Panulirus homarus species complex, distributed widely through the Indo-Pacific, from South Africa to the Marquesas Islands.Evolutionary divergence and speciation often occur at a slower rate in the marine realm due to the higher potential for long-distance reproductive interaction through larval dispersal. One common evolutionary pattern in the Indo-Pacific, is divergence of populations and species at the peripheries of widely-distributed organisms. However, the evolutionary and demographic histories of such divergence are yet to be well understood. Here we address these issues by coupling genome-wide SNP data with mitochondrial DNA sequences to test the patterns of genetic divergence and possible secondary contact among geographically distant populations of the highly valuable spiny lobster ST = 0.148, P < 0001), superficially in accordance with previous analyses. However, detailed demographic analyses supported a much more complex evolutionary history of these populations, including a hybrid origin of a North-West Indian Ocean (NWIO) population, which has previously been discriminated morphologically, but not genetically. The best-supported demographic models suggested that the current genetic relationships among populations were due to a complex series of past divergences followed by asymmetric migration in more recent times.After stringent filtering, 2020 SNPs were used for population genetic and demographic analyses, revealing strong regional structure (FOverall, this study suggests that alternating periods of marine divergence and gene flow have driven the current genetic patterns observed in this lobster and may help explain the observed wider patterns of marine species diversity in the Indo-Pacific.The online version contains supplementary material available at 10.1186/s12864-022-08984-w. In comparison to the terrestrial environment, historical population divergence and speciation in the marine environment is believed to have occurred at a relatively slow pace, with historical interactions poorly understood, due to difficulties in easily observing the most important barriers to gene flow in this realm. Past heterogeneous historical changes in the marine environment and currents, have affected connectivity and dispersal patterns over time, resulting in complex patterns of speciation that are not easily discerned from the present. A variety of speciation processes have been proposed to explain patterns of marine diversity in the Indo-Pacific, including speciation from a center of origin in the Indo-Australian Archipelago , vicariaExamining situations where there are historical patterns of lineage divergence and secondary contact in both core and peripheral populations, are important not only in understanding speciation process, but also for the current conservation and management of species in the marine environment. Spiny lobsters  with wide distributions and high species diversity can be viewed as an excellent model for understanding demographic patterns of marine diversity and speciation. Furthermore, their extended pelagic larval periods  could In marine organisms, genome-wide SNP discovery has been of considerable assistance in improving our understanding of the degree of population genetic differences , 13, detSpiny lobsters play a key ecological role as predators in marine ecosystems in many parts of the world. They are also a major economic resource for artisanal and commercial fisheries in tropical, subtropical and temperate ocean regions of the world, with combined annual landings valued at over US$1 billion . BecausePanulirus homarus  species complex has a strong genetic structure along with a very extensive distribution, despite its long larval period (~ 6 months). The species has three or four morphological subspecies  [P. homarus rubellus subspecies from South African (SA) waters, with the possibility of limited secondary contact with the adjacent P. h. homarus subspecies in the Indian Ocean [P. h. megasculpta samples from the coasts of Iran and Oman in Northwest Indian Ocean (NWIO). Instead, the genetic data indicated that P. homarus from each of the East African (EA), NWIO, and core region of the East Indian Ocean/Indo-Australian Archipelago all belonged to the P. h. homarus subspecies, with only a small degree of genetic divergence between each region. The small and isolated eastern peripheral population in the Marquesas Islands (MI) in the central Pacific Ocean had previously been identified as another potential subspecies (P. h. “Brown”), but was identified genetically as merely a divergent remnant population [P. h. rubellus from SA, and P. h. homarus from its “Central core” region of the East Indian Ocean/Indo-Australian archipelago, and its peripheral populations in EA, NWIO, and MI), or what are the drivers of these potentially complex patterns of divergence.Among Indo-Pacific spiny lobster species, the tergits) , 22  from which it could be anticipated the genetic variation and demographic size of a species’ decreases from the center to the edge of its geographic distribution, along with the degree of connectivity [Here we used genomic SNP and mtDNA sequencing data to test a variety of potential historical models of population divergence, admixture and demographic size change, to determine the most likely evolutionary histories of the ectivity  as obserP. h. rubellus), Tanzania in eastern Africa (EA) (morphotype described as P. h. homarus), Oman and Iran in the Northwest Indian Ocean (NWIO) (morphotype previously described as P. h. megasculpta), from the species’ genetically homogeneous “Central core” (C) distribution throughout the East Indian Ocean/Indo-Australian Archipelago (with representative samples from South India) (morphotype described as P. h. homarus), and from the Marquesas Islands (MI) isolated population in the mid-Pacific Ocean (morphotype described as P. h. “Brown”). The samples were either purchased from fishers or fisheries markets after confirmation of geographic location of capture or obtained by courtesy of research organizations as described in Lavery et al. [Muscle tissue was taken from the pleopods of lobster specimens sampled from all five major regions across the Indo-Pacific Ocean known to be genetically differentiated from previous studies  Fig. 1,,1. Samply et al.  and presy et al. . GenomicP < 0.00001) and linkage disequilibrium (r2 > 0.80) of loci were pruned in Plink 1.9 [After sequence data analysis, QC and SNP calling by the Diversity Array Technology proprietary pipeline (DArT PL)  genotyplink 1.9 . Finallylink 1.9  were alsE) and observed (HO) heterozygosity and inbreeding coefficient (FIS) were estimated by Genetix [Population genetic diversity indices including, expected  and pairwise Fe StAMMP  for the e StAMMP  to visuae StAMMP  and LEA e StAMMP  with sNMWHYBRIDS  using Je package .P. h. rubellus in SA versus the adjacent Indian Ocean/Central P. h. homarus; P. h. megasculpta in NWIO versus the adjacent Central P. h. homarus; and the Central P. h. homarus MI. The demographic scenarios compared included a standard neutral model (population split with constant population size over time), population size change, secondary contact, and division into different time periods (see Supplementary file ref migrants per generation) was included in the models. The best scenario was selected using log-likelihood and Akaike information criterion (AIC) and was confirmed by visually comparing the data and model allele frequency spectrum plots. Then the best model’s parameters were optimized using 100 simulations. Further details of model selection and analysis were as described in Silva et al. [ref, mutation rate and generation time) was carried out as per Silva et al. [Historical demographic patterns were evaluated using the ∂a∂i package in Python3.8 based on methods described in Gutenkunst et al.  using tha et al.  and Porta et al. . The cona et al. . For coma et al.  with theP. homarus mtCR lineage from SA (PhSa78) database failed in SNP genotyping and therefore was only included in mtCR analysis.The maximum likelihood tree haplotypic median-joining network and Bayesian clustering  using BAPS  of the mAfter filtering steps, a total of 2020 SNP loci for 191 individuals were retained for analysis  as well as inbreeding (FIS), while the MI population has considerably lower values for all these measures. Moreover, the highest values of diversity indices were observed for NWIO and then SA populations. Arlequin and BayeScan identified 181 and 121 outlier loci respectively and those SNPs shared between the results of these two methods (81) were considered as outliers for further analyses. No outlier loci were detected by outflank.Measures of heterozygosity and inbreeding across all surveyed populations Table  indicateST = 0.148, P < 0.0001, and outlier FST = 0.637 versus mtDNA ФST = 0.68, P < 0. 001) among the five regional groupings. Pairwise genetic differentiation tests showed the highest difference between MI and all other regions, and the smallest differences between the Central and EA regions, for both data sets  and all other locations except MI were lower than the divergences observed for the MI, in contrast to the pattern observed in mtCR DNA sequence data  sampled from both Oman and Iran  revealed a number of individuals with likely hybrid origins Table . Four inpe Fig. S from SA P. h. rubellus was the earliest population to diverge from the Central P. h. homarus lineage. The relative divergence times estimated from the demographic models were  generations ago; NWIO P. h megasculpta and P. h. homarus- 30*2Nref  generations ago, Marquesas – P. h. homarus- 0.2*2Nref generations ago. Each of the asymmetric migration rates were greater than ten times that estimated for the reverse direction. Each of the peripheral populations were estimated to have population sizes less than one half that of the Central population after initial demographic split, and both the NWIO and MI populations were estimated to have had recent population size reductions. Demographic analyses using DIYABC resulted in remarkably similar best-fitting demographic histories , undertaken in ∂a∂i, showed that a range of different demographic events best fitted the genomic SNP data Fig. , Table 4P. homarus complex has facilitated new insights into the evolutionary processes occurring in this species. Overall, the analyses reveal that this species complex exhibits a rich and dynamic pattern of historic and present divergences and introgression, shedding light on the diverse evolutionary processes that contribute to high marine species diversity in the Indo-Pacific. The study confirmed an instance of mitonuclear discordance using both genome-wide SNPs and mitochondrial DNA sequences, highlighing the advantage of using genome-wide SNPs coupled with