfdhfr on chromosome 4, pfmdr1 on chromosome 5, and pfdhps on chromosome 8. These latter differences were relatively stronger when isolates from Bougoula were compared to other geo-temporal populations.Differences in haplotypes and regions of selective sweeps between geo-temporal populations can be distinguished from comparing extended haplotype homozygosity between populations, captured by the cross-population metrics Rsb and cross XP-EHH. Both Rsb and XP-EHH metrics identified several regions of selective sweeps as differentiating geo-temporal populations . OverallPlasmodium exported protein) and PF3D7_1004800  were under balancing selection in most populations, while Duffy binding-like merozoite surface protein 2  had the highest Tajima’s D value but not for all populations. Amongst the top 15 common genes were the invasion ligands, pfdblmsp2 and pfsurfin8.2, as well as the drug resistance gene, pfcrt, and other loci involved in metabolic processes and environmental responses.Genetic variation patterns in antigenic loci can be indicative of balancing selection, identifiable with allele-frequency-based summary statistics such as Tajima’s D for each gene. The overall distribution of gene-wise Tajima’s D was skewed to negative values for most genes across all geo-temporal populations . The TajP. falciparum parasite. Though the country is divided by the River Niger, and there is heterogeneity in transmission levels, the analysis here showed that the malaria parasite populations across seven endemic sites share the same genetic structure. This provides evidence of temporal and geographic geneflow across the southern transmission belt of the country, similar to what has been described for the parasite from other malarial endemic settings in West Africa (FWS) varied between populations partly driven by transmission differences. Infections from the region of Kolle were the most complex (polygenomic), while those from Nioro and the metropolis of Bamako were predominantly clonal. The samples from Kolle were the oldest in the collection and the higher complexity likely reflected the higher transmission rates in 2007. The complexity of infection can track changes in transmission imposed by intervention, and this was evident when isolates from Faladje showed a slight but significant reduction in complexity between 2007 and 2017. The association between the complexity of infection and transmission intensity could also be driven by the difference in Anopheles vector densities between rural and urban dwellings , the strongest differences in allele frequencies were for SNPs within or around drug-resistance-associated and immunity-related markers. This includes codon position A220S in the pfcrt, a gene involved in parasite resistance to chloroquine  variant at Pfmdr1 codon 86 but the mutant  at codon 184 was at higher frequencies across all sites, probably selected for by lumefantrine  selects for non-vaccine variants in the population and the observed genetic variation in this study could further affect its efficacy in Malian populations , several genomic regions showed evidence of recent directional selection in Mali. This included strong signatures around the antimalarial drug resistance gene ethamine . Likewisethamine . Not allvaccines . Other delopment . Hence, ulations .Pfdblmsp2 gene harboring the strongest signature of balancing selection by most populations. It has been identified previously as a target of balancing selection despite a potential role in drug responses (pfcrt drug resistance gene, suggesting that dual selection from drugs and probably immunity may be acting on these loci as previously shown (Immune action also mounts selection pressure on parasite populations, which leaves signatures of balancing selection in the genomes. It was not surprising to find the esponses . Balancily shown . It is, Plasmodium falciparum across Mali are genetically similar but complex within a decade of sampling. The genetic difference between temporal or geographic populations targeted drug resistance and immune genes affected by drug interventions and differences in immunity imposed by variation in transmission intensities. As drugs and future vaccines are vital tools for elimination of malaria, the temporal and spatial population-specific differences in associated alleles should be monitored for exploitation as current and new malaria elimination strategies are being implemented.Infections of


Cucumis sativus L.). Based on a genomic variation map of individuals worldwide, we implemented a Fisher’s combination method by combining 4 haplotype-based approaches: integrated haplotype score (iHS), number of segregating sites by length (nSL), cross-population extended haplotype homozygosity (XP-EHH), and Rsb. Overall, we detected 331, 2,147, and 3,772 population-specific, recent positive selective sites in the East Asian, Eurasian, and Xishuangbanna populations, respectively. Moreover, we found that these sites were related to processes for reproduction, response to abiotic and biotic stress, and regulation of developmental processes, indicating adaptations to their microenvironments. Meanwhile, the selective genes associated with traits of fruits were also observed, such as the gene related to the shorter fruit length in the Eurasian population and the gene controlling flesh thickness in the Xishuangbanna population. In addition, we noticed that soft sweeps were common in the East Asian and Xishuangbanna populations. Genes involved in hard or soft sweeps were related to developmental regulation and abiotic and biotic stress resistance. Our study offers a comprehensive candidate dataset of population-specific, selective signatures in cultivated cucumber populations. Our methods provide guidance for the analysis of population-specific, positive selection. These findings will help explore the biological mechanisms of adaptation and domestication of cucumber.Population-specific, positive selection promotes the diversity of populations and drives local adaptations in the population. However, little is known about population-specific, recent positive selection in the populations of cultivated cucumber ( Locating population-specific, positive selection from genetic variation is a vital and considerably challenging task in evolutionary biology. Positive selection drives local adaptation  and may Arabidopsis thaliana  . To captHaplotype-based methods, as powerful tools to detect recent positive selection, have developed many relevant metrics, such as integrated haplotype score (iHS) (Cucumis sativus L.) is an important vegetable crop mainly cultivated in temperate and tropical regions of the world , the clade consisting of C.sativus, Cucumis melo, Citrullus lanatus, and Lagenaria siceraria was named the 4-way clade and the clade consisting of all 7 species was named the 7-way clade. Based on the phylogeny and whole genome alignments, we inferred the ancestral states. The ancestral allele state for each site in cucumber was determined if more than 2 species in the 4-way clade or 4 species in the 7-way clade were consistent with each other. In addition, the allele state in cucumber different from that in ancestors was set as “derived.” Sites with ancestral allele states that could not be determined by the above 2 clades were set as “missing.”First, we obtained 7-way, cucumber-based, pairwise alignments from the website r2 value as 1 block according to our analysis of the LD decay using PopLDdecay v3.40 . The Fisher’s combined test statistic is equal to:n degrees of freedom  . More signatures of selection were identified by the methods based on 2 populations than by the methods based on a single population , which was mainly because of 2 causes: (i) more SNPs used by the methods comparing 2 populations and (ii) the significantly higher sensitivity of the methods based on 2 populations compared with the methods based on a single population (Supplementary Table 5). The overlaps of 4 approaches in 3 cultivated populations were similar . The intersection of XP-EHH and Rsb was the biggest, followed by the intersection of iHS and nSL. The overlapping sites of all 4 approaches were 38, 23, and 8 SNPs in East Asian, Eurasian, and Xishuangbanna population, respectively.Filtered by the quality of SNPs and by the data requirements for each method, the genomic SNPs used in each method differed (Supplementary Table 4). For the iHS and nSL methods, the ancestral state for each variant was required; some SNPs were failed to determine the ancestral state, therefore, there were fewer SNPs than those used by XP-EHH and Rsb. For each SNP, the P values from the 4 methods provides a concise way to integrate the above findings . In the 287 intergenic sites, 70 sites located in potential promoter regions. These genes were related to reproduction, plant response and resistance, and biosynthesis (Supplementary Table 7).WRKY27 gene (Csa3G354510) with 3 selective nonsynonymous variants, influencing pollen viability (AQP) gene (Csa3G345890) which plays key roles in drought, flooding, nutrient availability, response to temperature and light conditions (UGT1) gene (Csa3G608710) with a selective nonsynonymous variant, disrupting the biosynthesis of littorine and its tropane alkaloids derivatives (hyoscyamine and scopolamine) . In the 1,921 intergenic sites, 743 sites located in potential promoter regions. We further found that the genes were involved in regulation of fruit length, development, response to environment, and DNA repair (Supplementary Table 9).MARCH2 (Csa3G186720), that regulates plant development, including root development, organ size decisions, flowering time, and plant–environment interactions, such as drought and ABA (TST) gene (Csa3G021130) defends against herbivores by their volatile terpenes (DREB) gene (Csa3G180260) enhances tolerance to freezing temperatures, drought, and high salinity . In the 2,960 intergenic sites, 960 sites located in potential promoter regions. The genes were involved in resisting stress and pathogens, regulating developmental processes, biosynthesis, and flesh thickness (Supplementary Table 11).TLP) gene (Csa3G003990), containing a selective synonymous variant located in the Thaumatin domain, enhances drought and salt stress tolerance (ABCG) gene (Csa6G434390) with 5 synonymous variants, of which 2 localized at ABC2 membrane family, is involved in ABA tolerance and plant resistance against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in Arabidopsis (WRKY6 gene (Csa7G328830) with a selective nonsynonymous variant and a synonymous variant was detected serving functions in plant senescence processes, pathogen defence mechanisms, abiotic stress responses, and regulation of the accumulation of fatty acids during seed development (DXS1) gene (Csa2G404880) with a selective nonsynonymous variant located in the MR_MLE_C domain, which has been proposed serving fruit carotenoid biosynthesis and is required for the development and survival of tomato plants (TOP1) gene (Csa3G063680), which modulates auxin-regulated root development in rice  were detected. Meanwhile, regions with the top 5% of XP-CLR values were identified. These shared regions between the analysis of genetic diversity and XP-CLR analysis were considered to be selective sweeps. In our analysis, we scanned the whole genome for each cultivated population by performing 4 haplotype-based approaches, including iHS, nSL, XP-EHH, and Rsb. Finally, we used Fisher’s combination method to combine the P values of the 4 methods, which can greatly increase the power to pinpoint the selected regions , nSL (B), XP-EHH (C), and Rsb (D) in East Asian. Supplementary Fig. 5: Manhattan plots of P values of iHS (A), nSL (B), XP-EHH (C), and Rsb (D) in Eurasian. Supplementary Fig. 6: Manhattan plots of P values of iHS (A), nSL (B), XP-EHH (C) and Rsb (D) in Xishuangbanna. Supplementary Fig. 7: Venn diagram for selective sites in East Asian. Supplementary Fig. 8: Venn diagram for selective sites in Eurasian. Supplementary Fig. 9: Venn diagram for selective sites in Xishuangbanna. Supplementary Table 1: Individuals and SNPs kept after quality filtering. Supplementary Table 2: Decay of linkage disequilibrium (LD), measured by r2 values, in 4 populations. Supplementary Table 3: The parameters in BEAGLE5.0 used to calculate haplotypes in each population. Supplementary Table 4: Signatures showing selection detected by the 4 methods in the cultivated populations. Supplementary Table 5: P values of the tests for equality of the proportion of selective signatures detected by each 2 methods in each cultivated population. Supplementary Table 6: States of selective SNPs only detected in the East Asian population. Supplementary Table 7: Functions of genes containing selective SNPs in the East Asian population. Supplementary Table 8: States of selective SNPs only detected in the Eurasian population. Supplementary Table 9: Functions of genes containing selective SNPs in the Eurasian population. Supplementary Table 10: States of selective SNPs only detected in the Xishuangbanna population. Supplementary Table 11: Functions of genes containing selective SNPs in the Xishuangbanna population.Supplementary Figs. 1–9 and Supplementary Tables 1–11 are available at figshare


Bombina orientalis) population in Beijing is purportedly descended from a single introduction of about 200 individuals translocated from Yantai, Shandong Province, China, in 1927.The successful establishment of a species population following a single introduction of a few individuals to a non-native area has been limited. Nevertheless, the oriental fire-bellied toad  sequences showed the two haplotypes found in Beijing nested within Yantai haplotypes, thus corroborating the historical record of the translocation source. The mtDNA and 11 nuclear microsatellite markers revealed both considerably lower genetic diversity in Beijing than in the source population and strong genetic differentiation between them. Although the current census population in Beijing may be in the range of a few thousand, the effective population size was estimated at only 20–57. Simulations also suggest that this population may have descended from 40–60 founders.B. orientalis population reveals the genetic footprints of a small population sustained in isolation for nearly a century. Our results provide an intriguing example of establishment success from limited founders and may inform ex situ conservation efforts as well as the management of biological invasions.The Beijing population’s genetic patterns were consistent with the consequences of a severe bottleneck during introduction followed by genetic drift. The introduction trajectory constructed for this The online version contains supplementary material available at 10.1186/s12862-022-02072-z. Genetic diversity is essential for maintaining the viability and adaptive potential of wild populations , 2. SmalHowever, some species have successfully, although rarely, established populations from a single introduction of a few founders, and those populations have persisted in isolation from other populations –13. SuchLeontopithecus rosalia ) [B. orientalis at the PKU site, but did find them at ZWY. With no known further introduction efforts and a lack of genetic exchange with other populations, the currently surviving Beijing population represents a case of successful establishment from a single introduction of a limited number of individuals. That has provided a rare opportunity to gain knowledge of the demographic and genetic consequences that befell a small, isolated vertebrate population over a time frame of approximately 90 years, or 30 generations     found noee years ).Fig. 1BB. orientalis in Beijing. We conducted field surveys of this species in Beijing  of the 12 sites that we surveyed in Beijing during 2015–2018, and we collected 13–49 toe clip samples from toads at each site, for a total of 150 samples  was about 1,500–4,000  and the alleged source population (Yantai) and a non-source native population (Qingdao) Fig. b.We successfully obtained the Cytb and D-loop sequences from all 261 samples. We found 38 and 10 haplotypes of the combined mtDNA sequences in the Yantai and Qingdao samples, respectively, but only two unique haplotypes in the 150 Beijing samples , number of private alleles, allelic richness (AR), and observed (HO) and expected (HE) heterozygosities  = 2, in which the Yantai and Qingdao samples clustered together and the Beijing samples clustered separately, was the best supported result  at all tested minimum allele frequencies showed consistently large values (from over 1000 to infinity) for the Yantai and Qingdao populations and substantially small values for the Beijing population  (Table Effective population size estimates (9) Table .Table 3RWe used mtDNA data with a simulation-based approach to estimate the number of female founders  that could retain the haplotype diversity of the Beijing population. The number of retained mtDNA haplotypes increased with an increasing number of females drawn randomly from the source population that was simulated using the observed mtDNA diversity in the Yantai population Fig. . Based oNa = 17.3; Table Na in the introduced population rapidly decreased during the bottleneck and continued to decrease because of subsequent genetic drift , the more rapidly the Na stabilized. Population genetic diversity loss trends were similar for different founder population size settings, except that a founder size of 20 resulted in population extinction in simulations with smaller λ values (1.02–1.06). The final Na values were higher with a larger founder size or a more rapid population growth  compared to that of the Yantai population [A recently published study also investigated the pulation . Using apulation  results pulation  identifiNc for the Beijing population was about 1,500–4,000 individuals, recent Ne estimates, under a range of parameter settings, were merely 20–57 (95% CI: 17–69)  Table . Thus, tound 0.1 ). Althou species , the conB. orientalis population in Beijing remain. The farthest Beijing location where we detected the species, YTS, was approximately 13 km from the original release site, ZWY  dispersal through continuously forested habitats [B. orientalis population has had the ability to further expand its range, but low habitat availability and connectivity in Beijing’s highly urbanized landscape have largely contained its spread. Although more distant populations than the ones we found in our field surveys likely exist, long-distance dispersal in the species’ Beijing range may be very difficult across fragmented habitat and human-dominated landscape and thus likely rarely occur. We also found a significantly positive relationship between the genetic distances among samples and their geographic distances from the ZWY site , a species native to South and Central America, were brought to Queensland in 1935 and, despite its low genetic diversity, their progeny spread along the Australian coasts, becoming a major invasive species in that country [The relationship between population genetic diversity and introduction success has long been a topic of heated debate –38. For  country , 44. OurB. orientalis diverged