Artemisia  are each endemic from a single archipelago , representing a perfect opportunity to study three similar but independent anagenetic speciation processes. By analysing plastid and nuclear DNA sequences, as well as nuclear DNA amount data, generated from a comprehensive sampling in all the islands and archipelagos where these species are currently distributed, we intend to find common evolutionary patterns that help us explain the limited taxonomic diversification experienced by endemic Macaronesian Artemisia. Our time-calibrated phylogenetic reconstruction suggested that divergence among the three lineages occurred in a coincidental short period of time during the Pleistocene. Haplotype and genetic differentiation analyses showed similar diversity values among A. argentea, A. thuscula and A. gorgonum. Clear phylogeographic patterns—showing comparable genetic structuring among groups of islands—were also found within the three archipelagos. Even from the cytogenetic point of view, the three species presented similarly lower genome size values compared to the mainland closely related species A. arborescens. We hypothesize that the limited speciation experienced by the endemic Artemisia in Madeira, Canary Islands and Cape Verde archipelagos could be related to their recent parallel evolutionary histories as independent lineages, combined with certain shared characteristics of seed dispersal, pollen transport and type of habitat.Anagenetic speciation is an important mode of evolution in oceanic islands, yet relatively understudied compared to adaptive radiation. In the Macaronesian region, three closely related species of  Artemisia  are each endemic from a single archipelago . By analysing plastid and nuclear DNA sequences, as well as nuclear DNA amount data, we found that the three single-endemic species showed similar genetic diversity, phylogeographic structure and cytogenetic patterns. We hypothesize that the limited speciation experienced by the endemic Macaronesian Artemisia could be related to their recent parallel evolutionary histories as independent lineages, combined with certain shared characteristics of seed dispersal, pollen transport and type of habitat.Anagenetic speciation in oceanic islands—where initial colonizers arrive on an island or archipelago and then diverge into a single new species, but without subsequent taxonomic differentiation—is relatively understudied compared to adaptive radiation. In the Macaronesian region, three closely related species of  These Macaronesian taxa have been considered the vicariant species of A. arborescens L., a species occurring in the Mediterranean region, but not present in these oceanic islands, where the habitats it usually colonizes—rocky scrubland influenced by humid sea winds—are occupied by the above-cited local endemics  is a native but non-endemic species, also present in Northwest Africa  is considered an introduced taxon . According to the phylogenetic reconstructions of Artemisia performed by et al. (2023), the three endemic Macaronesian species of Artemisia  constitute a monophyletic clade within the subgenus Absinthium, together with the continental—including continental islands as well—species A. arborescens. The evolutionary relationships among species within this clade differ between those former phylogenetic studies. However, they have in common that none of them infers A. arborescens as the sister species of the Macaronesian endemics, suggesting multiple colonization events from the continent. Everything considered, this Mediterranean–Macaronesian group of closely related species—showing very similar characteristics in terms of morphology, ecological preferences, reproduction strategies and life cycle—represents a perfect opportunity to study anagenetic speciation processes replicated in the three mentioned archipelagos.The three endemic Macaronesian tributed , most ofArtemisia by analysing plastid and nuclear DNA sequences, as well as nuclear DNA amount data, generated from a comprehensive sampling in all the islands and archipelagos where these species are currently distributed. More specifically, these data obtained at the population level will be employed to i) delineate from a molecular point of view the taxonomic identity—based to date just on morphological characters and geographic distribution—of the three species endemic from Madeira, Canary Islands and Cape Verde. Our genetic data will also be analysed to ii) infer the evolutionary relationship among the endemic Macaronesian Artemisia, examining the colonization history of these archipelagos. From a phylogeographic point of view, in all cases of anagenesis evaluated in recent studies, no geographical patterning of the observed genetic variation has been reported among populations of the endemic island species  explain the limited taxonomic diversification experienced by this group of insular endemic plants.In this paper, we will investigate the evolutionary patterns of the three endemic Macaronesian Artemisiasee . Sequencing of plastid DNA  was performed for 141 individuals from 29 populations  of A. thuscula, A. argentea and A. gorgonum , starting from 10 000 random initial conditions for each simulation, and chose the number of groups that gave the highest ΔFCT. Finally, we conducted analysis of molecular variance (AMOVA) in Arlequin v3.5 with 10 000 replicates ; 7 generations, sampling every 1000 generations. The program TRACER version 1.6 (http://tree.bio.ed.ac.uk/software/figtree).A matrix was constructed with a sequence of each of the Macaronesian haplotypes, as well as the sequences from seven t 2.1.10 . The choNuclear ribosomal DNA sequences were first edited to code intra-individual nucleotide polymorphisms—visualized in the chromatograms as two peaks close to equal in height—according to IUPAC terminology . Sequences were then phased using the Bayesian statistical method implemented in PHASE 2.1.1  with theArtemisia was