mitochondrial DNA to help understanding population admixture.The application of SNPs for the analysis of genomic diversity in the P. homarus from SNP data were similar to those observed in other spiny lobsters [IS values. The patterns across populations fit the overall population structure interpretations (see below), with both higher diversity and homozygosity in populations believed to be of mixed origin , and lower diversity and FIS in the isolated, remnant population (MI). Similar raised FIS has been observed in other crustacean studies, including on P. ornatus [P. homarus within NWIO [Portunus pelagicus [Overall, the genetic diversity values observed within lobsters , 44. The ornatus , P. homahin NWIO  and the elagicus .P. homarus is at its lowest in the eastern peripheral population (MI), but highest in two of the western peripheral populations (SA & NWIO), a pattern consistent with the CPH which predicts that genetic diversity should be higher in the central population than at the margins. However, the CPH is not always a reliable explanation for the patterns of genetic variation within populations including when there are a lower amounts of sample date from the margins of the population range for comparison, as in this study. Other factors are acting in the western periphery of this species, where secondary contacts appear to have occurred between core and peripheral populations may explain the observed genetic diversity pattern. Another marine species with similarly wide Indo-Pacific distribution, the pearl oyster (Pinctada margaritifera) shows similar partial support for CPH hypothesis, but instead at the western margins [The genetic diversity across the population groupings of  margins .ST analyses revealed very strong genetic structure in the P. homarus complex. The results confirmed the patterns of strong genetic divergence observed in mtDNA data in the previous study [P. h. rubellus, MI and the rest of P. homarus)  Fig. b. The reus) Fig. , 3, 4, oP. h. megasculpta genetic lineage, and explaining the inconsistencies in previous genetic data that questioned the genetic basis of this subspecies from this NWIO region. Previously, a distinct morphotype and subspecies (P. h. megasculpta) had been described [The new genomic analyses shed an entirely new light on the NWIO population, once again providing support for the existence of a distinct escribed , 22 in tescribed , 10. Insescribed , 48, 49.P. h. homarus and NWIO lineages. Both the ∂a∂i and ABC analyses indicate that the NWIO population has experienced a long period of genetic isolation from the remainder of P. h. homarus, with only very recent secondary contact driving the clear pattern of introgression observed.The historical demographic analyses provide a much stronger evolutionary context for interpreting the genetic introgression between the − 8) and generation time used by Silva et al. [Dating these evolutionary events accurately is difficult without a much stronger knowledge of rates of SNP mutation in this species. However, the mtDNA data indicates the (now apparent) “NWIO lineage” was estimated to have diverged between 0.6 mya  and 6 mya et al. , our demP. h. rubellus population and the adjacent P. h. homarus. From all the DAPC, admixture, network, demographic and hybrid analyses , which appears to have experienced comparatively little hybridization. The apparent relative isolation of this portion of the Indo-Pacific range of this species has been previously identified for this species [P. homarus to this NWIO region, followed by a long period of isolation, favored by oceanographic conditions in the Miocene and Pliocene, and then recent post-glacial conditions that may have favored reconnection and secondary contact.The introgression between the  species , 48, 49  species ). A seri species . It appeP. homarus. In addition, the marine environmental conditions throughout the OMZ in this region were considerably different from much of the surrounding Indian Ocean waters, likely providing strong selection pressures favouring the adaptive divergence of marine species in these waters. Both these factors are likely to have favored the divergence of a distinct lineage (or even subspecies) in this NWIO region. Evidence supporting the marine distinctiveness of the NWIO region includes its recognition as a distinct biogeographic province [The generally isolated nature of the NWIO over the long term was likely due to monsoonal upwellings (over the last ~ 13 mya) along the Somali and Oman coastlines, and is exemplified by the existence of a significant and persistent Oxygen Minimum Zone (OMZ) in the Arabian Sea over this long period . Althougprovince  and its province .P. homarus phyllosoma in the northern Indian Ocean and facilitating recent admixture between the Central (P. h. homarus) lineage and the NWIO (P. h. megasculpta) lineage in the NWIO region. There is recent evidence from fish species/subspecies with restricted distributions in the Arabian Sea, that these isolated lineages have occasionally mixed with adjacent endemic lineages to form hybrid populations in the NWIO [A substantial reduction of the monsoonal upwellings along the western Indian Ocean coastline  occurred during the Pleistocene glacial maxima, prompting a decrease in the intensity of the OMZ over these periods , and allthe NWIO , 57, 58.P. h. megasculpta subspecies could not be supported.In the mtDNA data, NWIO individuals from Oman and Iran had haplotypes scattered throughout the mtDNA phylogenetic tree/network, although a number of individuals sampled from Oman clustered together in their own clades –10. In cP. h. homarus populations, but a new, distinct lineage found only in NWIO, the “NWIO lineage”. The admixture of genetic material from these two lineages is mostly clearly revealed in the admixture plots , having a 50% SNP admixture of P. homarus and P. h. rubellus, confirming that is it very likely to be an F1 hybrid of a female P. h. homarus with male P. h. rubellus. Other SA individuals , indicating that they are progeny of male P. homarus and female P. rubellus. The demographic modeling provided additional evidence of the recent admixture of the P. homarus lineage into P. h. rubellus  or verylobster: ). Althouispersal .P. homarus is occurring at the peripheries of the distribution. The processes demonstrated here may be likely to occur in many other Indo-Pacific marine species with wide larval dispersal, although at different geographic scales, dependent on their maximum dispersal distances.Perhaps unsurprisingly for a species with such a long pelagic dispersal, most of the subspecies divergence in P. homarus complex, there is an apparently relatively simple example of rare dispersal, with a degree of genetic bottleneck, followed by allopatric divergence. The potential physical reasons for MI population’s colonization and subsequent isolation have been discussed previously, but in brief, rely on historical changes in current patterns in the central Pacific [P. homarus “Brown”; George, 2006). Although it appears to contain mainly a subset of the genetic diversity of the Central population, without further gene flow it is likely to form a new species in due time.At one extreme, in the eastern periphery of  Pacific . AlthougAt the other extreme, at the far western periphery, is a long-diverged population, that has apparently had time to evolve adaptations to cooler waters, and a degree of reproductive isolation. As discussed previously , this coP. h. megasculpta) population. With the aid of genomic SNP analyses, we now see that this population was likely founded through a rare colonization event more recently than that of P. h. rubellus. After a subsequent long period of isolation, sufficient for the emergence of a distinct genetic lineage, it has experienced recent secondary contact at a far earlier stage in its divergence than has P. h. rubellus. This has led to extensive introgression, and even though this lineage had diverged sufficiently to establish a distinct morphology, there is clearly little reproductive isolation. It remains to be seen whether this distinct lineage persists, or whether it becomes reabsorbed into the wider P. h. homarus population. Perhaps a close parallel can be seen in the largely sympatric and closely-related P. ornatus. In this species, there exists a distinct mtDNA lineage that predominates in the western Indian Ocean, where it likely originated, but is now also found at lower frequencies much more widely to the east [P. h. megasculpta lineage, if current levels of introgression persist.In between these extremes lies the NWIO (the east , 45. We Pinctada margaritifera [Portunus segnis [Panulirus penicillatus [P. homarus complex is likely to have been involved in these divergences, although it now difficult to discern in these species after long periods of divergence.There are a number of other examples of widely-distributed marine invertebrates with pelagic larval dispersal that have surprisingly divergent populations in the Western Indian Ocean, which were not predicted from morphology. These include the black-lipped pearl oyster, ritifera , swimmins segnis  and the cillatus  all withP. homarus complex is an excellent direct illustration of the active isolation and contact during the speciation process in widely distributed marine organisms. Similar patterns of isolation and secondary contract have been reported as occurred among Jasus spiny lobsters [P. homarus the processes are still in action and observable, which provides a better insight into some of the potential drivers of marine biodiversity in the Indo-Pacific.We suggest that the lobsters . For mosP. homarus complex appears to provide an excellent demonstration of a range of speciation processes that are likely to be common-place in other widely distributed marine invertebrates. Rather than support the simplistic predictions of theories such as the CPH, the genetic patterns observed here strongly suggest that relatively complex historical demographic events have driven the current patterns of marine biodiversity we see in this region. This study also has implications for the appropriate future management and aquaculture of spiny lobsters as a valuable fisheries resource. There is likely to be great value in using further genomic studies of this species complex to identify and understand the role of environmental factors in adaptive speciation. Lastly, it is likely there is similar complexity in the lineage divergence in this species in the South China Sea region and therefore further genomic investigations with larger samples sizes from this region are warranted.The Additional file 1. The result of supportive analysis from of mtCR sequences and SNPs in this study.Additional file 2. Script of models used in this study for demographic analysis.