from the European Bombina clade in the Miocene [B. orientalis, one in the Korean Peninsula and adjacent areas and the other in the Shandong Peninsula  may risk that species becoming invasive in that area. Such invasion risk may be particularly high for amphibian species with broad geographic distributions, short life cycles, frequent reproduction, and large clutch sizes—characteristics that give them superior environmental adaptability and the potential for rapid population growth and range expansions [B. orientalis [B. orientalis population will not only track its demographics and range expansion but also help us gain insights into both the founding process and the prospects for small and isolated amphibian populations.Our case study of the introduced pansions –52. Therientalis , the speientalis , 54, traB. orientalis population and demonstrate strong genetic signatures of founder bottleneck and genetic drift in that small population over time. Genetic data-based simulations suggest that the current Beijing population, which has expanded to thousands of individuals and spread at least 13 km from the initial introduction site, possesses low genetic variation and a small Ne, and that the population may have descended from a limited number of toads released in 1927. The relatively high reproductive output, as well as the similar climatic and environmental conditions between their native and introduced ranges may have facilitated B. orientalis’ successful establishment in Beijing. Further investigation would reveal the long-term fitness effects of low genetic variability on the reproduction, adaptability, and persistence of this and other populations that experience similar genetic trajectories (such as invasive species colonizing non-native ranges), as well as aid conservation using ex situ techniques.Our study provides an intriguing example of a non-native amphibian population that established from a single introduction of a small number of founders and has persisted in isolation for nearly a century. Our results corroborate the historical record of the source population for the Beijing B. orientalis (body length 38–45 mm) spend most of their time on land, breeding from May to July in waterbodies such as streams, ponds, ditches, and paddy fields in low-altitude (< 900 m above sea level) mountainous forests, bush lands, and swamps [B. orientalis toe clips of adult toads were collected and stored in 95% ethanol at − 20 °C until DNA extraction. To compare the population genetic diversity between introduced and source populations, we collected B. orientalis samples from the reported source, Yantai City  in Beijing, we conducted a one-time mark-recapture experiment in August 2015 at a waterhole at the XSP site and marked (toe-clipped) 81 toads. One week later, we returned and captured 51 toads, 21 of which were marked toads from the first visit. From that, we estimated that a total of 197 (81 × 51/21) toads visited that single waterhole during that one-week period. Based on this rough estimate, and observed population sizes at several other sampling sites, we estimated 150–200 toads visiting each site. Considering the probable existence of unidentified breeding sites  and based on the distribution pattern and density of the sampled sites, we estimated between 10 and 20 breeding sites in the population range within Beijing. Therefore, we expected the total Nc of the Beijing population to be about 1500–4000 individuals.To estimate the population size  to extract total DNA from the collected toe clip tissues. DNA quality and concentrations were checked by agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer . To confirm species identity for each sample, we amplified an ~ 430-bp segment of the mtDNA 16S rRNA gene, as described in Lin et al. . This se Beijing . The PCRWe analyzed the genetic diversity of two mtDNA sequences: an ~ 1071-bp fragment of the D-loop region with primers D-F1086 5’-AACCGAGTGATAACCTA-3’ and D-R2102 5’-AAATCTACAGCGTGAGT-3’ and an ~ 1170-bp fragment of the cytochrome b gene (Cytb) with primers Cytb-MNCN-GluF 5’-GAAAAACCACCGTTGTTATTCAACTACA-3’ and Cytb-Amp-P10R 5’-TTCAGYTTACAAGACYGATGCTTT-3’ . PCRs weB. bombina and B. variegata 8 and (GT)15 biotinylated probes following protocols described in Zheng et al. [We also used a panel of 12 loci to generate nuclear microsatellite genotypes for all samples . After g et al. . Except g et al. . All PCRg et al. , and verhttp://www.ncbi.nlm.nih.gov) with accession nos. MZ593176–MZ593237 , and nucleotide diversity (π) using DNASP v. 5.0 [Using CLUSTAL W , we aligP v. 5.0  for D-loP v. 5.0 , 66.To visualize the mutational relationships and geographic distributions of mtDNA haplotypes, we constructed a haplotype network using the combined mtDNA sequences  for each sample and using the median joining method implemented in NETWORK v. 5.0 .P values were adjusted using Bonferroni corrections [t-tests to compare each diversity parameter across loci between populations.For microsatellite data, allelic dropout, null allele, and scoring error occurrences were estimated in MICROCHECKER v. 2.2.3 , and Harrections . We usedrections ; allelicrections ; the obsrections ; and therections . To evalK, was set to 1 through 7, with the largest K equal to the number of sampled sites . We also ran the analysis on the Beijing samples alone with K set to 1 through 5. For each K value, we performed 10 independent runs using the admixture model with correlated allelic frequencies among clusters. Simulations were run for one million Markov chain Monte Carlo iterations with a burn-in of 100,000. The most likely K value was determined using the ΔK method [K value were combined using CLUMPP v. 1.1 [To infer population genetic differentiation based on microsatellite data, we first used a Bayesian clustering algorithm implemented in STRUCTURE v. 2.3.4 . The numK method  implemenK method . The resP v. 1.1  with defTo assess the genetic structure among individuals, we also conducted discriminant analysis of principal components (DAPC) using the R package ADEGENET v. 2.1.3 . SpecifiRST and ΦST values of our microsatellite and mtDNA data, respectively, using ARLEQUIN, and tested for genetic differentiations among the populations using GENEPOP.To evaluate the extent of genetic differentiation among inferred genetic populations, we calculated the pairwise rho to test the correlation between genetic and geographic distances of the samples from each site and the ZWY samples. Pairwise Nei’s genetic distance [distm in the R package GEOSPHERE v. 1.5–14 (https://rdrr.io/cran/geosphere/).Since ZWY was one of the initial sites of release and likely the founder population for the other populations we found in Beijing see “”, we usedistance  between Ne) by using the LD single-sample estimator in NeEstimator v. 2.1 [Ne based on the amount of LD created by random drift among unlinked loci and corrects for bias caused by small sample sizes [Ne reflects the population size in the last few generations [Ne estimation methods, Gilbert and Whitlock [Using the microsatellite data, we estimated the sampled populations’ contemporary effective population sizes  due to the initial founding bottleneck and subsequent genetic drift over 100 years using BOTTLESIM v. 2.6 [Na as the contemporary Yantai population, we ran the simulation for 1000 iterations with the following parameters: life-span = 4 years, age of sexual maturation = 2 years [B. orientalis toads [Nc in Beijing was possibly in the range of a few thousand. As the species’ inhabitance and range expansion are highly dependent on suitable habitat, we excluded the possibility of an exponential growth pattern and instead used a logistic growth pattern to model population growth with the population capacity set to 5000. Liu’s record of the introduction event stated that a portion of approximately 200 toads were released at each of the two sites (ZWY and PKU), but he gave no accurate numbers [B. orientalis introduced at PKU did not seem to propagate, that site was not included in our population simulations. Assuming that a roughly even number of individuals were introduced at each site, we set a pre-bottleneck population of 100 individuals for the simulated founder population at ZWY. Those individuals’ genotypes were randomly assigned using the Yantai population genotype frequencies, and new mutations were assumed to be negligible. We tested four values for the founding population (bottleneck) size: 20, 40, 60, and 80. The post-bottleneck annual population growth rate (λ) was set to 1.02, 1.04, 1.06, 1.08, and 1.10.We simulated the loss of genetic diversity in the microsatellite data , an numbers . Since tAdditional file 1: Table S1. Site and sample size information for the Bombina orientalis surveyed in this study. Table S2. Mitochondrial DNA haplotype information. Table S3. Primer information for microsatellite loci. Table S4. Results of the Hardy–Weinberg equilibrium tests for individual microsatellite loci in each population. Table S5. Results of paired t-tests of genetic diversity parameters between the Beijing and Yantai samples. Fig. S1. Complete graphs of the STRUCTURE analysis of Bombina orientalis population genetic differentiation based on microsatellite data. Fig. S2. Correlation between genetic distance and geographic distance among sites in Beijing. Fig. S3. Simulation of the loss of genetic diversity over 100 years in the introduced Beijing population of Bombina orientalis at different initial sex ratios in the founder population.Additional file 2: Data S1. Individual mitochondrial DNA haplotypes and microsatellite genotype data.