estimated using flow cytometry at the Unitat de Citometria, Centres Científics i Tecnològics, Universitat de Barcelona (CCiTUB), following the protocol described in A. argentea, five populations of A. thuscula and three populations of A. gorgonumsee . As for the sequencing procedures, samples from all the islands where these species are distributed were included in genome size analyses. Vouchers of each population were also deposited in the herbarium BCN. We compared the measures of qDNA among the Macaronesian species to check whether significant differences occurred between taxa from different archipelagos. These genome size data were also compared with qDNA values of A. arborescens sampled from mainland and continental islands published in A. arborescens. To analyse possible differences in the amount of DNA between the species of the three archipelagos, a one-way ANOVA test with a post hoc Tukey’s test for comparison of means between archipelagos was carried out.The nuclear DNA amount or genome size of Macaronesian endemic A. argentea, A. thuscula and A. gorgonum samples were aligned in three matrices (one per region) containing 841, 905 and 725 nucleotides, respectively. All these plastid DNA regions showed a moderate level of polymorphism among the 141 specimens of Macaronesian endemic Artemisia analysed in this study. Specifically, 13, 16 and 6 polymorphic segregating sites (including indels) were, respectively, observed in ndhC-trnV, rpl32-trnL, rps16-trnK regions. Based on analysis of the concatenated matrix—including nucleotide polymorphisms and indels—we were able to identify 18 haplotypes across the 29 Macaronesian populations . The species from Madeira, A. argentea, is represented in an early diverging branch of the MCC tree, this position also lacking statistical support. According to the strict-clock time calibration of the tree (A. arborescens and the three Macaronesian endemic species) diverged from its sister A. absinthium lineage ca. 3.83 million years ago (Mya). Diversification among species/archipelagos lineages was estimated to occur 1.36 Mya  to 0.84 Mya (95% HPD 0.36–1.78 Mya) ago. Genetic differentiation within the species started from ca. 0.56 Mya ago in A. argentea to ca. 0.12 Mya ago in A. gorgonum.The sequences of ndhC-trnV, rpl32-trnL, rps16-trnK intergenic spacers obtained from ulations . Accordiulations , these hthe tree , the MacArtemisia thuscula is the species showing the largest number of haplotypes (H = 7), while A. gorgonum from Cape Verde shows the highest haplotype diversity (Hp = 0.749). In contrast, A. argentea sequences present the largest number of segregating sites (S = 9) and the highest nucleotide diversity (π = 0.00136), despite showing the lowest number of haplotypes (H = 5) as well as the lowest haplotype diversity (Hp = 0.589). Most populations harbour a single haplotype, with only one out of seven populations in A. argentea, four out of 16 populations in A. thuscula and two out of six populations in A. gorgonum presenting some degree of haplotype diversity (i.e. two or three haplotypes within the same population).The results of haplotype and nucleotide variability analyses revealed similar genetic diversity levels among Macaronesian taxa . ArtemisA. argentea, where haplotypes were separated by three to five mutation steps. In all species, frequent haplotypes  were combined with relatively rare haplotypes . Phylogeographic pattern in A. argentea showed haplotype Hm1 widely distributed in Porto Santo and Desertas Islands, while populations in Madeira Island exclusively presented other three haplotypes . In A. thuscula, haplotype Hc2 dominated in populations from Tenerife and Gran Canaria , while Hc3 was the main haplotype in La Gomera, La Palma and El Hierro . The rest of Canarian haplotypes were less frequent, mainly found in El Hierro or La Palma and being always exclusive from a single island. In A. gorgonum, northern Santo Antão Island harboured three exclusive haplotypes , each of them dominating in a different population. In contrast, in southern islands of Santiago and Fogo, one main haplotype (Hv4) was widely distributed in all three studied populations, the other two haplotypes Hv5 and Hv6 being only found in a couple of individuals from Fogo.The parsimony haplotype networks, together with the geographic distribution of haplotypes, showed clear phylogeographic patterns in each Macaronesian species . HaplotyFCT values occurred at K = 2 for the three Macaronesian species see . In all cases, the genetic structure showed by K = 2 clustered populations according to a geographic pattern. For larger values of K, the same clustering pattern recovered for K2 was always maintained, but each time one additional population split into an independent group. Therefore, we considered K = 2 as the most informative number of groups to explain phylogeographic structure of endemic Macaronesian Artemisia. In A. argentea, the genetic structure showed by K = 2 clustered the populations from Porto Santo and Desertas in one group and the populations from Madeira in another group see . SAMOVA analysis for A. thuscula split populations from Gran Canaria and Tenerife in one genetic group and populations from La Gomera, La Palma and El Hierro in a separate genetic group see . Regarding A. gorgonum, the spatial genetic analyses differentiated one group for Santo Antão populations and another group for Fogo and Santiago populations see . The results of AMOVA analyses studying the partitioning of genetic diversity are summarized in Supporting Information—Table S4. Genetic differences among K = 2 clusters defined by SAMOVA explained a significant proportion of the variance in all species , while genetic differences among islands represented lower percentage values in all cases.Spatial genetic analyses of plastid DNA haplotypes using SAMOVA indicated that the largest increase of see  showed a complex structure where ribotypes from the different species