Oncorhynchus kisutch) from 149 sampling locations and a total of 24,542 high‐quality SNPs obtained using genotyping‐by‐sequencing and mapped to the Coho salmon reference genome to (1) evaluate the current delineation of CUs for Coho in Canada and (2) compare patterns of population structure observed using neutral and outlier loci from genotype–environment association analyses to determine whether separate CUs that capture adaptive diversity are needed. Our results reflected CU boundaries on the whole, with the majority of sampling locations managed in the same CU clustering together within genetic groups. However, additional groups that are not currently represented by CUs were also uncovered. We observed considerable overlap in the genetic clusters identified using neutral or candidate loci, indicating a general congruence in patterns of genetic variation driven by local adaptation and gene flow in this species. Consequently, we suggest that the current CU boundaries for Coho salmon are largely well‐suited for meeting the Canadian Wild Salmon Policy's objective of defining biologically distinct groups, but we highlight specific areas where CU boundaries may be refined.Conservation units (CUs) are important tools for supporting the implementation of standardized management practices for exploited species. Following the adoption of the Wild Salmon Policy in Canada, CUs were defined for Pacific salmon based on characteristics related to ecotype, life history and genetic variation using microsatellite markers as indirect measures of local adaptation. Genomic data sets have the potential to improve the definition of CUs by reducing variance around estimates of population genetic parameters, thereby increasing the power to detect more subtle patterns of population genetic structure and by providing an opportunity to incorporate adaptive information more directly with the identification of variants putatively under selection. We used one of the largest genomic data sets recently published for a nonmodel species, comprising 5662 individual Coho salmon ( At a smaller scale, management units (MUs) may be defined as local populations that are demographically independent and thus should be managed separately  based on neutral loci, but a total of five stocks were ascertained based on putatively adaptive (i.e. outlier) loci. In another example, Sandoval‐Castillo et al.  in southern Australia, whereas neutral genetic markers identified a single metapopulation. These studies support defining separate categories of CUs that reflect different processes underlying genetic distinctiveness between groups. For example, MUs may be defined as genetically distinct groups that arise due to connectivity (or lack thereof) detected using neutral markers, while adaptive units (AUs) represent adaptively differentiated groups defined using markers that are putatively under selection, as proposed by Funk et al. , Chum (O. keta), Chinook (O. tshawytscha), Sockeye (O. nerka) and Pink (O. gorbuscha) salmon, are an important social, cultural and economic resource in Canada .Pacific salmon, all of which are semelparous anadromous species and which include Coho . These sampling sites were located within 35 of the 44 established CUs (Table https://www.ncbi.nlm.nih.gov/assembly/GCF_002021735.2/), as detailed in Rougemont et al. (https://github.com/enormandeau/stacks_workflow). SNPs with a read depth >10 and <120 , with a minor allele count (MAC) >15 (which translates to a MAF threshold of ~0.15%), and that were not missing in at least 95% of the data set were retained. We filtered on MAC rather than MAF because of the large size of the data set, following Rougemont et al.  using the R package ade4  and among populations within regions (ΦSC), as well as among CUs (ΦCT) and populations within CUs (ΦSC). We used the pegas implementation of AMOVA  in each sampled population using adegenet v2.1.3  to estimate the components of variance explained by genetic differentiation among regions  and genetic differentiation (βST) and the distance from the southernmost site to assess the potential confounding effect of latitude on subsequent genotype–environment association (GEA) analyses.Previous studies identified a strong signal of latitudinal isolation by distance (IBD) across the entire North American distribution of Coho salmon due to the northward postglacial population expansion  of 19 bioclimatic variables related to temperature (°C) and precipitation (mm) between the years 1970 and 2000 . The number of latent factors to retain was chosen using a scree plot. Then, we used the lfmm_ridge function in the lfmm v1.5 R package  method . For the candidate SNP data set, we used two subsets of GEA outliers: (1) combined unique outlier SNPs detected by both RDA and LFMM and (2) only outlier SNPs detected by RDA, according to recommendations by Forester et al. .We evaluated the extent to which genetic clusters differed if defined using neutral loci or candidate loci under selection, indicating the delineation of distinct management  and AUs. We used two approaches to assess neutral and putatively adaptive genetic groups: (1) distance‐based clustering and (2) discriminant analysis of principal components (DAPC).2.4.1p‐value <0.05 suggests that the correlation is stronger than expected based on randomized matrices.Distance‐based cluster analyses were performed to group sampling locations based on the genetic distances between all pairs of sites and to assess whether CU boundaries correspond with genetic clusters. Pairwise genetic distances between sampling locations were calculated using Cavalli‐Sforza and Edward's chord distance. Unrooted neighbour‐joining trees based on pairwise chord distance were generated using the ape v.5.5 package . We then visualized the separation of groups based on a linear discriminant analysis, which aims at maximizing variation between groups while minimizing variation within groups.Discriminant analysis of principal components is a model‐free clustering approach that aims at maximizing between‐group discrimination while minimizing within‐group discrimination  after filtering individuals for missing data . In total, our final data set included 5581 samples from 146 locations across 35 CUs  were excluded from further analyses due to low sample size .A PCA on individual genotypes from across all sampling locations revealed a split between the Thompson River (including the upper Fraser) and the rest of BC Figure . These tK = 2 clusters, ancestry coefficients clearly indicate a split between sampling locations in the Thompson River basin from all other sampling locations in BC, with KAW exhibiting a high degree of admixture between the two major regional clusters , while 8.6% of variation was found among populations within regions  and excluding KAW. As a result, the BC data set consisted of 4555 individuals across 119 sampling locations in 31 CUs with a total of 24,109 SNPs. The Thompson data set consisted of 1000 individuals in 26 sampling locations within four CUs and 10,009 SNPs.CT = 0.0569, p < 0.001) and differentiation between populations within CUs explained 4.97% of variance , while differentiation between populations within CUs accounted for 3.83% of the variance , with mean FST = 0.0551 in BC (range = 0.0005–0.1637) and mean FST = 0.0402 in Thompson  and a linear increase in genetic differentiation (βST) with increasing distance from the southernmost site , and all predictor variables significantly contributed to the model , and RDA6 was marginally significant  so we retained the first six axes , of which 375 were associated with temperature, 266 were associated with precipitation and 155 were associated with normalized distance. LFMM did not detect any outliers associated with geology. In total, 1117 unique outlier SNPs were detected by RDA and LFMM, and 129 were shared between both methods.The first three axes of the PCAs on the temperature and precipitation variables at sampling locations within the BC group were significant and cumulatively explained 85.3% and 88.9% of the total variation, respectively Figure . The ful Figures  and S5,  Figures . With LFp < 0.05) and explained 28.6% of the total genetic variation. We then incorporated this PC axis as a conditioning variable in a partial RDA (pRDA). All predictor variables, including the conditioning variable, had a VIF < 10, indicating no multicollinearity among predictors. The pRDA was significant . The first two axes were significant , we lowered the threshold to ±2.5 SD to increase the number of candidate loci for subsequent clustering analyses (see below). With this threshold, the RDA yielded 106 candidate SNPs associated with environmental predictors on the first two axes: 50 were associated with temperature, 29 with precipitation, 12 with normalized distance and 15 with geology , none of which overlapped with the SNPs detected by RDA (124 unique outliers with both methods).For the Thompson region, the first three axes of a PCA on temperature variables and the first two axes of a PCA on precipitation variables were significant, capturing 80% and 82% of the explained variation, respectively Figure . These fy Figure . Previou3.4All GEA candidate SNPs were excluded, resulting in a neutral data set consisting of 22,992 SNPs for the BC group and 9885 SNPs for the Thompson group. For the sampling locations in BC, the outlier data sets consisted of (a) 1117 unique SNPs detected by either RDA or LFMM and (b) 450 outliers detected by RDA. In the Thompson group, we performed analyses on two outlier data sets consisting of (a) 124 unique SNPs detected by either RDA or LFMM and (b) 106 SNPs detected by RDA.Using the neutral SNPs, the neighbour‐joining tree showed geographic clustering of populations within CUs in both regions Figure . In BC, r = 0.56, p = 0.001; neutral vs. RDA: r = 0.57, p = 0.001), indicating concordance among trees built with different marker types. Altogether, populations in 15 of the 18 CUs that clearly grouped together within their designated CUs showed the same unambiguous clustering with GEA outlier loci. However, we detected additional subdivisions among populations within CUs that were not observed with neutral loci .In the Thompson region, genetic clusters also typically corresponded with CUs regardless of marker type used Figures , which chttps://axuereb.shinyapps.io/Coho_DAPC/) and corroborated results from the neighbour‐joining clustering. In BC, populations in the Middle Skeena CU  and Lower Skeena CU (CO‐34 Sustut/Damshilgwit [SUS/DAM]) strongly separated from the rest of the BC populations, as did populations in Lillooet  and Lower Fraser . Additional axes revealed strong separation of West Vancouver Island populations  from other BC populations  and steelhead (O. mykiss) after strong genomic associations with adult migration timing that may not be reflected in the current configuration of CUs were identified  and interior Fraser (CO‐48) populations from all other populations, including those in the lower Fraser River : the three coastal populations in Burrard Inlet  formed a cluster that was distinct from the remaining four sampled populations in the same CU , all of which are tributaries to the Squamish River draining into Howe Sound. The Burrard Inlet populations were more genetically similar to populations in East Vancouver Island (CO‐13) across the Strait of Georgia than to the other populations in CO‐10. In the Nahwitti Lowland (CO‐15) in northern Vancouver Island, another genetic group consisting of the west coast Vancouver Island populations  and Quatsese River was clearly separated from populations in the same CU on the east coast of Vancouver Island across both methods. Marble Creek Coho may also be differentiated from other populations in this group. A similar pattern has also been observed for Chinook salmon, where individuals from Marble Creek were distinct from those in nearby locations . Previous work on Coho salmon population structure using microsatellites also failed to detect clear population structure in the central coast of BC  in this population compared with other sampled populations in this CU and across all populations.Patterns of genetic structure in the Fraser River drainage basin showed that all sampled populations in the Lower Fraser (CO‐47) clustered strongly together. Within the Thompson region, we detected well‐supported genetic clusters separating the Lower, South and North Thompson, and interior Fraser populations corresponding to CUs . One exception was Salmon River (SAL) in South Thompson (CO‐08), which was clearly separated from the remaining South Thompson populations with DAPC and formed a unique branch on the neighbour‐joining tree. This distinction of Coho from Salmon River has not been reported previously, suggesting that our data set was able to identify more subtle differentiation than was previously detectable. Although significant enhancements were made in Salmon River to mitigate precipitous declines in Coho populations in the Thompson River drainage, these efforts involved releasing hatchery‐reared juveniles that originated from the same location  in marine and freshwater habitats , with the aim of reducing false positive detections and increasing confidence in the retained SNPs  genetic distinctiveness as indirect measures of reproductive isolation that serve as proxies for local adaptation