Hemiculter leucisculus is an invasive fish and widely distributed in the Xinjiang Tarim River. In this study, RAD-seq was used to explore the genetic diversity and population subgroup structure of H. leucisculus in the Tarim River and develop relevant Simple Sequence Repeat (SSR) markers. The study collected 40 samples distributed at four different sites of the Tarim River. A total of 7,291,260 single nucleotide polymorphisms (SNPs) were obtained. The genetic diversity results showed that the population genetic diversity level of H. leucisculus was low. The population pairwise FST values ranged from 0.231 to 0.258, indicating that there was moderate genetic differentiation among these populations. AMOVA showed that the genetic variation within populations accounted for 92.31% of the total variation. The principal component analysis (PCA) and neighbor joining (NJ) tree revealed that the four populations could be separated into two clusters (upper-middle and downstream populations) and the individuals from Taitema Lake (TTMH) showed differences and had a bigger geographic distance than the others. There is the probability that the H. leucisculus from Bosten Lake entered Taitema Lake to breed and then expanded into the Tarim River due to the water diversion projects in location. In addition, 147,705 SSRs loci were detected and 22,651 SSR primer pairs were developed. This study will contribute to providing valuable molecular data for the management of wild populations, marker-assisted selection and resource exploitation of H. leucisculus. Hemiculter leucisculus  is a small cyprinid fish. It has a wide distribution in East Asia, living in the upper middle layer of water and likes to live in clusters. It is an invasive fish in the Tarim River of Xinjiang. In the past, the economic value of H. leucisculus was considered low, so its potential value has not been exploited. However, in some areas where stable populations have been established, it has become an important economic fish [H. leucisculus of Fenhe Reservoir and the growth characteristics of H. leucisculus [H. leucisculus from Dalai Lake [H. leucisculus mainly focuses on classification, nutrition and biology. At the same time, there is less research on the genetic background of H. leucisculus, which makes it difficult to gain strong theoretical support for the population dynamics of H. leucisculus. Therefore, it is important to study the genetic diversity of H. leucisculus.cisculus . Li Baollai Lake . CurrentRAD-seq (Restriction site-associated DNA sequencing) is a series of sequencing technologies based on restriction endonucleases characterized by simple operation, low application cost and wide coverage. It provides an accurate and efficient technique for studying population genetic structure and diversity at the genome level and has been widely used in these fields. Microsatellites, also known as simple sequence repeats (SSR), are abundant and dispersed broadly in the coding region and non-coding region of eukaryote genome and are composed of short 2–5 bp) variable tandemly repeated arrays. SSR markers are very practical genetic markers widely used in many fields, such as paternity testing, molecular marker-assisted breeding, genetic map construction, quantitative trait loci location and population genetics ,5. bp variaH. leucisculus in the Tarim River. The objectives of our study were to use SNP genotyping to investigate and evaluate the genetic diversity, genetic differentiation and population structure of H. leucisculus and develop SSR markers. These data are the basis for the formulation of effective management strategies for this invasive fish, providing essential information both for its control and its potential exploitation as a fishery resource.In this study, we used the RAD-seq method to unravel the population structure of Along the 1321 km long main course of the Tarim River, fish were sampled in four sectors corresponding to the upper, middle and lower reaches of the river. Fish samples were taken from four locations in the Tarim River basin from May to October 2019. In total, 40 specimens were collected from four sample sites in the Tarim River basin . The finEcoRI and incubated at 37 °C with T4 DNA ligase and EcoRI adapter. Using NEBNext dsDNA fragmentase, the reaction mixtures were segmented into 50–1000 bp. The samples were purified with AMPure XP beads . The fragmented DNA was used to conduct end repairing, dA-tailing and adapter ligation. Subsequently, the PCR productions were purified and pooled to obtain the lengths of 300–400 bp digested fragment sequences for library construction. PCR amplification was performed on the size-selected fragments  using NEBNext high-fidelity 2X PCR master mix. Finally, the PCR products were purified using a High Sensitivity DNA assay Kit  and sequenced on the Hi-Seq 2500 platform . The restriction site-associated DNA sequencing library was constructed using the recommended method described by Willing E M et al. (2011) . About 1The raw data generated from the Illumina sequencer were filtered with the fastp program. Low-quality data filtering included removing reads with adapter, reads containing more than 10% of N and reads containing more than 50% bases with a quality value of less than 20 to obtain clean reads for subsequent bioinformatic analysis. To ensure the quality and quantity of the data, the minor allele frequency (MAF) was below 0.01. With the population RAD-tags set as a reference, the Burrows–Wheeler Aligner (BWA) was used to compare reads of each sample in the population, and GATK, a mutation detection software, was used to detect SNP in the population.oH), expected heterozygosity (eH), and Wright’s inbreeding coefficient (FIS). Pairwise F-statistics (FST) among the four populations were performed using the Arlequin v3.5 [SNPs obtained by sequencing were used to evaluate population genetic statistics, including average nucleotide diversity (Pi), observed heterozygosity per locus (uin v3.5 . In addiuin v3.5 .A neighbor-joining (NJ) tree was constructed to cluster the populations using TreeBest software. Based on SNP differences between individuals, principal components analysis (PCA) can cluster individuals into different subgroups according to different traits. PCA analysis was conducted by software GCTA v1.93.2  in R. Thhttp://pgrc.ipk-gatersleben.de/misa/ (accessed on 9 November 2020)) was performed using the Microsatellite (MISA) identification tool, Primer3 V2.3.6 [Microsatellite mining  of the populations ranged from 0.000964 to 0.001144, which was highest in TTMH. The observed heterozygosity (Ho) across all populations ranged from 0.0593 to 0.0774, with an average of 0.0672. Ho was lowest in YBZ (0.0593) and highest in TTMH (0.0774). Expected heterozygosity (He) ranged from 0.1925 to 0.2055. eH was lowest in YBZ (0.1925) and highest in TTMH (0.2055). The individual inbreeding coefficients (FIS) of the populations ranged from 0.402 to 0.7016 . The CV error showed the lowest value with K = 9 and indicated that the optimal cluster number was nine genetic clusters B. When tThe study detected a total of 147,705 potential simple sequence repeats in 1,533,492 fragments . The uniThe Primer3 V2.3.6 was used to develop SSR primer pairs using 147,705 sequences containing SSRs . FinallyH. leucisculus individuals. Among the 10 primers, 6 pairs of H. leucisculus genomic DNA were successfully amplified by PCR, while the other 4 pairs of primers could not obtain PCR products  [H. leucisculus in the Huaihe River basin was low, while that in the Yangtze River was high (Pi = 0.03289) [H. leucisculus may have been affected by the founder effect during the invasion of the Tarim River, resulting in lower genetic diversity than in the Yangtze River basin [FIS in populations can directly indicate the loss or surplus of heterozygotes. The FIS values for the four populations were greater than 0, indicating that the H. leucisculus population had different degrees of heterozygote loss. Combined with oH < eH, there was a certain degree of inbreeding. The FST value has three levels of different genetic differentiation: moderate differentiation was 0.05 to 0.15, larger differentiation was 0.15 to 0.25 and high differentiation was greater than 0.25 [FST [H. leucisculus may be at a moderate level. Combined with low genetic diversity, the high FST values (FST = 0.243) among these populations may be caused by habitat fragmentation in the Tarim River basin [H. leucisculus was lower in the lower reaches than in other regions. This could be that the upstream terrain is more rugged than the downstream. Rugged reaches promote genetic differentiation, and gentle reaches are more conducive to gene flow [Genetic diversity research can reveal the status quo of species germplasm resources, genetic diversity and phylogenetic evolution of species . Due to < 0.005) . Genetic< 0.005) . It is t< 0.005) . We exam0.03289) ,24. The 0.03289) . In addier basin ,26. The han 0.25 . In fact.25 [FST . Therefoer basin ,30. In aene flow .Combined with the results of NJ tree and PCA, the individuals from TTMH showed differences and had a bigger geographic distance than the others. The genetic differentiation pattern of fish is usually consistent with the water system pattern of distribution ,33. It cH. leucisculus is an invasive species in the Tarim River system of Xinjiang. According to the population structure analysis, it was inferred that the origin of the four sites was the same species. Compared with the others, TTMH had some genetic differences. Dong et al. showed that human impact index (HII) was the most important factor affecting H. leucisculus invasion among the determinants of H. leucisculus invasion [Cyprinus carpio, Carassius auratus, Hypophthalmichthys molitrix and Hypophthalmichthys nobilis directly or indirectly from the Yangtze River basin for economic benefits and H. leucisculus and Bosten male fish in the pond dominated [H. leucisculus from Bosten Lake entered Taitema Lake to breed and then expanded into the Tarim River.invasion . The watominated ,43. Highominated . TherefoDatnioides Pulcher), and a total of 26,359 SSR loci were detected through sequence analysis [Pelteobagrus Wallis [H. leucisculus SSR markers. In this study, 22,651 SSR primers were designed to meet the primer development conditions by analyzing the simplified genome sequence of H. leucisculus. These results provide a basis for the development of H. leucisculus new primers.SSR markers are one of the most widely used molecular markers and play an important role in genetic research. Traditional SSR development methods often needed to construct the genome enrichment library, and the hybridization screening and sequencing processes were time-consuming and laborious. Today, the development of high-throughput sequencing technology and the sharp reduction of cost create better conditions for the development of SSR markers, which have the advantages of short development cycle, high yield and excellent flexibility . In receanalysis . Wang ets Wallis . At presH. leucisculus from Bosten Lake entered Taitema Lake to breed and then expanded into the Tarim River due to the water diversion projects in location. In addition, 147,705 SSRs loci were detected and 22,651 SSR primer pairs were developed. This study provides valuable genetic information for the management of wild populations, marker-assisted selection and resource exploitation of H. leucisculus.In the study, low levels of genetic diversity were observed among four populations in the Tarim River. Major genetic variations originated from within populations variation and moderate genetic differentiation among these populations was observed. The four populations were able to be separated into two clusters (upper–middle and downstream populations), and the individuals from TTMH clustered and had a bigger geographic distance than the others. There is the probability that the