appeared intermingled without a clear taxonomic pattern. Neighbour-net network see  showed better-defined groups corresponding to the taxonomic assignation of the samples, but sequences from different species were also intermixed in largely reticulated groups.Regarding nuclear DNA regions, we were only able to obtain good quality sequences from short fragments of ITS (202 bp) and ETS (346 bp) nuclear DNA regions. From that nucleotide positions onwards, double messy signals in the sequence chromatograms made impossible the base calling. Multiple intra-individual nucleotide polymorphisms visualized in the chromatograms as two peaks close to equal in height were detected along the fragments of the sequences that could be correctly read. After aligning these nrDNA sequences, 11 intra-individual polymorphic sites for ITS and 10 intra-individual polymorphic sites for ETS were counted. Apart from these polymorphic sites showing sequence variation within individuals, no other genetic variability between the samples was detected. Parsimony TCS network A. argentea, A. thuscula and A. gorgonum are basically the first obtained in natural populations, as the data had so far come from specimens grown in botanical gardens—with only one accession per species—except for one population of A. thuscula from Tenerife . These differences in genome size between continental and Macaronesian species were statistically significant . There were also small but significant differences in the amounts of DNA among the Macaronesian species . Tukey test revealed that the populations of A. gorgonum (Cape Verde) presented an average genome size (10.52 pg) significantly higher than those of A. thuscula  and A. argentea . Populations of Canary Islands and Madeira did not show significant differences among them.Artemisia are distributed—confirmed that A. argentea, A. thuscula and A. gorgonum constitute well-defined taxonomic units from a genetic point of view, each species being circumscribed to a single archipelago . These endemic Artemisia taxa represent an outstanding example of independent anagenetic speciation processes  show a 2C value significantly lower than A. arborescens populations, although all those taxa share the chromosome number 2n = 18 (http://ccdb.tau.ac.il/home). In contrast, A. arborescens from continental islands show higher 2C values than on the mainland  than to the lineage divergence patterns of the group.Our phylogenetic analyses based on plastid DNA sequences—including a comprehensive sampling in all islands where endemic Macaronesian Artemisia using nuclear DNA sequences . Numerous biogeographic studies have proposed that drastic changes in sea level during specific periods of the Pleistocene could have led to the emergence of seamounts facilitating the colonization of Macaronesian archipelagos  occur on very arid and near sea-level habitats, potentially similar to those found in the emerged seamounts. Therefore, we can speculate that the ancestors of Macaronesian Artemisia species could have taken advantage of narrow windows of opportunity during emergence of paleo-islands to colonize in parallel the different archipelagos at a similar time.As in our results based on plastid DNA data, previous phylogenetic reconstructions of our tree , the splArtemisia showed very similar values among species both in terms of haplotype and nucleotide diversity  during their early evolutionary history at Pleistocene could have blurred the phylogenetic signal of molecular markers, homogenizing the genetic diversity levels as well among the species .Recent reviews on anagenetic diversification in oceanic islands have concluded that geographical patterning of genetic variation is unexpected among populations of endemic species originating anagenetically , where it is the main haplotype, present in all analysed individuals excepting one  are usually inferred as the centres of diversification and as the origin of stepwise dispersal to the smaller islands of La Palma and El Hierro in the west  and another one for western islands of La Gomera, La Palma and El Hierro . This genetic split is also suggested by genome size results, where Tenerife and Gran Canaria populations show the lowest qDNA values of the archipelago  Liede & Meve, Globularia amygdalifolia Webb and Umbilicus schmidtii Bolle, Euphorbia tuckeyana Steud. ex Webb, Echium, C. jacobaea, G. amygdalifolia, U. schmidtii and E. tuckeyana) showed an evident north-south differentiation pattern at the molecular level. Our results in A. gorgonum—a species also distributed in western islands of Cape Verde—support as well the same phylogeographic structure. Three related haplotypes were found in Artemisia populations from the northern Santo Antão Island  and southern (Fogo and Santiago) islands also suggest this geographic pattern  and the rest of the populations of this species occupying—in some cases dominating—coastal sites close to sea level. This potential taxonomic diversification process occurring within Madeira archipelago would support the role of habitat type and niche conservation/migration as driving forces for speciation in insular Artemisia.Low rate of speciation in some island endemics may also be influenced by the type of habitat occupied by these plants (Artemisia species—together with their recent evolutionary history as independent lineages—could be key factors to explain the limited speciation ability within this group of plants. Further genetic studies using highly variable nuclear markers (e.g. high-throughput sequencing approaches) would elucidate the role of gene flow via pollen or seeds in the evolutionary history of these island endemics. Additional experiments regarding their reproductive biology (e.g. the occurrence of self-compatibility or self-incompatibility) would also be helpful to fully understand these textbook examples of anagenetic diversification in oceanic islands.Overall, we hypothesize that characteristics of seed