DFO, . Using SOverall, our analyses of population genetic structure based on outlier loci generally revealed concordant results to those based on neutral loci. These findings are analogous to previous results reported in anadromous Atlantic salmon  and Chinook salmon (O. tshawytscha) populations, for which overall genetic differentiation based on genome‐wide SNP data largely reflected geography, but a single locus (GREB1L) strongly associated with migration timing distinguished populations characterized by premature and mature migration phenotypes, and called into question the need for CUs aimed specifically at conserving this important component of adaptive diversity , phenotypic, and ecological information, our aim was not to define a new set of CUs for this region. Instead, we aimed at validating and providing recommendations for potentially refining existing CU boundaries using a larger and more powerful genomic data set compared with that which was previously available. Following the framework proposed by Funk et al. , we firsIn the BC ESU, we sampled from Coho populations that are currently managed in 31 of the 40 existing CUs. Overall, our results largely reflected CU definitions, suggesting no major restructuring. However, there are three areas in which additional MUs may be warranted based on evidence of substructure within the current CU boundaries. We propose: (1) separating Howe Sound and Burrard Inlet (populations currently managed together in CO‐10), (2) separating the west and east coasts of Northern Vancouver Island (populations currently managed together in CO‐15) and (3) separating the south‐west coast of Vancouver Island including Sooke [SOO] from the remaining East Vancouver Island‐Georgia Strait populations (CO‐13). Moreover, we suggest that boundaries may be refined between CO‐30 , CO‐27 (Hecate Strait) and CO‐29 (Douglas Channel‐Kitimat Arm), whereby coastal populations in CO‐30 and populations in CO‐27 may be combined in a single MU, and the remaining CO‐30 populations could be combined with the CO‐29 populations to create another MU. Observed differentiation of Martin River [MTI] in CO‐30 also suggests that it may belong to a distinct MU, but more sampling in the lower portion of CO‐30 and in CO‐26  would help to resolve this boundary.Despite the generally similar patterns of population genetic structure using either neutral or outlier loci, we did observe a few inconsistencies that may warrant the definition of distinct AUs in BC. In the Johnstone Strait/Southern Fjords CU (CO‐12), all three sampled populations were differentiated, potentially indicating a need for distinct AUs. However, support for their distinction is unclear and sampling of other populations within CO‐12 would help elucidate genetic groups. We also suggest that while populations on Haida Gwaii clustered within three distinct MUs that match the current CU definitions , populations in Haida Gwaii East (CO‐23) may warrant distinction as two AUs: the first one comprising Pallant [PAL] and Deena [DEE] and the second one comprising Copper [COP] on the east coast of Haida Gwaii along with Northern Haida Gwaii‐Graham Islands Lowlands populations (CO‐25).Overall, our findings suggest that the current definition of CUs as genetically distinct entities was generally accurate despite the relatively limited genetic data available. In addition to genetic differentiation based on microsatellite markers, ecological, morphological and life history characteristics were also incorporated into the approach used to define CUs and the overall concordance observed with groups detected using putatively adaptive genetic variation suggests that they largely reflected local adaptation. The widespread agreement between patterns observed with either neutral or outlier SNPs also suggests that natural selection is an important driver of population structure for Coho salmon, where gene flow is restricted among populations that differ in environmental conditions. However, identifying those few cases that support the definition of AUs that are different from MUs is critical for making appropriate management decisions, such as ensuring that adaptively divergent populations are not used to supplement declining populations. Moreover, while CUs represented biologically meaningful groups with a high degree of differentiation among them, pronounced differentiation was also generally observed among populations within CUs. Strong site fidelity and homing behaviours exhibited by Coho and other salmonids Quinn,  is likelWe declare no conflict of interest.Appendix S1Click here for additional data file.


Cephalorhynchus commersonii, has two subspecies, one of which is endemic to South America (C. commersonii commersonii) and little is known about its population genetics. Our objective was to investigate the population genetics of this subspecies throughout its distribution. Using 70 skin samples and information available in GenBank, 308 mitochondrial DNA sequences and 28 species-specific microsatellites were analyzed. The species presented low genetic diversity when compared to other dolphin species, but was consistent with other species within the genus. Strong population structure based on mitochondrial DNA was exhibited throughout its entire distribution, a pattern consistent with female philopatry. However, this pattern was not detected when using microsatellites, suggesting male-mediated gene flow. Demographic tests suggested a population expansion beginning approximately 15,000 years ago, after the Last Glacial Maximum. In a climate change scenario, we recommended considering each sampling location as an independent population management unit in order to evaluate the impact of possible environmental changes on the distribution of genetic information within the species.A key in species conservation is understanding the amount and distribution of genetic diversity and how environmental changes that occurred in the recent past may have influenced current patterns of population structure. Commerson’s dolphin,  As we improve our understanding of the structuring of natural populations, it becomes essential that researchers also focus on understanding the processes underlying these patterns2. Most species are divided into populations, even in apparently continuous environments such as the marine environment, where barriers to gene flow are not always clear and can be associated with currents, temperature, salinity or primary productivity6.Understanding population structure is vital for studying the ecology of endemic species and planning or designing conservation strategies10. Populations may respond differently to a common threat11. Their susceptibility to changes will largely depend on genetic variability, i.e. their capacity to cope with environmental changes13. The largest threats to marine mammals currently include global warming14, and other anthropogenic impacts, especially fishery bycatch, resource overfishing, oil exploration, and pollution16. Predicting how populations will respond to these threats is complex and may depend on factors that shape their distribution patterns (such as prey availability) and their genetic diversity17. However, distribution of current genetic diversity also reflects the influence of long-term historical processes. By examining environmental changes that took place over the course of the Pleistocene and Holocene (including climate change), we gain insight into how similar factors will impact contemporary populations18. The Last Glacial Maximum (LGM) at the end of the Pleistocene is one of the largest global environmental changes and had significant impact on the genetic diversity of many natural populations of top predators19. After the LGM, many populations of marine mammals showed demographic expansion both in the northern and southern hemispheres, such as narwhals Monodon monoceros20, belugas Delphinapterus leucas21, bowhead whales Balaena mysticetus22, South American fur seal Arctocephalus australis23, South American sea lion Otaria flavescens25, southern right whale Eubalaena australis26, dusky dolphin Lagenorhynchus obscurus27, and spectacled porpoise Phocoena dioptrica28. These historical climatic fluctuations, characterized by periods of contraction and retraction, have been suggested to be the primary driving factor behind the origin and radiation of some species from the southern hemisphere, e.g., those that are involved in the genus Cephalorhynchus29.Despite their broad range and their dispersal capabilities, marine mammals usually exhibit strong population structure at some geographic scaleCephalorhynchus includes four dolphin species widely distributed in cool temperate waters of the Southern Hemisphere, with each species endemic to a different region30. Among them, Commerson’s dolphin , has the largest distribution and comprises two subspecies: C. commersonii commersonii (South America) and C. commersonii kerguelenssi (Kerguelen Islands), which differ from each other both morphologically and genetically31. Along the southeastern coast of South America, the species is distributed between 40° and 56° S, including Strait of Magellan and the Falkland  Islands33. It is found frequently in shelf waters (< 200 m deep) and near shore  Islands), where the continental shelf is wide and flat, there are large tidal cycles, and the waters are relatively cold as they are influenced largely by the Malvinas Current39. This species is present in different coastal habitats, with the highest densities in areas in close proximity to river mouths40.The genus 42. However, there is still a gap of information regarding their northern distribution and contemporary genetic variation. Increasing the sampling effort will lead to a better understanding of how genetic diversity is distributed throughout the species range. Also, the information obtained here is crucial for the development of effective and sustainable management plans for the species43. To achieve these goals, we used mitochondrial and microsatellite DNA variability to investigate the population genetic diversity and structure of Commerson’s dolphin throughout their distribution in South America.The population structure of Commerson’s dolphins is poorly known, with existing information primarily focused on the southern range of the species. There, some degree of genetic differentiation at regional scale was detected using mitochondrial markers suggesting site fidelity by femalesCephalorhynchus commersonii were collected between 1999 and 2019 from five localities along the Patagonian coast: Playa Unión , Bahía Camarones , Caleta Olivia , Golfo San Jorge , and Puerto Deseado  control region sequences available in GenBank  were included for further analysis, increasing the sampling location to the south of our sampling area . Before extraction, tissue samples were digested at 55ºC for 2 days, adding 4 μl of Proteinase K  approximately every 8 h. DNA quality and quantity were evaluated by electrophoresis on 1% agarose gel. Sex of the individuals sampled via skin biopsy was identified molecularly following Bérubé and Palsbøll46 method for the amplification of ZFX and ZFY regions.Total genomic DNA was extracted following a glass milk protocol modified from Elphinstone et al.47. The reaction was performed in a 20 μl final volume including: 0.5 units of TSG; 2.5 mM MgCl2, 200 μM dNTPs, 10 mM buffer , 0.3 μM of each primer and 1 μl of DNA template. The PCR profile was: 3 min at 93 °C; then 30 cycles of 1 min at 92 °C, 1 min at 48 °C and 1 min at 72 °C; then a final extension phase of 5 min at 72 °C. Each PCR product was purified with enzymatic PCR cleanup at 37 °C for 30 min and then at 80 °C for 20 min, prior reaction using: 0.025 μl of Exonuclease I (NEB), 0.25 μl Antarctic Phosphatase (NEB), 2.5 μl Antarctic Phosphatase buffer (NEB), and 7.225 μl of H2O. For all samples, a target fragment of ~ 900 bp was sequenced at MacrogenUSA , followed by visual checks and alignment on MEGA748.The mtDNA control region was amplified using primers MTCRf (5′-TTCCCCGGTCTTGTAAACC) and MTCRr (5′-TTTTCAGTGTCTTGCTTT)49. Briefly, the protocol consisted of tagmentation genomic DNA, followed by PCR amplification. Purification of the PCR products was conducted using in-house made speedbeads follow Faircloth, et al.50. DNA library sequencing was performed using a MiSeq Benchtop Sequencer . Forward read output data from the MiSeq were imputed into Msatcommander 1.0.8-beta51 for the detection of microsatellite loci, with final product size, including primers and flanks, between 50 and 120 bp. A total of 34,973 microsatellites and 5017 sets of primers were identified following this criterion, and 70 were chosen at random for amplification/testing. Amplification was carried out in two multiplex PCR reaction (35 microsatellites each one) per individual. Further details on the molecular protocol to primer amplification are available in Ruzzante et al.49 and references therein. Summarized, this step consisted of diluting the multiplex PCR products and indexing them via a second PCR. PCR products were then pooled in equal proportion and subsequently cleaned via speedbeads. After quantification, the library was diluted to 15 pM and sequenced in a single direction using an Illumina MiSeq Benchtop Sequencer . Post-sequencing data were automatically genotyped using Megasat52 followed by manual verification.A single