Drosophila buzzatii exposed to a hallucinogenic columnar cactus, also consumed by humans, to identify ortholog genes exhibiting adaptive footprints of alkaloid tolerance. Genomic analyses in human populations revealed a suite of ortholog genes evolving under recent positive selection in indigenous populations of the Central Andes. Our results provide evidence of selection in genetic variants related to alkaloids toxicity, xenobiotic metabolism, and neuronal plasticity in Aymara and Quechua populations, suggesting a possible process of gene-culture coevolution driven by religious practices.Cultural transformations of lifestyles and dietary practices have been key drivers of human evolution. However, while most of the evidence of genomic adaptations is related to the hunter-gatherer transition to agricultural societies, little is known on the influence of other major cultural manifestations. Shamanism is considered the oldest religion that predominated throughout most of human prehistory and still prevails in many indigenous populations. Several lines of evidence from ethno-archeological studies have demonstrated the continuity and importance of psychoactive plants in South American cultures. However, despite the well-known importance of secondary metabolites in human health, little is known about its role in the evolution of ethnic differences. Herein, we identified candidate genes of adaptation to hallucinogenic cactus in Native Andean populations with a long history of shamanic practices. We used genome-wide expression data from the cactophilic fly  Populations responded both culturally and genetically to the specific environmental conditions, resulting in further dramatic changes with the advent of agricultural and horticultural societies ~ 8,500 years ago2. One of the best pieces of evidence is the ability to digest novel foods such as lactose of dairy cattle3 and carbohydrates from crops4, or to tolerate potentially toxic substances like alcohol5, salt6 and arsenic7. Despite these examples illustrating how lifestyles and dietary factors can shape global patterns of genetic variation in human populations, the role of ancient traditions has received little attention.The dispersal of human populations out of Africa almost 100,000 years ago has been accompanied by the colonization of almost every terrestrial habitat, resulting in conspicuous ethnic differences across regions8. Shamanic practices employing hallucinogenic plants are found in almost every indigenous group around the world; from the Siberian Koryaks using fungi of the genus Amanita, the Australian aborigines using Pituri herbs (Duboisia hopwoodii), to the popular use of Cannabis by Mongols, Indians and Chinese, the ingestion of Iboga roots (Tabernanthe iboga) in west Africa, or the consumption of Mandrake (Mandragora spp.) in Europe9. In the New World, archaeological investigations have shown that psychotropic cacti have been used in shamanic rituals for almost 10,000 years12. The use of cacti was both common and widespread across the Americas, ranging from southern United States to the northern regions of Chile and Argentina. These plants played a vital role in religion, medicine, ritual life and folklore of major ancient civilizations such as Aztecs, Mayans, and Toltecs, which remain until now e.g.,9. In South America, the consumption of giant columnar cacti of the genus Trichocereus, rich in phenylethylamine alkaloids, is common throughout the cultural continuum of the Andean region that can be traced back almost 6,000 years15. In the regions of Ecuador and Perú, T. pachanoi and T. peruvianus are among the most consumed species, whereas T. bridgesii is also consumed in central Bolivia16, and T. terscheckii is the most commonly ingested cactus at higher latitudes between southern Bolivia and northern Argentina18. All these four species have been equally named “San Pedro” in Spanish, or "Achuma" and other similar variants derived from the Quechua term “Kachum” (Cactus)20 and likely used indistinctly due to their high mescaline content21.Shamanism is an extremely ancient system of religious practices that predominated throughout most of human prehistory, starting at least 30,000 years ago23. The use of these cacti has been especially important to consolidate the politico-religious organization of early Central Andean civilizations. Dominant cultures often implemented cosmological beliefs and shamanic rituals aided by psychotropic plants in sophisticated religious complexes as expansionist strategies24. However, the use of these plants was not limited to religious activities, since its consumption was widely spread and practiced by all members of the society, with a special role of the female gender20. The mode of consumption included brews, dried buttons, raw tissues, cigars, enemas or even through the ingestion of cactophilic snails of the genus Scutalus, which concentrates the hallucinogenic alkaloids14.Common representations of San Pedro and its use are consistently found in stone sculptures, reliefs, ceramics and textiles throughout major cultures of the Central Andes: Caral (3000 BCE), Chavin (1500 BCE), Cupisnique, (900 BCE), Wari (600 BCE), Moche (400 BCE), Nazca (200 BCE), Salinar (200 BCE), Lambayeque (800 CE), Chimu  and Inca 25, including the teratogenic effects of mescaline in hamsters27, it is uncertain if the ancient use of psychotropic cactus has left detectable genetic footprints in Central Andean populations. Currently, alkaloids are a health problem of global importance, with authorities interested in avoiding the exposure of consumers to these toxins in food products28, and as expected, the religious use of psychotropic plants by children and pregnant women in Native communities have raised important concerns30. Notably, early cytogenetic studies in the Huichol Native Mexican population suggested the possibility that their long cultural tradition of consuming hallucinogenic cacti may have selected against inherited cytogenetic abnormalities31 and recent analyses have revealed an important genetic predisposition for drug responses and addictions33. However, the identification of genetic variants related to drug susceptibility has been challenging in human studies.Despite several studies have addressed the cytotoxicity of alkaloids not only in humans, but in several vertebratesDrosophila , and the possibility to perform complex physiological experiments in the latter, provides a valuable tool to identify candidate genes, that would otherwise be difficult to pinpoint. For instance, several studies have utilized D. melanogaster to detect genes implicated in human diseases, to understand the genetic architecture of quantitative traits, or to explore the genetic basis of alcoholism and drug addiction35. However, an important drawback of D. melanogaster is its poorly understood ecology36, hindering the extrapolation of ecological traits to other species.One powerful approach to address physiological adaptation in human genetics is to gain experimental insights from genetic model species. The highly evolutionary conservation among genetic pathways of humans and Drosophila, on the other hand, provide a well-known model of evolutionary ecology, especially useful to elucidate the role of chemical adaptation39. The evolution of cactophily in Drosophila was a major ecological transition resulting in the ability of many species to exploit cacti with high levels of secondary metabolites. In North America, for example, D. mettleri exhibit genomic signatures of positive selection in P-450 gene family related to the detoxification of isoquinoline alkaloids present in Senita (Lophocereus schottii) and Saguaro (Carnegiea gigantea) cacti, while adaptation to Agria (Stenocereus gummosus) and Organ Pipe cacti (Stenocereus thurberi) in D. mojavensis also included GST and UGT gene families38. In South America, the cactophilic D. buzzatii stands as an emerging model species in adaptation genomics41. Originally from the arid zones of central South America, this species utilizes prickly pear cacti of the genus Opuntia as main hosts and columnar cacti of the genus Trichocereus as secondary hosts, representing an interesting transition between tolerance and adaptation to the use of columnar cacti, especially useful to elucidate signatures of natural selection associated to generalized detoxification systems43. Moreover, the main host of D. buzzatii  is a widely consumed food by ancient and contemporary human populations across the Americas, allowing to assess the genetic consequences of switching from a nutritional to a hallucinogenic cactus with relevance in humans. Recently, we have shown that the transcriptional response of D. buzzatti to host shifts is mostly modulated by the phenylethylamine alkaloids of T. terscheckii41. We found that D. buzzatti deploys a wide array of genetic products to mitigate the harmful consequences of ingesting alkaloids, of which many were shown to be under positive selection39.The combined genomic and transcriptomic data of D. buzzatii related to the use of hallucinogenic cacti, also consumed by humans, provides an excellent opportunity to explore selection signatures in Native populations of South America. Herein, we characterized the alkaloid profile of T. terscheckii and used whole-genome expression data related to alkaloid tolerance in D. buzzatii to provide insights into the potential evolutionary role of the ancient shamanic lifestyle of indigenous human populations of the Central Andes  analysis revealed consistent retention times in all samples, with detectable levels of nine identified phenylethylamine alkaloids  spectra confirmed the presence of mescaline and trichocereine as the major components of the chloroform and ether fractions, respectively , and in higher doses of alkaloids. i.e., T. terscheckii vs O. sulphurea (DEG I); and T. terscheckii 2 × alkaloids vs T. terscheckii (DEG II). We found a total of 127 protein-coding genes differentially expressed across both comparisons (Table D. melanogaster (showing informative annotations) for downstream analyses. Specifically, 23 genes were over-expressed and 58 genes were under-expressed in comparison DEG I, while 33 genes were over-expressed and only 3 genes were under-expressed in the comparison DEG II  were enriched in Gene Ontology (GO) terms mostly related to the detoxification metabolism , neuronal processes such as neurotransmitter clearance and serotonergic mechanisms (Aldh), or related to general metabolism   and ATP synthesis (ATPsynC)  Fig.  and S5. nC) Fig. .Figure 3Homo sapiens , nervous system development , oxidative stress , exogenous drug catabolic