dispersal, pollen transport and type of habitat in endemic Macaronesian The following additional information is available in the online version of this article – Biological traits in endemic Macaronesian Artemisia species and A. arborescens. Information obtained from the floras of the territories where these species are distributed (see the main text for the references). Sampling details of the populations studied from the genetic and cytogenetic point of view. GenBank accession numbers of the sequences generated for the populations studied from the genetic point of view. Results of analyses of molecular variance (AMOVA) in the geographical and genetic groups of populations defined in the study. Nuclear DNA content of the species studied, where the 2C values of the three Macaronesian species (with a representative of each island where the taxa live) listed below are the first obtained in natural populations. Asterisks (*) indicate data from Garcia  Values of ΔFCT used to estimate the most likely K from SAMOVA analyses. Geographic distribution of the populations according to the spatial genetic partitioning defined by SAMOVA for K = 2 in (A) A. argentea, (B) A. thuscula and (C) A. gorgonum. Statistical parsimony allelic networks for nuclear (ITS and ETS) phased sequences. Different colours in the pie charts (ribotypes) correspond to the different species. Sequences constituting each inferred ribotype are specified next to each pie chart. Neighbour-net network inferred from nuclear ribosomal DNA sequences. Tip names correspond to the sample codes shown in plad057_suppl_Supplementary_MaterialClick here for additional data file.


Canis lupus familiaris) and wild canids like gray wolves (Canis lupus) and golden jackals (Canis aureus). Wolf–dog hybridization may lead to a significant reduction of specific adaptations in wolves that could result in the decline of wolf populations. Detection and genetic discrimination of hybrids between dogs and wolves are challenging because of their complex demographic history and close ancestry. Citizen scientists identified two phenotypically different‐looking individuals and subsequently collected non‐invasive samples that were used by geneticists to test wolf‐dog hybridization. Genomic data from shed hair samples of suspected hybrid individuals using double‐digest restriction‐site‐associated DNA (ddRAD) sequencing resulted in 698 single nucleotide polymorphism (SNP) markers. We investigated the genetic origin of these two individuals analyzed with genetically known dogs, wolves, and other canid species including jackals and dholes . Our results provide the first genetic evidence of one F2 hybrid and the other individual could be a complex hybrid between dogs and wolves. Our results re‐iterate the power of next‐generation sequencing (NGS) for non‐invasive samples as an efficient tool for detecting hybrids. Our results suggest the need for more robust monitoring of wolf populations and highlight the tremendous potential for collaborative approaches between citizens and conservation scientists to detect and monitor threats to biodiversity.Human demographic expansion has confined wildlife to fragmented habitats, often in proximity to human‐modified landscapes. Such interfaces facilitate increased interactions between feral or domesticated animals and wildlife, posing a high risk to wild species. This is especially relevant for free‐ranging dogs ( Free‐ranging dogs can severely impact wildlife, especially other canid species through predation, scavenging, disease transmission, and hybridization. Detection and genetic discrimination of hybrids between dogs and gray wolves are challenging due to complex demographic history and close ancestry. In this study, we report the first‐ever detection of a wolf‐dog hybrid using genetic and photographic evidence in peninsular India with the help of citizen science. Moreover, wolves mostly occupy savanna grassland and scrub habitats outside designated protected areas, beyond the jurisdiction of the state forest departments. In such scenarios, involving citizen scientists can offer innovative and effective avenues for monitoring wildlife populations and habitats that require conservation interventions . Prior to extraction hair samples were rinsed with 0.1X commercial bleach, washed with ethanol, and nuclease‐free water in order to remove any DNA on the surface  using BWA mem Li, . These s2.2Freebayes  collected by the citizen scientists, and followed it up with constructing and sequencing ddRAD libraries. The number of reads obtained and mapped to the dog reference genome varied slightly between both the samples .The first set of filtering using all 35 samples resulted in 1573 SNPs. These were used to perform PCA and Admixture to identify the origin of H1 and H2, with several canid species. We observed clear segregation of Dhole, Jackal, and the wolf‐dog cluster Figure . To bettK values between two and four and supported the observations of PCA analysis. Using all 35 individuals, four clusters were identified by both methods (best support for K = 4). The clusters included dhole, jackal, Indian wolves, and rest of the world wolves  with confirmatory genetic analysis. However, mating events between dogs and wolves have been detected . But very few studies have explored monitoring of wildlife populations through a combination of citizen science and non‐invasive sampling ; data curation (lead); formal analysis (lead); investigation ; methodology (lead); visualization ; writing – original draft (lead); writing – review and editing . Mihir Godbole: Data curation ; writing – review and editing . Abi Tamim Vanak: Conceptualization ; resources ; writing – review and editing . Uma Ramakrishnan: Conceptualization ; funding acquisition (lead); project administration (lead); resources (lead); supervision (lead); writing – review and editing .The authors declare no conflicts of interest.Data S1.Click here for additional data file.