sample with the highest quality and quantity DNA was selected for shotgun sequencing and microsatellite design by means of visualization on 1% agarose gel stained with GelGreen . Our sequencing library was developed using 1 ng of genomic DNA following the Illumina Nextera XT Sample Preparation Kit protocol 53 were estimated with DNAsp v554, considering each sampling location as a separate population. The analysis of molecular variance (AMOVA) based on FST (using haplotypes frequencies) and ΦST  was performed in Arlequin56, and significance was tested using 10,000 permutations. The substitution model (Kimura 2-parameter) was selected under Akaike Information Criterion (AIC) generated with jModelTest v2.1.1057. These analyses were tested under two scenarios: using all datasets and a data subset that includes sampling sites with microsatellite information  avoiding related individuals (see next section: “ST) in Arlequin58. A haplotype network applying the median-joining algorithm and default parameters was built in Network 5.0.1.059. Demographic tests were explored through the Fu neutrality test60 and mismatch distribution analysis61 using Arlequin 3.5.2.258. A Mantel test was performed in Genalex 662 to examine isolation by distance through the use of linearized Φst63. BEAUTi v1.8.3 and BEAST v1.8.364 were used for Bayesian skyline plot reconstruction, assuming a piecewise-linear Bayesian skyline tree prior. The substitution model selected was Hasegawa–Kishino–Yano (HKY) based on AIC generated with jModelTest v2.1.1057. In addition, we used a lognormal relaxed clock rate (uncorrelated) with a mutation rate for the control region of 6.3 × 10–7 s/s/year derived for Lagenorhynchus obscurus65. Ninety million iterations of the MCMC (Markov Chain Monte Carlo) were applied, discarding the first 9,000,000 steps as “burn in” and sampling every 1000 iterations. Finally, convergence was checked through the effective sample size (ESS > 200) and a Bayesian skyline plot was built, both using Tracer v1.666.Haplotype and nucleotide diversity67. Tests for Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) between pairs of loci were conducted in Arlequin 3.5.2.258, applying 1,000,000 Markov chain steps (MCMC) with 100,000 dememorization steps and 30,000 permutations, respectively; p values were adjusted using false Discovery rate adjustment68. Duplicates among the samples were tested using Genalex 662, removing one individual from each resampled pair. Allelic richness (AR) for each location were estimated with FSTAT 2.9.369, and Arlequin 3.5.2.258 was used to estimate observed (Ho) and expected heterozygosity (He). Effective population size was estimated with LDNe70. Population structure was estimated through AMOVA using Arlequin 3.5.2.258. Also, hierarchical population structure analysis was performed in Structure 2.3.471 under the admixture model, using 5,000,000 MCMC, 1,500,000 burn-in steps, and 10 replicates for each genetic cluster (K = 1–8). The Evanno method72 implemented in Structure Harvester73 was used to estimate the most likely K. Both genetic differentiation tests were conducted using two datasets: first using all samples and, second a data subset without related individuals. To account for filial relatedness, Queller and Goodnight 1989 estimator74 was applied using Genalex 662. Those individuals with a relatedness coefficient ≥ 0.5  were removed. Contemporary gene flow among sampling locations was estimated using BayesAss Version 375, with a run setting of 150,000,000 iterations, 10% burn-in, and the rest of parameters as default. The model convergence was checked through the effective sample size (ESS > 200) using Tracer v1.666. Finally, a Mantel test63 was performed in Genalex 662 to examine isolation by distance among sampling sites using linearized FST values.Potential null alleles were assessed using Microchecker 2.2.3We amplified the mtDNA control region in 55 of 70 individuals sampled, with the per individual sequence length ranging from 326 to 698 bp; the shortest sequences belong to the oldest samples. Three duplicate individuals (identified with microsatellite loci) were removed from the analysis. We used a 423 bp consensus sequence to align all sequences available in GenBank. From a total of 308 sequences used for population analyses, we found 26 polymorphic sites, defining 27 distinct haplotypes distributed throughout the species distribution Fig. . Five ofST = 0.139 and ΦST = 0.194, suggesting female philopatry , Fry Table . Different Table . Data sunt Table , S2. FinThe minimum spanning network reflects the geographic distribution of the different haplotypes with a star-like configuration suggesting demographic expansion Fig. . Haplotyp = 0.002, α = 0.02), suggesting a population expansion. Also, the mismatch distribution showed a unimodal distribution suggesting a recent population expansion event . Furthermore, Bayesian skyline reconstruction also revealed a recent demographic expansion around 15,000 years ago  per locus, which is independent of sample size, ranged from 1.132 to 8.003 and the highest mean over loci AR was found in Caleta Olivia (3.053). Average Ho and He ranged from 0.315 and 0.320 to 0.348 and 0.341, respectively. No significant difference was found in AR, Ho and He among populations .From the 70 microsatellites/primer sets tested, 30 polymorphic loci were amplified successfully in 50 individuals from four sampling locations only . No evidence of linkage disequilibrium was detected, nor were null alleles detected. Two loci showed significant departure from Hardy–Weinberg equilibrium after false Discovery rate adjustment and were removed from the analysis. Moreover, three individuals were eliminated following their identification as duplicates. Summary statistics for the remaining 28 microsatellites loci in the four populations from north-central Patagonia are presented in Supplementary 1 Table ST = 0.032, p = 0.023); this result is based in only two small significant FST values between the two most geographically distanced populations: Playa Unión (43° S) and Bahía Camarones (45° S) with Puerto Deseado (47°S)  are prone to population fragmentation and isolation, even in the short-term and along the contiguous coastline without physical barriers, which is probably due to natal fidelity78. Through the use of mtDNA and species-specific microsatellites we analyzed the population genetics of Commerson’s dolphins to understand how its genetic information is distributed along their range in southern South America. Our results showed that Commerson's dolphin represents a single panmictic population, with a strong female philopatry along the entire distribution based on mtDNA data and gene flow mediated by males at least in the northern of its distribution where microsatellite data could be analyzed.In marine mammal population genetic studies, a common assumption is that the species under study represent a single genetic population, with likely some degree of gene flow between predefined "populations"79. Also, males dispersal is expected to improve reproductive success and avoid inbreeding depression18. Although it is best known in terrestrial mammals79, several studies with marine mammals, especially with cetaceans, support this hypothesis showing male-mediated gene flow82. However, it does not appear to be a common pattern for the species in the genus Cephalorhynchus. Based on mitochondrial markers, Heaviside’s dolphin, C. heavisidii, did not show evidence of population structure83, whereas Chilean dolphin, C. eutropia, showed genetic differentiation only through microsatellite analysis84. In contrast, Hector’s dolphin, C. hectori, displayed significant genetic differentiation and limited gene flow among populations using mtDNA and microsatellites86. Particularly with Commerson’s dolphin from South America, previous genetic analysis of mtDNA population structure found high levels of genetic differentiation indicating a strong site fidelity by females42. This was also confirmed in the present study, which covers the entire Commerson’s dolphin distribution in South America and extensive survey of microsatellite data, not reported before.Genetic structure analysis based in mtDNA and nuclear DNA markers exhibited contrasting patterns. Considering the mtDNA control region analysis a strong genetic structure among Commerson’s dolphins sampling locations was detected, suggesting a strong female philopatry which is not biased by kinship relationships as when kin pairs were detected one member of the pair was eliminated from the analysis. Female dispersal was low (nM < 5), which is consistent with significant breaks in dispersal. In contrast, no structure among northern populations was detected using microsatellite data, supporting the idea of male-mediated gene flow at least in the northern region of Patagonian continental shelf. In genetic populations studies of mammals, is common to assume the existence of sex-biased dispersal behavior, which is based on female philopatric behavior due to the requirements needed to breed and parental care87.The genetic variability estimated with both molecular markers is distributed homogeneously suggesting, also, a single genetic population of Commerson’s dolphin. Furthermore, those estimated through mtDNA is similar to previously reported for the species in the south of its distribution (between 47 and 56° S) and for the genus88. In this context, the Commerson´s dolphin population of the coast of Argentina might have experienced periods of population expansion and contraction coupled with glaciation events that occurred during the Pleistocene. Thus, population growth and geographic expansion occurred, likely with rapid diversification of haplotypes with minimum nucleotide differences. The haplotype network in a star-shape coincides with the results of the paired differences curve (mismatch distributions) and Fu values, suggesting recent population expansion for the species.A combination of high h (> 0.5) and low π (< 0.005) is characteristic of species/populations that suffered a population bottleneck followed by a rapid demographic expansion and the consequent accumulation of mutations28 likely associated with the LGM and sea ice retreat, around 15,000 years ago . Habitat changes including changes in the distribution and abundance of prey are likely to have taken place during this period. These changes in primary productivity and relative abundance of prey played a key role in the history of some coastal dolphin populations. An example is dusky dolphin historic demography which was coupled with fluctuations in anchovy populations, its main prey17. Marine mammals were favored by these changes, leading to a sudden demographic expansion along the coast of the Southwestern Atlantic Ocean89.Other marine mammals from the region also exhibited recent demographic expansionCephalorhynchus radiation, based on the west-wind drift mechanism, suggests that Commerson’s dolphin is one of the latest species to establish its current distribution, after setting up the New Zealand population29. Once established in South America, the species appears to have expanded northward across the south-western Atlantic Ocean coasts. As can be visualized in the haplotype network  and the northern as a sink (with the lowest density)39. Although the species' range has not been estimated, it was reported individual movements of up to 250 km in distance34. This range of movement is higher than the home range estimated for other species of the genus (< 60 km)30, and it may explain the lack of a clear pattern of population structure, such as "stepping-stone model" suggested for Hector's dolphins population85. Consequently, the Commerson´s dolphin population on the coast of Argentina might respond to a meta-population model. Therefore, it is necessary to increase the analysis with nuclear markers analysis using samples from southern localities to have a better understanding of the population structure.Population expansion from the southern to the northern ranges, with strong female philopatry and contemporary male-mediated gene flow, can produce the isolation-by-distance pattern (IBD) observed in our dataThe condition of panmixia does not necessarily mean that the species should be considered as a single management unit. If indeed the species is structured according to a metapopulation, any impact at the local level could disrupt the dynamic of the populations and generate isolation between them. Taking into account the effects caused by past climatic fluctuations and in a current climate change scenario, alterations in sea surface temperature could produce changes in the habitat and affect the population structure of the species. To assess whether the pattern of genetic differentiation in the species conforms to a metapopulation model, an increased sampling effort in terms of both sample size and geographic coverage is recommended. The analysis of molecular markers in the present study is geographically uneven, with the analysis of autosomal markers restricted to the northern part of the species distribution. Hence, based on the precautionary principle, we recommend that the sampling areas analyzed here should be considered as single management units, where future evaluations and technical reports consider them independently.Supplementary Information 1.Supplementary Information 2.