processes, , alkaloid detoxification (CYP3 and CYP4 family genes), general metabolism  and response to narcotics  , nervous system development , muscle contraction  and response to toxic substances . Overall, our analysis of enrichment of biological pathways in H. sapiens showed similar results to those obtained for D. melanogaster , we searched orthologs sequences in the Human genome. Our analysis revealed a total of 70 genes exhibiting moderate to high orthology confidence with ens Fig. D. The GO) Tables  and S7. er Table  and S8.To investigate potential signatures of recent selection for alkaloids tolerance in human populations, we analyzed the extent of genetic differentiation and haplotype homozygosity in Native communities of the Central Andes exhibiting a long history of consumption of hallucinogenic cacti. For this, we used two genomic datasets including our target groups (within the area of influence of Andean shamanic practices) and reference populations . Data-set 1 included the Aymara and Quechua as target populations and Yukpa and Bari as reference populations, while our Data-set 2 consisted of Aymara and Quechua as target groups, and Wichi and Yanesha as references Fig. -S9. NotiP-values into a Fisher’s combination score (ZF). We identified ten candidate genes showing evidence of evolution under the influence of positive selection in at least one comparison; i.e., genes with identified signal either one , two , three (ALDH2), or four comparisons . Our results were highly consistent when considering either the median or mean values to summarize at the gene level the |iHS| and LRT scores estimated at the variant level, especially when comparing against Bari, Yukpa and Wichi reference populations  for Extended Haplotype Homozigosity, and Likelihood Ratio Test (LRT) score for genetic differentiation . Then, we combined the indexes into gene-level summary statistics (mean and median) to finally merge the resulting ons Fig. . Finallyons Fig. .Figure 4Fst test revealed low genetic differentiation respect to Central Andean groups , while our admixture analysis confirmed a certain degree of shared ancestry with the Andean population44  responsible of alkaloids detoxification and clearance of psychotropic medication in humans54. These findings are in line with our detection of several psychotropic and toxic alkaloids in T. terscheckii. Mescaline, for example, the major hallucinogenic alkaloid of the genus Trichocereus, and 2-phenylethylamine, a precursor of mescaline with mild psychotropic properties55, have both been associated with lethal and teratogenic effects in rodent embryos56. Although little is known on the action of the mescaline derivative alkaloids , early studies suggested no or mild psychoactive effects57, whereas tyramine, N-methyltyramine, hordenine and dopamine are likely precursors of these alkaloids, displaying neuromodulatory properties with possible adverse effects58. In particular, tyramine was reported to exert oxidative changes in the brain of rats similar to that of narcotics59, while excessive intake in humans results in a toxicological response known as the “cheese reaction”, leading to hypertension, migraines, neurological problems and respiratory disorders that can induce heart failure and brain hemorrhage60. Further, hordenine and 3,4-dimethoxyphenethylamine exhibit inhibitory effects on monoamine oxidases responsible for alkaloids degradation, potentiating the physiological effects of cactus alkaloids 62. Moreover, the preparation of San Pedro is usually performed by boiling the plant for several hours to potentiate its effects9, implying that even minority alkaloids could accumulate in significant quantities, influencing both toxic and psychotropic properties50. Indeed, although the hallucinogenic experience is the most relevant attribute of San Pedro cacti, other common physiological effects include respiratory failure, diffuse anxiety, motor dysfunction, partial or total cardiac arrest, or even death50. Further, regular use of hallucinogenic plants by children, pregnant, and breastfeeding women has been widely reported in Native American populations, raising important health concerns30. For instance, medical investigations reported significant associations between drug abuse and maternal and fetal morbidity63, while recent family studies suggest an important inherited predisposition driving the variability to drug response and addiction33. Taken together, our results suggest that the detection of subtle but significant shifts in allele frequencies of genes implicated in alkaloids metabolism could likely be related to their fitness value in the Central Andean region where humans have been consuming San Pedro cacti for millennia.Our in vivo study of transcriptomic responses in the genetic model of cactophilic 66. In fact, dietary adaptation has been a major contributor to human evolution, especially important during the transition from generalized plant diets of early hominids to the increased meat consumption and mastery of fire in Homo, leading to reduced ingestion of plant toxins68. Later cultural transitions to intensive agriculture, horticulture and animal domestication starting ~ 10,000 years ago resulted in further changes of the selective pressures on the human genome. Some common examples of modern instances representing gene-culture coevolution include lactase persistence during adulthood3, tolerance to alcoholic fermentation5, and protection against plant secondary metabolites69. Thus, the standing genetic variation derived from our early hominid diet may have provided the basis for rapid selective responses in shamanic societies with a deep history of consumption of plant secondary metabolites. Our findings are consistent with previous population genomic analyses combining ecological information, which found subtle shifts in allele frequencies of genes related to diets rich in roots and tubers in boreal human populations, suggesting that several SNPs may be involved in the detoxification of plant secondary metabolites65. Indeed, an increasing number of studies are discovering ethnic distributions of selected detoxification genes70, likely associated to novel toxins derived from modern lifestyles71, and recent advances in the field of pharmacogenomics have revealed important variation across populations in SNPs associated with opioid dose variability32. Our finding of signatures of selective sweeps associated with CYP3A43 and especially in CYP3A4 is in agreement with several studies where signals of positive selection in CYP3A genes were found in African, Asian and European populations, suggesting that this locus is sensitive to natural selection66. Moreover, CYP3A43 has been associated with the clearance of neuroleptic drugs (antipsychotics), while CYP3A4 is considered the most important drug metabolizing CYP enzyme in humans , and a major responsible for the metabolism of opioids and other alkaloids with important clinical implications in drug addiction73. Although little is known on the phenotypic effect of most polymorphisms, previous studies have provided evidence of higher activity of the wild-type CYP3A4*1 than that of most isoforms when exposed to the quinine alkaloid72. Thus, the higher metabolic rate associated to the wild-type found in Quechua and Aymara populations could have provided a selective advantage to detoxify cactus alkaloids, contributing to the allele fixation in the Central Andes.It has been argued that genetic differentiation between ethnic groups is largely related to the ingestion of substances and their detoxification24. More than twenty years of archaeological research in northern Peru revealed that the civilization of Caral, the oldest in the Americas , exhibited one of the largest early urban complexes in the world probably based on communal spiritual exaltation74. Detailed studies on the archaeological site have found evidence of cactus trade and numerous shells of the cactophilic snail Scutalus proteus used for concentrating the alkaloids of San Pedro14, suggesting the deep and widespread history of cactus use in the region . Shamanic traditions extended far in time, to the recent Inca Empire, and even survived the suppression of European colonization, which failed to eradicate the use of San Pedro that still persists today50.Elucidating the antiquity and extent of the use of psychotropic cacti in the Andean culture is a difficult task, if not impossible. The lack of writing systems in early Andean societies only allows us to infer the relevance of shamanic traditions through the indirect evidence left in archaeological settlements or in the inertia of the cultural legacy. Notwithstanding, most studies agree that the use of San Pedro seems to have been a critical element of the cultural evolution throughout most of the prehistory of Andean civilizations, even prior to the first expansion associated with the religious power and theocratic rule of Chavın and Cupisnique cultures almost 3,500 years ago76. Notwithstanding, our study has several methodological limitations that require discussion. First, we cannot rule out that the signals of selection detected in our analysis could have been driven by adaptive evolution in response to a complex combination of selective pressures that could also be potentially important for population health. For instance, missense of CTSF has been related to the Kufs neuronal disease47 while variants of COX5A result in lactic acidemia, pulmonary arterial hypertension and failure to thrive77. Some mutations in ATP2A1 can result in a rare autosomal recessive myopathy (Brody disease), characterized by exercise-induced muscle stiffness78, whereas variations in FADS1 have been associated with ethnic differences and medical phenotypes such as metabolic syndrome, abnormal lipid metabolism, and attention deficit disorder/hyperactivity79. In particular, CYP3A is not only a critical genetic contributor to drug clearance, but also has important implications in the endogenous metabolism of several hormones such as estrogens, cortisol and corticosterone associated with cancer risk and sodium retention related to hypertension and pregnancy complications80. The diversity of exogenous and endogenous substrates acting on CYP3A suggests that multiple and even antagonist selective pressures have likely shaped pleiotropic functions and potential trade-offs, resulting in complex genotype–phenotype relationships. Thus, further studies on the endogenous-exogenous metabolism interaction are needed to better understand their relative importance for phenotypic associations and ethnic considerations. Secondly, despite recent studies in Drosophila are significantly increasing our overall understanding of the genetic basis to the susceptibility to psychotropic drugs, such as cocaine, morphine, amphetamine and methamphetamine82, it is worth to note that several genes are likely to be overlooked, partly due to the divergent functionalities and physiological differences between flies and humans. Third, we acknowledge that many human orthologs of our candidate genes might not be shared across our studied datasets, resulting in the underestimation of the number of genes. Indeed, recent comparative studies of flies and humans recognize that the overall low number of