Ruditapes philippinarum) is one of the most commercially important bivalves along the coast of China. The increasing expanding of clam culture may result in some serious problems. In this paper, we investigated the genetic diversity and differentiation of R. philippinarum populations and tested the hypothesis that clam population differentiation is influenced by the southern breeding and northern culture. The present findings will provide useful information for natural resource conservation and genetic breeding of the Manila clam in China.The Manila clam (Ruditapes philippinarum) is one of the most commercially important bivalves along the coast of China. With the continuous expansion of clam farming scale, it may lead to some serious problems, including loss of genetic variation, inbreeding depression, and reduced effective population size (Ne). In the present study, eleven microsatellite markers were used to investigate the genetic diversity and differentiation among 13 clam populations along the coast of China. As a result, 150 alleles were detected according to the genotyping results of eleven microsatellite loci. The observed heterozygosity (Ho) was estimated to be ranging from 0.437 to 0.678, while the expected heterozygosity (He) was calculated to be varying from 0.587 to 0.700. Fst values between populations ranged from 0.0046-0.1983. In particular, the Laizhou population had the highest genetic variability, which was significantly different from the others . For all the clam populations, there was no significant linear regression between genetic and geographic distance, indicating that these populations do not follow a pattern of isolation by distance (IBD). Genetic structure was estimated according to NJ, principal coordinates (PCoA), and structure-based clustering. Estimates of effective population size range from dozens to thousands among different populations, based on linkage-disequilibrium and molecular coancestry methods. The results reveal the genetic diversity of clams and verify the hypothesis that clam population differentiation may be influenced by the mode of southern breeding and northern culture, providing guiding information for natural resource conservation and genetic breeding of clams.The Manila clam ( Ruditapes philippinarum) is an important marine bivalve living in the intertidal zone and has the second largest production among bivalve mollusks [The Manila clam , ridgetail white prawn , Silond catfish (Silonia silondia), Pacific abalone , blood clam (Barbatia virescens), and crab (Portunus trituberculatus) [Genetic variation can affect the ability of aquatic animals to adapt to environmental changes . Examinaculatus) ,13,14,15culatus) . SSRs arculatus) ,18,19. HR. philippinarum) along the coast of China. The examination of population genetic structure and differentiation of the clams aims to verify the hypothesis that clam population differentiation may be affected by the mode of southern breeding and northern culture. The present findings will not only provide useful information for genetic structure in a wide range of populations, but also help to promote natural conservation and genetic breeding of clam R. philippinarum.In this study, the new multiplex SSR method has been performed by using labelled M13-tails, providing a cost-effective method for SSR genotyping in clams. Eleven polymorphic microsatellite markers were selected to analyze the genetic diversity and differentiation of the Manila clam (R. philippinarum) were collected from the northern and southern coast of China .Eleven pairs of microsatellite markers with stable amplification were selected from the previous reports ,21. The o), and the expected heterozygosity (He) [r) and inbreeding coefficient (Fis) were calculated through FSTAT 2.9.3 [is values indicate inbreeding within populations (excess of homozygotes), whereas the significant negative Fis values represent an excess of heterozygosity. The differences of allelic richness among different groups were compared by the Kruskal–Wallis test of SPSS 26. Furthermore, differences in the allelic richness for each population at each locus was tested using a Kruskal–Wallis rank sum analysis [st) were calculated by Genepop 4.0 [The software MSAnalyzer 4.05 was used to calculate number of alleles (N), the observed heterozygosity (Hity (He) . The allAT 2.9.3 . The siganalysis . Hardy–Wepop 4.0 . For theepop 4.0 . The genepop 4.0 . An analepop 4.0 . A princepop 4.0 ,31.r, GD, Ho, and He) were shown for each locus and population in r) varied from 3.2 (Rpt100 and Rp-07) to 5.5 (Rpt106). At the population level, the average of observed heterozygosity (Ho) was calculated to be ranging from 0.437 to 0.678, while the expected heterozygosity (He) was estimated to be varying from 0.587 to 0.700. Among the eleven loci, the highest He value (0.700) was detected in the selected population of XY, while the lowest value (0.587) was found in the QD population. The number of alleles per locus in each population ranged from 2 to 11, and allelic richness per locus varied from 1.7 to 5.5. For all these populations, the LZ population had the largest number of alleles (6.9), as well as the maximum of allele richness (4.1). In contrast, the least number of alleles (5.1) and the minimum of allele richness (3.3) were found in the ZH population. Despite this, no significant difference in allelic richness was detected among these populations . The