Juglans regia L.) is a famous fruit tree species valued for its nutritious nuts and high-quality wood. Although walnut is widely distributed and plays an important role in the economy and culture of Pakistan, the genetic diversity and structure of its populations in the country remains poorly understood. Therefore, using 31 nuclear microsatellites, we assessed the genetic diversity and population structure of 12 walnut populations sampled across Pakistan. We also implemented the geostatistical IDW technique in ArcGIS to reveal “hotspots” of genetic diversity. Generally, the studied populations registered relatively low indices of genetic diversity , and eight populations had positive inbreeding coefficient (FIS) values. Low among-population differentiation was indicated by AMOVA, pairwise FST and DC. STRUCTURE, PCoA and neighbor joining (NJ) analysis revealed a general lack of clear clustering in the populations except that one population in Upper Dir was clearly genetically distinct from the rest. Furthermore, the Mantel test showed no correlation between the geographic and genetic distance , while barrier analysis suggested three statistically significant genetic barriers. Finally, the spatial interpolation results indicated that populations in Ziarat, Kashmir, Dir, Swat, Chitral, and upper Dir had high intrapopulation genetic diversity, suggesting the need to conserve populations in those areas. The results from this study will be important for future breeding improvement and conservation of walnuts in Pakistan.Persian (Common) walnut ( Juglans regia L.; Juglandaceae) is a monoecious, wind pollinated, fruit tree with animal-based seeds dispersed. It is self-compatible but achieves a high rate of cross-pollination [J. regia is valued for its nutritious and medicinal nuts as well as high-quality timber [The common walnut , morphoividuals ) fruit pividuals ) and RAPividuals ). Morphoividuals . On the ividuals . Additio genomes , as show species . Juglans have proved effective for the analysis of genetic diversity and population structure of both cultivated and wild walnut species [J. regia based on SSR markers in Pakistan [J. regia, and (2) identify ‘hotspots’ of genetic diversity that deserve conservation. The results reported herein will provide a more detailed information on the genetic diversity and structure of walnut in Pakistan, and hence might promote effective and sustainable utilization and conservation of walnut germplasm in the country. SSR loci in  species ,28,29,30Pakistan . TherefoJ. regia from 12 populations majorly distributed in natural forests along river sides and mountainous areas across Pakistan, even though some populations such as HCR were found in a settlement area . The 15 µL multiplex PCR mix comprised 1 µL of each reverse and forward primer, and 2 µL of the DNA template with the remaining volume topped up with an appropriate amount of Golden Star T6 Super PCR mix . The following thermocycling regimen was employed: initial denaturation at 98 °C for 2 min, 35 cycles 98 °C for 10 s, primer annealing temperatures , observed heterozygosity HO), expected heterozygosity (HE) and unbiased expected heterozygosity (UHE) across loci was calculated in GenAIEx. Any significant shift from Hardy-Weinberg equilibrium with respect to heterozygosity deficit was evaluated by testing for inbreeding coefficient (FIS) among the populations, with 5000 randomizations using FSTAT v2.9.3.2 [hierfstat [For each population, the number of alleles (v2.9.3.2 . Number v2.9.3.2 . The priierfstat .FST) and genetic distance (DC) between pairs of populations, we used FreeNA v11.0 [FST values were calculated based on the corrected ENA  procedure, and corrected values of Cavalli-Sforza and Edwards algorithm [DC) followed the INA  algorithm. Subsequently, the data was represented on a heatmap generated in the Origin v8.0 program . Analysis of molecular variance (AMOVA) was conducted in Arlequin v3.5.1.3 [J. regia populations, the non-rooted tree was generated using the neighbor-joining (NJ) procedure with 1000 bootstrap replicates in POPTREE v2.0 [K), population structure was tested at K values ranging from 1 to 10, each with 10 replicates based on 100,000 Markov Chain Monte Carlo (MCMC) iterations following a burn-in period of 10,000 steps, followed by evaluation of optimal K using STRUCTURE HARVESTER v6.94 [FST matrix was obtained in GenAIEx. We also performed genetic barrier analysis using BARRIER v2.2 [DA); [To examine the pairwise genetic differentiation . PrinciER v6.94  in GenAIER v6.94  to generIER v2.2  followinIER v2.2 , and witIER v2.2  with a b.2 [DA);  was usedNA = 3.793, HO = 0.563, HE = 0.558). At population level, mean observed heterozygosity (HO) ranged from 0.523 (DIR) to 0.649 (ZTR) while the expected heterozygosity (UHE) was lowest in DUB (0.465) and highest in HDR (0.629). The average number of alleles for the entire dataset was 3.793, while the total number of alleles per population ranged from 83 (DUB) to 142 (HDR). Generally, the mean value for UHE was slightly higher than the HO (FIS) ranged from −0.134 in DUB to 0.120 in HDR, with 7 of 12 populations showing positive inbreeding values  were reported in populations located in the southwestern and northern areas, whereas two populations in Dir 1 (DIR) and upper Dir (DUB), both in the northern region, registered the lowest UHE values  and 2.39 (HDR). The highest values of AR were mostly in the northern region, although two populations, one from the southwestern and the other from the Northeastern also registered high values of AR. As with the case of UHE, two populations from Dir and upper Dir also had low AR levels in the range of 1.93–1.94  DC . On the other hand, certain population pairs  appeared to be genetically close to one another in terms of both genetic differentiation and genetic distance. AMOVA analysis indicated that 9% of the total variation was partitioned among populations, 2% among individuals within populations, while the remaining 89% resided within individuals  0.50) DC a. Populaividuals .J. regia populations into two genetic clusters: cluster 1 comprising only one population (DUB) and cluster 2 comprising all other populations  tree partitioned the ulations b. Populawed by 3 b, hence wed by 3 a. Consisanalysis a. PCoA lanalysis c, furtheJ. regia   . However = 0.22) . The firJ. regia from Pakistan harbor a low to moderate level of genetic diversity  [Spondias purpurea [Our results show that populations of iversity , which i regions . However (0.227) , which w (0.227) ,60. This (0.227)  and Sponpurpurea .J. regia populations. Three possible causes are the presence of null alleles, inbreeding, and population sub-structuring [Heterozygosity deficit were detected in eight ucturing ,64. Nullucturing . Even thucturing . HoweverUHE and AR revealed high intrapopulation genetic diversity in the northern, eastern and southwestern regions suggesting that these geographical areas might be centers of genetic diversity for J. regia in Pakistan. The high levels of UHE and AR in the northern region indicate a possible center of genetic diversity, perhaps originating as a glacial refugium, as corroborated by the presence of private alleles in all the populations. Indeed, northern Pakistan might encompasses the Pleistocene refugial to where many plant species retreated during the ice ages; this area is therefore expected to harbor high genetic diversity [J. regia. Alternatively, considering the long-standing economic and cultural importance of walnut cultivation in Asia [The interpolation of iversity ,68. Howe in Asia , the hig in Asia . HoweverFST and DC which suggested low to moderate FST and DC within populations, similar to results obtained for Calotropis gigantea and J. regia from Kyrgyzstan [The AMOVA analysis revealed higher among-population than within-population partitioning genetic variation, a rare scenario particularly for species with outcrossing mating system such as walnut. Therefore, this could be as a result of habitat fragmentation as discussed above, and possibly the influence of artificial selection on landraces, if it occurs. Fragmentation of habitats limits or prevents long-distance pollination events, resulting in pollination only within clumps of close relatives. The results from AMOVA were consistent with the differentiation depicted by pairwise rgyzstan ,71.STRUCTURE, PCoA and NJ tree results clearly demonstrated that population DUB was delineated from the rest of the populations, suggesting that gene flow from DUB to other populations was very limited, though HCR and HDR might have limited gene flow with DUB according to STRUCTURE. A normal explanation would be geographical barriers, but DUB is situated in the middle of a cluster of populations in northern Pakistan, whereas ZTR and KAR are far more distant. Therefore, the distinctness of DUB likely reflects its history, not its current geography. Hence DUB might have been genetically altered by gene flow from cultivated material or local land races, or possibly derive wholly from a distinct region . Hence further research is needed on this population for better determination of its nature. The regional intermixing of populations other than DUB, as confirmed by STRUCTURE, PCoA and NJ tree appears inconsistent with a naturally fragmented distribution. The close proximity of many northern populations might explain the genetic similarity between them, but UKR and KMR are >300 km away from the main cluster, whereas KAR and ZTR are each >700 km away from all others. Hence the genetic similarity of these distant populations is difficult to explain in terms of the current distribution. Therefore, other factors must be considered, such as a common historical gene pool and previous connections in the range that have now been lost, or possible recently human or natural transport of propagules.J. regia is self-compatible [The Mantel test showed no correlation between geographical and genetic distance, thus further supporting our assumption of frequent gene flow and potential long-distance dispersal of propagules. Considering the topographical complexity of Pakistan, it is likely that walnut populations in the country are geographically isolated by the geographic barriers. Our analysis identified three significant genetic barriers, which might coincide with mountain ranges such as the Hindu Kush, Karakoram, and Himalaya in the northern Pakistan. Barrier B1 ran north to south, whereas B2 ran Northwest to Southeast. The third barrier, B3, might have been simple distance, or the varying relief (probably part of Chaghi hill) separating ZTR from the other populations . The prempatible . Thus, impatible ,74,75.J. regia in Pakistan is vital to devise appropriate measures for conservation [in situ and ex situ conservation strategies as a way of maintaining the adaptive potential of this