reported genes associated to drug response, is likely due to the current low representation of human studies 33. This is especially critical in our case, as the reduced genotyping effort of South American Native communities implied a limited sampling for both our target and reference populations84. Thus, a larger collection of whole-genome sequences is necessary to supply larger sample sizes and SNP density to capture a larger number of genes and discriminate whether positive selection preferentially acted on protein-coding or regulatory regions.Although it is important to note that the effects of selection and demography are difficult to disentangle, the examination of multiple loci should be informative of these processes because population fluctuations result in similar random effects across loci, while positive selection tends to be locus-specific. Furthermore, our outlier approach and the combination of our two tests considering both genetic differentiation and linkage disequilibrium should be robust to the confounding effects of demographyAlthough more efforts are certainly needed to test our hypotheses, this study provides a first step in addressing the complex interplay between cultural and genetic co-evolution in the Central Andes, that would otherwise be extremely difficult to investigate. Our identification of candidate genes for alkaloid tolerance in Andean populations not only contributes to a better understanding of how ancient practices may have contributed to recent human evolution, but also provides insight on the genetic basis of ethnic differences in many disease risks. Several genetic variants related to alkaloid responses are also implicated in addictions, neurological disorders, and the metabolism of many important drugs. Therefore, the identification of ancient adaptive footprints to natural drugs across different ecosystems is fundamental for human health and especially important for the development of personalized genomic medicine. A larger representation of South American genomes combined with functional validation studies will be key to demonstrate the putative role of shamanism in the recent evolution of the human genome.40. Briefly, we collected individuals of D. buzzattii from a wild population of Northwestern Argentina to generate nearly isogenic lines of three conspicuous genotypes . We also collected fresh pieces of their hosts present in the area: the giant columnar cactus Trichocereus terscheckii and the prickly pear cactus Opuntia sulphurea. To assess the effect of the columnar cactus and the hallucinogenic alkaloids on the genomic expression of D. buzzattii, we exposed 1st instar larvae to each cactus, including the addition of the alkaloid fraction of T. terscheckii (2 × the native concentration). Thus, we employed three rearing media  to perform two pairwise comparisons  for differential gene expression analyses (see below). We extracted total RNA from 10 batches of 3rd instar larvae for each genotype , using a TRIzol/RNeasy protocol specific to Drosophila. We used the Illumina paired-end library (insert size: 150–450 bp) and sequenced in a HiSeq 2000 platform with 101-cycle reads, obtaining a mean of 16 Gbp of raw reads per genotype and treatment (NCBI Accession: PRJNA314520).The experimental set up has been previously described in detailT. terscheckii, we collected fresh samples from five individuals at different times of the year to account for temporal variation in alkaloid concentration. The plant material was collected in the northwest of Argentina, in the Valle Fertil Natural Park (public land of the province of San Juan) and the identification of the cactus was performed by Alejandro Saint-Esteven et al85. We isolated alkaloids from plant tissues through the alkaline-CH2Cl2 extraction method40. The chemical profile of the extracted fraction was accomplished by GC–MS (Thermo Scientific EM/DSQ II—Trace GC Ultra AI3000). Alkaloids identification was performed by comparing the retention times and mass spectra with reference standards and database spectra (UMYMFOR and NIST). The relative variation and abundance of each alkaloid was quantified based on the integration of the peaks area across the gas chromatograms. We analyzed five GC quality standards  of the most representative alkaloids present in the genus Trichocereus21: Tyramine; Hordenine; 3,5-dimethoxy-4-hydroxyphenethylamine; 3,4-dimethoxyphenethylamine; 3-methoxytyramine. Given the occurrence of dopamine as a major biosynthetic precursor of mescaline and other substituted phenethylamines in giant columnar cacti21, we performed an acid extraction to pH 3 with 0.1 M HCl solution. Dopamine identification was performed by comparing the retention time and mass spectra of the acidic extract and a dopamine reference standard  through HPLC–tandem Mass Spectrometry (Waters Quattro Premier XE spectrometer). To identify mescaline and trichocereine  alkaloids, we used their solubility differences, extracting with chloroform and ether, respectively86 and purified the solutions by column chromatography (silica gel G 60 Merck) with a mixture of CH2Cl2:MeOH. Major compounds were determined by thin-layer chromatography and identified via 1H-NMR in a Bruker AM-200 MHz spectrometer (details in Supplementary Information). All methods were carried out in accordance with relevant guidelines and regulations. The holotype of the cactus collected in our sampling region is housed at the Herbarium of the Institute of Botany Darwinion in Buenos Aires (voucher No. 8958). Collection permits were issued by the Environmental Ministry of the government of San Juan Province (Argentina), under permit No. 1300–0236.To thoroughly characterize the alkaloid fraction of D. buzzatii, we analyzed differential gene expression between flies reared in: T. terscheckii vs O. sulphurea (DEG I); and T. terscheckii 2 × alkaloids vs T. terscheckii (DEG II). Given that Native communities do not isolate the alkaloids, but rather concentrate all the characteristics of the plant, our first comparison (DEG I) allowed us to account for the differential expression caused by the columnar cactus , while our second comparison (DEG II) allowed us to elucidate the specific effects of high doses of hallucinogenic alkaloids . For the search human orthologs, we combined the significantly differentially expressed genes of both comparisons to contemplate the distinctive effect of the columnar cactus and especially its alkaloid fraction. Raw reads of our nine transcriptomes (three genotypes per treatment) were quality controlled using FASTQC v.0.10.187 and filtered for quality scores ≥ 25 and minimum lengths ≥ 25 bp, resulting in a mean of 12 Gbp per genotype in each treatment. We used the program RSEM v1.2.3088 to estimate gene expression levels in fragments per kilobase of transcript per million mapped reads of protein-coding genes using the reference genome of D. buzzatii39. To analyze the differential gene expression, we used the NOISeqBIO method of the R package NOISeq v2.18.089 with a false discovery rate (FDR) < 0.01, and applied a posteriori filter using a custom script to further reduce possible spurious “noise”. The statistical strategy of NOISeq considers the differences in both the mean expression level and in the order of magnitude to measure changes in gene expression between conditions, and thus identify significantly differentially expressed genes. We searched human ortholog genes from our filtered gene list through the DRSC Integrative Ortholog Prediction Tool (DIOPT) v6.0.190 which facilitates the identification of orthologs by calculating a score for the support of a given orthologous gene-pair relationship, as well as by a weighted score based on the functional assessment of molecular function annotation of all fly-human orthologous pairs predicted by each tool and algorithm. We retained the genes with moderate (best score in the forward or reverse search and DIOPT score >  = 2 or DIOPT score >  = 4) and high confidence rank (best score in both forward/reverse search and DIOPT score >  = 2). Because this tool uses D. melanogaster genes as input, we first performed a cross-species analysis to identify homologous genes with D. buzzatii and carried out a detailed exploration of the functional annotation using g:Profiler91. To interpret the biological information in the context of Homo sapiens , we analyzed our ortholog genes in the Reactome Knowledgebase92.To evaluate the effects of the columnar cactus and its alkaloid fraction on 93) and two reference populations from Northern Andes/Caribbean (Yukpa and Bari from Venezuela94). Data-set 2 consists of three focal ethnic groups of the Central Andes , and two reference populations from the Gran Chaco in Argentina (Wichi), and western Amazonia (Yanesha) in Peru44. While Aymarans, Quechuans and Uros represented our target groups, the reference populations represented our “control” groups  as they constitute putatively culturally divergent and relatively isolated populations95 outside the distribution range of Trichocereus species , resulting in a best model of K = 8 for both data sets  data Table . Data-seies Fig. . We filts Figure . We remo97, after phasing the data using the 1000 Genomes haplotypes as reference96. This analysis revealed the genetic clustering of all Central Andean individuals (both data sets), Bari, Yukpa (Data-set 1), Yanesha and Wichi (Data-set 2) individuals , we performed an additional local fine-scale analysis of genetic structure using FineStructure v4s Figure . We furt Figures  and S8 t Figures . Altoget98 to contrast whether allele frequencies in any of the evaluated populations are significantly differentiated from the putative ancestral genetic background. We performed the TreeSelect test in the Central Andean population, using the reference population of Han Chinese96 in all cases, and one reference population of South America  as summary statistics at the protein-coding gene level. In order to exclude possible stochastic signals of non-selective events, such as genetic drift or demographic fluctuations, we implemented a genome scan approach100 by taking into account the gene-level background for protein coding regions. We computed the median and the mean summary statistics for both |iHS| and LRT scores and estimated gene-level empirical distributions for each genomic background as previously implemented76. Empirical P-values were calculated using the gene-level score distributions generated from the genes in the background genome set. Finally, LRT and |iHS| based scores were combined using the Fisher combination test101:Pi stands for the empirical P-value obtained from the test i. Gene-level formal P-values were finally derived from the X 2 distribution with 4 degrees of freedom101. We thus obtained a total of eight ZFscores per gene for both the mean or the median as summary statistics: two for Data Set 1 (with either Yukpa or Bari as the 2nd reference population for the TreeSelect test), and two for Data Set 2 .For the analyses of positive selection, we employed two alternative approaches: the degree of genetic differentiation and the extended haplotype homozygosity. For the genetic differentiation test, we used the method implemented in the TreeSelect softwareSupplementary Information 1.Supplementary Information 2.