positive values of Fis were consistently found in all the populations, except for HY population (p < 0.005).For the eleven microsatellite loci, the genotyping results of 408 individuals were derived from 13 clam populations with sample sizes ranging from 24 to 40. The descriptive genetic statistics , whereas the highest differentiation was found between the QD and LZ populations . The genetic differentiation between the LZ population and other populations is relatively high, varying from 0.1020 to 0.1983 (p < 0.01). The genetic distances (DS) among populations were also displayed in Pairwise Fo 0.1983 . The lowst value was ranging from 0.0357 to 0.1729 (p < 0.01), with an average of 0.0663 . The lowest values of Ne were found in Chaozhou and Donggang, having extremely low Ne of less than the critical value (Ne = 50). Generally, the Ne values estimated from the Cn method were relatively lower than those from the LD method.Two single-sample methods were used to estimate Nstimates . AccordiR. philippinarum populations revealed a relatively higher level of genetic diversity (He = 0.636) than those estimates from allozymic analysis and other DNA-based analyses, such as mtDNAs, AFLP, and RAPD [Crassostrea gasar . In addition to clams, multi-locus heterozygosity deficiencies have been previously widely reported in many other bivalves [Departures from Hardy–Weinberg equilibrium (HWE) were measured through the significance of permutation tests for the null hypothesis, Fis = 0 . In the bivalves ,41. Earlbivalves . Howeverbivalves ,44. It ist = 0.066, p < 0.001),indicating the existence of the genetic heterogeneity among populations. As the wild population in Laizhou Bay , the LZ population remains the population with the highest level of genetic variability, showing great differentiation with other populations. This is consistent with the previous studies, supporting the natural status of clam populations with high levels of genetic variability [The overall genetic differentiation among these populations was moderate but highly significant , a key parameter in evolutionary biology, determines the rates of genetic drift and loss of genetic variability and modulates the effectiveness of selection [e reduction, known as the Ryman–Laikre effect [e would lead to a collapse of local genetic adaptation, which could expose local populations to adverse effects [The effective population size  and retention of genetic variation for future adaptations (Ne = 500) can be used as a guiding principle to indicate the short- and long-term genetic viability of populations [The previous studies have indicated that Niversity . The smaulations . As a ruthan 500 . The estulations ,56.e values (less than 50) have been obtained in several populations  according to LD and Cn methods. The small Ne values may be caused by inadvertent selection of the best offspring produced by a few parents and asymmetric reproduction [e values [e values from the LD method have been detected in multiple populations of clams, probably due to the linkage disequilibrium generated by the sampling process and inadequate correction [e is very large or limited data are available, by chance 2  can be smaller than the sample size correction, resulting in the negative estimates of Ne [e [e [e values. The future estimation of Ne needs an extensive evaluation in larger sample sizes using increased numbers of loci and alleles. Despite uncertainties related to the small sampling size, Ne estimates obtained by the two applied methods can provide useful complementary information for conservation programs to prevent inbreeding depression and loss of genetic variation. According to the present findings, the small Ne values, as well as the low differentiation, may be caused by few broodstock used in southern hatcheries, with offspring transferred to the northern coast for culture at the mode of southern breeding and northern culture.In the present study, small Noduction . Despitee values ,22. Thuse values ,60. Surprrection . If Ne ies of Ne ,62. Therof Ne [e . This isNe [e [e . DespiteR. philippinarum (Manila clam) populations from the coastal areas of China. The multiplex PCR using the labelled M13-tails was shown to be a cost-effective method for SSR genotyping in clams and mollusks, provided that the sufficient sampling size is ensured. The present findings support that the genetic population structure of clams may be influenced by the mode of southern breeding and northern culture. The assessment of the genetic diversity of R. philippinarum populations is of considerable importance for the optimal development of programs aimed at the conservation of cultivated and wild genotypes in the ecosystems. The present findings will provide guiding information on natural resource conservation and genetic breeding of the Manila clam in China. The highest level of genetic variability and greatest differentiation with other populations was confirmed for the wild Laizhou population. It was suggested that multi-locus heterozygote deficiency and segregation distortion in such populations may be caused by high genetic load. No relation was found between genetic and geographic distance, implying clam aquaculture may be served as one of the major factors influencing clam population genetic structure. Despite uncertainties related to the small sampling size, Ne estimates obtained by the applied methods can provide useful complementary information for conservation programs to warn about inbreeding depression and loss of genetic variation, thereby serving the needs of natural resource conservation.In this study, genetic diversity and differentiation were investigated by 11 microsatellite loci for