species. We found that population DUB has a small population size, showed the least genetic diversity, and is also the population most distinct from others. Hence, it requires urgent attention through interventions by both in situ and ex situ conservation. Additionally, spatial interpolation of genetic diversity indices revealed the need to conserve populations HDR, HCR, KAR, SHR, ZTR and KAR on the basis of their high genetic diversity. Finally, we identified three significant barriers, which are constraints to migration and natural gene flow in the region. We recommend deliberate augmentation of geneflow within and between walnut populations through artificial dispersal strategies, except population DUB so as to avoid outbreeding depression.The ultimate aim of conservation study is to guarantee the continuous existence of populations and to ensure the maintenance of their evolutionary potential . Knowledervation . GeneticJ. regia in Pakistan. Our results suggested that the genetic diversity of J. regia in Pakistan is somewhat low. Additionally, our results revealed high genetic diversity in six populations, and genetic distinction in DUB therefore, conservation attention should be given to those populations to ensure ultimate germplasm improvement for future breeding. As a future research direction, we propose wider sampling of J. regia populations from adjacent regions, for better understanding of the genetic relationship of J. regia in Pakistan and adjacent regions. Moreover, we recommend seeking local knowledge regarding human intervention into apparently wild populations. Despite the limitations of our study, the data presented herein is a strong basis for the germplasm improvement and conservation management of J. regia in Pakistan. The current study is the first attempt to comprehensively explore the genetic diversity and structure of


Amaranthus retroflexus L. and Chenopodium album L. (Amaranthaceae) are weedy plants that cause severe ecological and economic damage. In this study, we collected DNA from three different countries and assessed genetic diversity using inter-simple sequence repeat (ISSR) markers. Our analysis shows both weed species have low genetic diversity within a population and high genetic diversity among populations, as well as a low value of gene flow among the populations. UPGMA clustering and principal coordinate analysis indicate four distinct groups for A. retroflexus L. and C. album L. exist. We detected significant isolation-by-distance for A. retroflexus L. and no significant correlation for C.album L. These conclusions are based data from 13 ISSR primers where the average percentage of polymorphism produced was 98.46% for A. retroflexus L. and 74.81% for C. album L.These data suggest that each population was independently introduced to the location from which it was sampled and these noxious weeds come armed with considerable genetic variability giving them the opportunity to manifest myriad traits that could be used to avoid management practices. Our results, albeit not definitive about this issue, do not support the native status of C. album L. in Iran. Amaranthus retroflexus L.) and lamb’s quarters  are problematic cosmopolitan weeds that inhabit a wide variety of habitats across the globe  geographic distance and resistance of gene flow to altitudinal differences drive population genetic differentiation, i.e. isolation-by-distance (IBD), both of which would manifest as higher levels of genetic diversity when grown in common garden.The objective of this study was to characterize the genetic diversity of Iranian, French and Spanish l traits . Regardi. retroflexus and 17 C. album populations were collected in 2016 and 2017 from different provinces of Iran, Spain, and France  markers. ISSR markers are highly reproducible and accurate tools that generate highly reproducible banding patterns from a single polymerase chain reaction (PCR) amplification . Althougvia 0.8% (w/v) agarose gel electrophoresis. 52 ISSR primers  from the University of British Columbia’s UBC set no. 9  were screened for PCR amplification and thirteen primers that produced clear, reproducible banding patterns were chosen (Genomic DNA was isolated from the young leaves of plants according to the cetyltrimethylammonium bromide (CTAB) method described by e chosen Table 2.Bio-Rad T100™ thermal cycler  started with 4 min at 94°C, and 40 cycles of 1 min at 94°C, 75 s at each primer’s annealing temperature Table 2 Among the 52 primers tested, 13 produced clearly and reproducibly amplified ISSR fragments. These were scored based on a binary matrix for presence (1) or absence (0) of bands. Discriminatory power of the primers was evaluated by means of resolving power (Rp), mass resolving power (MRP), polymorphic information content (PIC), marker index (MI). Rp of each primer which is the ability of each primer to detect level of variation between individuals was calculated according to :where bI (band informativeness) takes the values of: 1–[2|0.5–p|], where p is the proportion of individuals containing the band. Further, mean resolving power (MRP) for each primer was calculated viafollowing . PIC valwhere fi is the frequency of fragments present in that locus and (1−fi) is the frequency of the null allele. MI, a measure of overall utility of a molecular marker technique, for each primer was calculated as a product of two functions, the polymorphic information content and effective multiplex ratio (EMR) , i.e.,The effective multiple ratio (EMR=npβ ) is the product of the number of polymorphic loci (np) in the population analyzed and the fraction of markers that were polymorphic (β) .The binary data matrix was analyzed using POPGENE version 1.32  to examiCorresponding estimates of gene flow (Nm), i.e. the average per generation number of migrants exchanged among populations, was calculated based on :To examine the genetic relationship among populations, unbiased genetic distance and genetic identity  were alsA. retroflexus L. and 74.81% for C. album L. within analyzed materials. These differentiating loci are therefore suitable for evaluating the genetic variability of these populations. Moreover based on PIC values, it can be concluded that the capacity of the marker system to detect polymorphic loci in a single amplification was very efficient; the average value of this coefficient amounted 0.78 for A. retroflexus L. and 0.71 for C. album L.. These results demonstrate this technique can be conveniently used for the genetic characterization of these populations of A. retroflexus L. and C. album L. Use of ISSR markers are also recently reported as a functional markers elsewhere  to 13 (UBC810). The ISSR pattern obtained with UBC810 primer is demonstrated in C. album: These 13 selected primers generated 49 ISSR bands in the 17 C. album populations, 3 to 8 bands per primer, of which 37 were polymorphic. The number of polymorphic bands varied from 1 in Al2 and UBC811 to 7 in UBC810. The ISSR pattern obtained with UBC810 primer is demonstrated in A. retroflexus L.  was found among populations. Additionally, our data indicate that there is little genetic diversity within a given population of A. retroflexus or C. album. Theory predicts that colonization of new areas will be associated with population bottlenecks that reduce within population genetic diversity and increase genetic differentiation among populations. This should be especially true for weedy A. retroflexus and C. album  was the result of independent and multiple introductions because of data showing their populations exhibited different genetic fingerprints and lacked a correlation between genetic and geographic distances. C. album populations are most commonly found on disturbed areas (The Mantel tests we conducted show isolation-by-distance (IBD) and therefore positive correlations between genetic distances and geographic distances among ulations Figure 3ulations , clonal ulations , fast-grulations  and highulations . This waed areas , and theed areas . As a reed areas .A. retroflexus is a neophyte , as inhabiting most of Europe. Plants thought to be native to Eastern Asia are included under C. album, but often differ from European specimens  it is likely the Iranian, French and Spanish populations of A. retroflexus L. and C. album L. were established by individuals from multiple different sources and (2) isolation-by-distance (IBD) has occurred particularly in A. retroflexus L. where the likelihood of gene flow is inversely related to distance. However, we show no evidence of isolation by distance among the C. album L. populations, indicating geographic distance or geographic barriers may not be the only factor affecting gene flow. Our results show genetic diversity between populations of A. retroflexus L. and C. album L., which may help explain their diverse phenotypic and biochemical traits and help to explain their success as noxious weeds. Our data supports the theory that in both species, the populations we have sampled have been genetically isolated and multiple introduction events occurred giving rise to these weedy populations.Analysis of ISSR markers in this set of A. retroflexus L. and C. album L. using ISSR primers. We were able to obtain an efficient and effective assessment of genetic diversity in A. retroflexus L. and C. album L. populations. While a large number of molecular markers (dominant and co-dominant) would have improved our analyses as would increased sample sizes or ranges, the amplification of many polymorphic loci indicated the set of ISSR primers we used was sufficient to assess the genetic diversity among the existing populations. Here, we demonstrate that ‘weedy’ traits, such as selfing and clonal growth may result in populations that have distinct phenotypic and genetic fingerprints depending on the selecting conditions. The low genetic variation within populations and maladapted gene flow among populations seen in our results indicates that every population is a unique, evolutionarily-significant unit and should be considered as an independent management unit for weed population control.Knowledge about genetic relatedness within and between populations is crucial for understanding how the populations came to be established as well as for designing successful weed management schemes to deal with them. Herein we evaluate the genetic diversity of Iranian, French and Spanish populations of The original contributions presented in the study are included in the article/SH performed the experiments, data collection, data analysis, figure preparation, and writing of the manuscript. MA conceived the original data, formulated the research plan, oversaw the research, and writing of the manuscript. MM and DM contributed to data analysis and writing of the manuscript. All authors contributed to the article and approved the submitted version.