Penaeus plebejus) is endemic to eastern Australia and is of high commercial and recreational value. As part of a recreational fisheries enhancement initiative, hatchery reared juveniles from Queensland were released into two, more Southern New South Wales (NSW) estuaries between 2014 and 2015. Responsible stock enhancement programs rely on knowledge of the population structure of the released species. Previously, in consideration of fisheries data, it was assumed the king prawn populations in Australia are one single breeding stock. In the present study, our first aim was to test this posit of no genetic differentiation using mtDNA control region (mtCR) sequences from the wild samples collected from four estuaries ranging from Queensland/NSW border  to Southern NSW. The second objective was to test for signals of hatchery-released animals in the two stocked estuaries. All four surveyed populations had an extremely high level of haplotype diversity (average h = 99.8%) and low level of haplotype sharing between populations. Estimates of PhiPT values were <0.01 or close to zero and AMOVA test did not indicate any significant differences among populations. Further, phylogenetic analysis and principal coordinate analysis did not support division of samples by population. Collectively these results suggest that eastern king prawn populations along the NSW coast can be considered as a single stock and stocking from the Queensland samples will not necessarily impact the genetic composition of the overall stock. After stocking of two estuaries, sharing of haplotypes was moderate to very high in the stocked sites (>80% in some collections) but negligible in the two unstocked estuaries . Moreover, some haplotypes present in the hatchery broodstock were detected in stocked sites, but not in unstocked sites. The highest stocking signal was detected in the estuary which becomes isolated from the sea by sand barrier suggesting such “lakes” maybe more favourable for stocking than estuaries directly open to the sea. Findings in the current study should assist in designing and implementation of future prawn stocking programs.Eastern king prawn ( Penaeus plebejus is a high value species in commercial and recreational fisheries and an endemic species to the eastern waters of Australia, ranging from a northern limit at Swain Reefs in Queensland down to Northeast Tasmania in the South  varying from 4% to 35.6% (n = 27)  . The hig(n = 27)  highlighReliable methods for identification of hatchery individuals at recapture is crucial for the precise assessment of stock enhancement programs. For some prawn release programs, stocked animals have been identified using coded wire tags and uropod clipping, as in Japan , and somA 2-year release program for eastern king prawn was initiated in recruitment limited estuaries in Australia in 2014, to enhance recreational fisheries for the species. The associated monitoring program collected prawn samples and assessed abundance from stocked and unstocked estuaries and this statistical information was reported to provide an initial non genetic determination of the potential contribution of the releases to prawn populations within the estuaries . The objWild broodstock consisted of female prawns that had already received spermatophores. They were collected by commercial trawlers and transported to hatchery facilities at Rocky Point . In the Four estuaries  consistiGenomic DNA extraction was conducted using QIAGEN DNeasy Blood and Tissue Kit, following manufacture’s protocol. The integrity of extracted DNA was tested using 1% agarose gel electrophoresis. PCR amplification of mtDNA control region (mtCR) was achieved using a published primer pair . PCR assDNA sequencing of PCR products was conducted using DNA Sanger sequencing  from both directions using the specific PCR primer pair mentioned above. Sequences were evaluated for the sequencing errors where there was disagreement between the forward and reverse sequences, the electropherograms were inspected manually, or when one of the two sequences corresponded to the consensus sequence, the consensus sequence was used.ST analogue which calculates population differentiation based on the genotypic variance  and tested for DNA sequence similarity within and between sample locations using BioEdit Sequence alignment editor V7.0.5.3 . Haplotyvariance  and analvariance . Phylogevariance . The patvariance  based onh) and average nucleotide diversity (π) was 99.8% and 2.3%, respectively.A stretch of 716 bp sequence fragment was recovered from 871 samples. Considering all samples, 294 polymorphic sites were detected along the 716 bp length, and a total of 750 mtCR haplotypes were identified among 871 samples and five populations (GenBank accessions ON804899—ON805769). The average haplotype diversity , each separate population had at least some specific types of haplotypes shared between individuals within that given population , with thp < 0.05  and all the variation (100%) was attributable to within population variation. DNA sequence divergence estimates were very similar for within populations and among populations comparisons, varying between 2.25%–2.29%  analysis of the mtCR sequences generally did not support division of samples based on the populations  with somFor the unstocked estuaries Corunna and Durras, nearly every animal had its own unique mtDNA haplotype, specifically 99% and 98% of haplotypes were unique respectively. By contrast, only 63% of the samples from Wallagoot, and 94% of the samples from Tabourie had unique mtDNA haplotypes . A totaln = 46) selected randomly irrespective of the sample collection time from each site, Wallagoot samples had the lowest number of haplotypes among all the populations , there was a high level of DNA similarity across the ∼700 bp with an average of 98% similarity for all sample pairs. DNA sequence divergence between sites were very similar to the within site divergence. Consequently, most of the tests between sites  were not statistically significant. None of the pairwise comparison of PhiPT values were significant between sites for pooled temporal data, and 100% of the molecular variation was attributable to within population variation. Moreover, neither phylogenetic analysis, nor PCoA analysis identified segregation of samples by populations.Collectively, these results suggest that the eastern king prawn populations in the east coast of Australia can be considered as a single panmictic genetic population, a conclusion which agrees with previous considerations based on adult migration, spawning patterns and dispersal of larvae along the coast , and alsWe posit that, given the high level of haplotype diversity, that shared haplotypes within sites result from kinship . For Wallagoot, a stocked site, a very large number of samples (∼37%), share a haplotype with at least one other sample. Indeed, within about the 6 months of the first stocking for Wallagoot, when the estuary was closed to the ocean, there is genetic evidence that nearly all samples were from stocked animals, based on the criteria of sibship. Approximately 6% of samples at Tabourie shared haplotypes with at least one other sample from this site. However, for the unstocked sites the respective values were very low (∼1% Durras and ∼2% for Corunna), which provides an estimate of natural background sharing. Given background sharing is around 1%, the values for the stocked sites could guide our estimates of the upper range of contribution rates for released prawns. Since eastern king prawn do not reproduce within estuaries, and rely upon a marine migratory phase to complete their life-cycle , isolatiIt is noteworthy that eastern king prawn collected from stocked estuaries following the second release event, when broodstock samples were available, haplotypes that were found within the hatchery broodstock were also detected in the stocked estuaries, although they were detected at low frequencies (1%–2%). The low detection rate may be due to the unknown number of missing broodstock samples. Moreover, it has been reported there is a high variance in prawn fecundity between females in the hatchery , so it cThere was a substantial difference in the proportion of shared haplotypes , between Wallagoot and Tabourie. In particular, over the 5 months following the 2014 release, over 80% of eastern king prawn collected from Wallagoot appear to be stocked. This strong signal at Wallagoot can be attributed to the long-term closure of the estuary to the sea, creating a type of marine lake, limiting the emigration of the animals out to sea and eliminating natural recruitment and dilution of the proportion of restocked animals into the system. Given this, releasing prawns in similar closed estuaries may be more successful than releasing in more open estuaries.Our results for Wallagoot highlight the potential impacts of stocking in severely recruitment limited estuaries, where stocked eastern king prawn can provide benefits to anglers for at least 5 months post-release, and these findings align with previous surveys of pilot release in these systems . The othCollectively, these findings show that eastern king prawn releases contribute to estuarine fisheries even where natural recruitment is occurring. Last, our results demonstrate that given the likely absence of broodstock information, the kinship approach is an effective method of identifying stocked animals.Acanthopagrus butcheri  and red snapper Lutjanus campechanus (per se (they carry their own genetic tags)  . Bravingvailable . Should The present study reports several novel results and conclusions from an attempted prawn stock enhancement program (which in itself is a rather novel undertaking) and also novel results from population genetic assessments. Firstly, we conclude nearly all wild samples have unique mtDNA haplotypes, perhaps reflective of large population sizes. Second, notwithstanding the diversity, we conclude no significant differences among populations suggesting a panmictic stock along the NSW coast. Third, to compensate for incomplete broodstock records and samples, we used a novel approach of comparing the level of haplotype sharing to infer restocking success assuming shared haplotypes likely indicate siblings. The restocked estuaries had much higher frequencies of shared haplotypes than the unstocked estuaries suggestive of a stocking signal. Lastly, we conclude that the strongest stocking signal was at a location isolated from the sea, effectively a type of lake, and this novel knowledge may assist further stocking programs.