Batagur affinis subspecies in Malaysia is part of a conservation genetics effort aimed at long-term sustainability. As the conservation status of this species is critically endangered, we found only six different haplotypes from four study areas and low genetic diversity using D-loop markers. Nevertheless, using genetic markers, we revealed the presence of one of the Southern River terrapin subspecies (B. affinis edwardmolli) in Kedah, Malaysia.A novel comprehensive population genetics study of two Batagur affinis) from four places in Peninsular Malaysia: Pasir Gajah, Kemaman (KE), Terengganu; Bukit Pinang (BP), Kedah; Bota Kanan (BK), Perak; and Bukit Paloh, Kuala Berang (KB), Terengganu. The goal of this study is to identify genetic differences in two subspecies of B. affinis in Malaysia. No previous reports were available on the genetic diversity, phylogenetic relationships and matrilineal hereditary structure of these terrapin populations in Malaysia. The sequencing identified 46 single nucleotide polymorphisms that defined six mitochondrial haplotypes in the Southern River terrapins. Tajima’s D test and Fu’s Fs neutrality tests were performed to evaluate the signatures of recent historical demographic events. Based on the tests, the B. affinis edwardmolli was newly subspecies identified in the west coast–northern region of Kedah state. In addition, the B. affinis edwardmolli in Bukit Paloh, Kuala Berang (KB), Terengganu (Population 4), was shown to have a single maternal lineage compared to other populations. Low genetic diversity, but significant genetic differences, were detected among the studied Southern River terrapin populations.A population genetics study was carried out on the Southern River terrapin ( Batagur affinis) [B. affinis (The Southern River terrapin (affinis)  , with Binding Column technology. The input volume of the EDTA whole blood sample yielded a final extraction volume of 200 µL.Nucleic acids were isolated from 200 µL of each EDTA whole blood sample. Following cell lysis and protein denaturation, extractions were carried out using an automated system, the ReliaPrepw/v) agarose gel with molecular markers.The amounts of extracted DNA were determined using a Thermo Scientific™ NanoDrop 2000c spectrophotometer . The automated system’s competence to extract nucleic acids of great purity was verified by direct gel loading. Following NanoDrop quantification of the isolated nucleic acids, the results were loaded directly into the 1% . Denaturation at 94 °C for 3 min was followed by 25 cycles of denaturation at 94 °C for 35 s. Meanwhile, the primer annealing stage was completed at 60 °C for 1 min and 30 s, followed by a 2 min extension at 72 °C and a final 2 min extension at 72 °C. The purified PCR products were shipped to a private company  for D-loop sequencing with only the forward primer. The reverse primer was then used in a sequencing procedure on selected samples (haplotype (HAP)) to confirm the polymorphism in the mtDNA sequence with the forward primer. Finally, mtDNA D-loop sequences from different haplotypes were then sent to GenBank under the numbers MZ555651–MZ555656.A primer set of 5′-TTTTTCCCCTAGCATATCACCA-3′ (forward) and 5′-AGTTGCTCTCGGATTTAGGG-3′ (reverse) designed previously by  was usedThe ClustalW algorithm in MEGA X   was usedSTF (STcF) values in AMOVA and Mantel tests [STF), and the probability (Chi-square test (χ² test)) [STF) and pairwise Tamura–Nei results are needed to assess the degree of geographic structuring of genetic variability. Then, we used DAMBE 6  [The Analysis of Molecular Variance (AMOVA) ,37 was uel tests , includi² test))  for all  Canada)  to test CTF) variance determined the best number of groups, k (the maximum was k = 3). To account for the family-wise error rate of = 0.05, the False Discovery Rate procedure was used [A Spatial Analysis of Molecular Variance (SAMOVA) was performed in SAMOVA 2.0  to find the groups of populations that are spatially homogeneous and maximally dissimilar from each other and confirm the genetic barriers between these groups . The higwas used .p-values were calculated using 1000 simulations under the selected neutrality model. Using the DnaSP 6.12.03 software, more mismatch frequency graphs were made to show the difference between nucleotide site differences and those that stayed the same over time [Two statistical tests, Tajima’s D ,45 and Fver time ,45. Secover time  was usedver time  to measuver time  between ver time . In the The tree was made using the Maximum Likelihood (ML)  and MaxiThe best-fitting evolution model for each sequence studied was estimated by employing the Akaike information criterion (AIC), with a correction for sample size implemented using jModelTest2 on XSEDE 2.1.6 . In the Batagur affinis affinis and B. a. edwardmolli mtDNA D-loop sequences were aligned using ClustalW in MEGAX [in MEGAX  softwarein MEGAX  analyseshttps://www.phylo.org/portal2/login!input.action; accessed on 14 May 2021) [The phylogenetic reconstruction was performed by using the IQ-tree  on XSEDEay 2021) . The treay 2021) . Firstlyay 2021) ,59. The BEAUti 2,  program Using Bayesian Markov Chain Monte Carlo (MCMC) simulations for 