POLA1, POLE, POLG, POLQ, and REV3L genes in Amerindian populations from the Brazilian Amazon, comparing these findings with genomic data from five continental populations described in the gnomAD database, and with data from the Brazilian population described in ABraOM. We performed the whole exome sequencing (WES) of 63 Indigenous individuals. Our study described for the first time the allele frequency of 45 variants already described in the other continental populations, but never before described in the investigated Amerindian populations. Our results also describe eight unique variants of the investigated Amerindians populations, with predictions of moderate, modifier and high clinical impact. Our findings demonstrate the unique genetic profile of the Indigenous population of the Brazilian Amazon, reinforcing the need for further studies on these populations, and may contribute to the creation of public policies that optimize not only the quality of life of this population, but also of the Brazilian population.Due to their continuing geographic isolation, the Amerindian populations of the Brazilian Amazon present a different genetic profile when compared to other continental populations. Few studies have investigated genetic variants present in these populations, especially in the context of next-generation sequencing. Knowledge of the molecular profile of a population is one of the bases for inferences about human evolutionary history, in addition, it has the ability to assist in the validation of molecular biomarkers of susceptibility to complex and rare diseases, and in the improvement of specific precision medicine protocols applied to these populations and to populations with high Amerindian ancestry, such as Brazilians. DNA polymerases play essential roles in DNA replication, repair, recombination, or damage repair, and their influence on various clinical phenotypes has been demonstrated in the specialized literature. Thus, the aim of this study is to characterize the molecular profile of  We kn factors . Errors  factors ,10,11,12DNA polymerases play essential roles in DNA replication, repair, recombination, or damage repair, and their influence on various clinical phenotypes has been demonstrated in the specialized literature. Despite this, no research has reported the frequency or biological value of mutations in these genes in Amerindian populations. In fact, genetic studies in these populations are extremely rare, and to date their genomic data are unavailable in international and Brazilian project databases, such as the 1000 Genomes Project, The Exome Aggregation Consortium (ExAC), The Genome Aggregation Database (gnomAD), Arquivo Brasileiro Online de Mutações (ABraOM), and the Brazilian Initiative on Precision Medicine (BIPMed). Given that genomic ancestry is a determining factor in complex disease association studies, the unavailability of genomic data from Amazonian Indigenous populations limits the validation of previously consolidated data in other continental populations, besides compromising the clinical applicability of such findings and also the discovery and characterization of new markers unique to these populations ,14,15. TPOLA1, POLE, POLG, POLQ, and REV3L genes in Amerindian populations from the Brazilian Amazon, comparing these findings with genomic data from five continental populations described in the gnomAD database, and with data from the Brazilian population described in ABraOM.Additionally, recent studies have shown that Amerindian ancestry can modulate the predisposition to certain diseases, which means that knowledge of their molecular profile has a relevant impact not only for these populations, but also for admixed populations with strong Amerindian ancestry, as in the case of the Brazilian population ,24,25,26http://conselho.saude.gov.br/comissoes-cns/conep/ accessed on 5 October 2022) and by the Research Ethics Committee of the Tropical Medicine Center of the Federal University of Pará (CAE: 20654313.6.0000.5172). Because we are dealing with Amerindian groups, the leaders of the investigated communities were contacted, and they signed an informed consent form for participation in the study.The study was approved by the National Research Ethics Committee .https://gnomad.broadinstitute.org/; accessed on 15 February 2022). Thus, we included data from 8128 individuals from Africa (AFR); 56,885 from Europe (EUR non-finish); 17,296 from the Americas (AMR); 9197 from East Asia (EAS) and 15,308 from South Asia (SAS).For comparison analyses we also used data from 5 continental populations available in the Genome Aggregation Database , which represents the population of southeastern Brazil, specifically residing in the state of São Paulo.Additionally, we included the population described in the Brazilian Online Mutation Archive  using the NextSeq 500 High-output v2 Kit 300 cycle .Whole Exome sequencing reactions were performed on the NextSeq 500http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 25 June 2021) in order to filter out and exclude all low-quality reads (below 10 reads) we used the tool Fastx_tools v. 0.13 .Reads in FASTQ format were analyzed for quality using FastQC v.0.11 , and indexed the generated file .We aligned the sequences found with the GRCh38 genome, which is commonly used as a reference in next-generation sequencing studies. For the alignment, we used the BWA v.0.7 tool  and GATK v.3.2 did the mapping quality recalibration and local realignment . GATK v.3.2  also performed the search for variants present in the 18 genes selected from the reference genome for analysis. Variant annotation was performed with the variant viewer software, Viva® .Picard Tools v.1.129 was used to remove duplicate reads  was analyzed by Fisher Test. All statistical analyzes were performed using the R Studio v.3.5.1 program . The False Discovery Rate (FDR) was used to correct the multiple analyses.https://pcingola.github.io/SnpEff; accessed on 25 June 2021); (c) the difference in allelic frequency of the variants between NAT populations and continental populations should be significant . After applying selection criteria, 55 genetic variants were statistically analyzed.We selected 5 component genes of the DNA replication and repair pathway in humans. The following selection criteria were applied to the exome sequencing result to perform the pairwise analysis: (I) the read should be high-coverage, with a minimum of 10 reads coverage; (II) the predicted impact should be “modifier”, ”moderate” or ”high” according to SNPeff . Our results identified 432 variants in total, with only 45 remaining after our selection criteria. We emphasize that the frequencies of some variants identified in this work and attributed to the Indigenous population have never been calculated or described for other populations. Thus, REV3L gene, the rs462779 variant in the Indigenous population differed significantly from the AMR, SAS and ABraOM populations. Notably, INDEL rs3087377 of the POLG gene stood out among all the variants analyzed, as its frequency in the Amerindian population is statistically different from four of the five continental populations investigated in the 1000 Genomes database  and the Brazilian population of ABraOM , thus being the marker that most demonstrated genetic differentiation of the population studied when compared to the others.Regarding the pairwise comparison of frequencies of the mutations described between NAT and each of the populations investigated , we obtained a total of 14 variants that met our selection criteria. POLE gene; rs3087377 in the POLG gene). On the other hand, the population that differed least from the Amazonian Indigenous was the African population, with only rs11573344, present in POLA1 . It is worth noting that all variants described here show modifier or moderate impact, according to the prediction of the SNPeff software, which can signify an important phenotypic impact in the individual with these variants. Regarding the distribution of these markers among populations, we visualized that the population with greatest difference from the NAT population was the Europeans, with a total of seven markers, all with high statistical significance  and one INDEL type had high clinical impact, present in the POLQ (Polymerase θ) gene, involved in translesion DNA synthesis (TLS), an important pathway that contributes to cell survival by bypassing DNA lesions that have not been repaired by other processes [POLQ and REV3L, investigated here [In this present study, we also found new variants never described in other populations, possibly unique to NAT populations. Among them, three had clinical impact predicted as modifier by SNPeff software  is an essential enzyme for successful replication to occur, and also has a 3′–5′ exonuclease activity, which corrects errors in DNA synthesis and helps maintain genomic stability [Our results also showed that the modifier variants rs4883544 and rs4883613 of the tability ,11,49.POLG gene was the SNV that stood out the most among our results, as it was differentially distributed in the NAT population when compared to four of the five continental populations investigated , as well as when compared to the Brazilian population described in the ABraOM . POLG (Polymerase γ) is considered the replicative as well as the mitochondrial DNA repair polymerase [Finally, rs3087377 of the lymerase . Differelymerase ,52,53.POLA1, POLE, POLG, POLQ and REV3L genes in Amerindian populations from the Brazilian Amazon, a population whose genetic component has not yet been described in any database of human genetic variability. Molecular epidemiology studies such as this are the basis for inferences about human evolutionary history, so the lack of such data creates a gap in the understanding of several processes investigated by population genetics [This is the first study to characterize the molecular profile of genetics ,54,55. UDespite the importance of our data, new studies should be carried out in order to broaden the understanding of the genetic profile of these populations. We also suggest case–control studies to validate the investigations performed in Amerindian populations or uncover new insights regarding the clinical impact of the genetic variations described here.Ethnic groups located in the Brazilian Amazon have differential distribution of important molecular markers present in DNA replication and repair pathways. Our findings may contribute to future studies of association between complex diseases in these populations and in the Brazilian population itself. The Amerindian populations investigated present a unique genetic profile, with genetic variants never previously described in other populations. We hope that these data can help establish public policies for this group and for populations admixed with them, such as the Brazilian population.