100,000,000 generations with a sampling frequency of 5000, two simultaneous analyses were conducted. The nucleotide substitution model for ML was set as TN93 empirical. The bootstrap analysis provided branch support (1000 pseudoreplicates), and all other parameters were set to default values. Finally, FigTree v1.4.4 was used to plot the phylogenetic trees. Batagur affinis edwardmolli (MN069309) and Batagur kachuga (MZ156025) were selected from the GenBank online database as out-groups [To construct the phylogenetic trees, the mitochondrial sequences of t-groups ,61. A Met-groups .Batagur affinis samples were divided into four population regions. From these samples, 30 samples were assigned to each population region. Six distinct D-loop haplotypes were found, each determined by 46 SNPs or substitution numbers inside the 651 bp fragments. These SNPs contained 43 transitions and three transversions at positions 1, 11, and 23, respectively  is defined as the average number of nucleotide differences per site in pairwise comparisons between DNA sequences [The number of haplotypes varies among different population regions, ranging from one to three . Among tequences . The Hd CTF was used to characterise the best population arrangement in SAMOVA with the number of groups (K). However, in this study, SAMOVA demonstrated that CTF decreased with population partitioning into the most group in all the analyses performed. It should be emphasised that population structure analysis using one or more single population groupings cannot determine group structure [CTF was chosen [CTF (0.9439). According to the SAMOVA analysis, the division of four population regions into two groups based on the majority of two subspecies (Group 1 (Batagur affinis affinis): Bota Kanan, Perak, and Bukit Pinang, Kedah; Group 2 (B. affinis edwardmolli): Kemaman and Kuala Berang, Terengganu) resulted in even higher and significant genetic variations between groups .In the current analysis , within-tructure . When mos chosen . Based oBatagur affinis using Tamura–Nei genetic distances and the fixation index (STF). The data is presented in a matrix format with pairwise comparisons of genetic distance (Tamura–Nei) below the diagonal and STF values and probabilities of population differentiation (based on a chi-square test with 110 permutations) above the diagonal.STF values between each pair of populations, and the probability of population differentiation based on a chi-square test with 110 permutations. Pairwise STF analysis showed highly significant genetic differentiation between populations. The pairwise STF values varied from 0.034 to 0.993, and the highest genetic differentiation was perceived between Population 3  and Population 4 . The average STF value in the present study was found to be 0.645.The pairwise values above the diagonal in the matrix indicate the Batagur affinis edwardmolli BP1). The average Tamura–Nei in the present haplotype study was 4.7%.Additionally, pairwise Tamura–Nei analysis showed highly significant genetic differentiation between haplotypes . Tamura–p-values. Tajima’s D values were negative except in Populations 3 and 4, indicating an excess of rare nucleotide site variants. On the other hand, Fu’s FS test results, based on the distribution of haplotypes, showed positive values for all the studied populations except for Population 1, signifying an excess of rare haplotypes.Tajima’s D and Fu’s Fs tests were conducted to infer the demographic history and detect the past population growth of the Southern River terrapin. The Tajima’s D and Fu’s Fs test results are given in In general, negative scores from the two tests (Tajima’s D and Fu’s FS) indicated an excess of rare mutations among the populations. In contrast, positive scores of two tests (Tajima’s D and Fu’s FS) suggest an excess of high-frequency variants, which can be consistent with population contraction or balancing selection. It is hard to find research that shows that the mitochondrial genome in natural populations has been changed by direct or indirect selection (via hitchhiking) .p-value less than 0.05), the observed data did not fit the model of population expansion well and may be due to factors other than neutral evolution. Moreover, the data indicated departures from neutrality that may be due to factors such as demographic events or natural selection. For the other populations, however, the observed data was consistent with the model of population expansion and did not show evidence of departures from neutrality [We also considered the SSD and RI under the demographic expansion model for each population. As shown in utrality ,47.By examining the frequency distributions of pairwise differences across sequences, it is possible to gain insight into historical demographic expansions ,71,72. IA unimodal peak was observed in both lineages of Population 1  and Population 3 . These fBatagur affinis . However, cluster 1 was thought to consist only of the population from the Terengganu region (B. a. edwardmolli). Surprisingly, B. a. edwardmolli was found in a minority population in Kedah, Malaysia, where it was discovered together with HAP_2 (B. a. edwardmolli KE09) and related to HAP_4 (B. a. edwardmolli BP31). This indicates that the B. a. affinis individuals that are most common in Kedah and Perak are in cluster 2 .A phylogenetic tree and a Bayesian analysis were undertaken to study the relationships and divergence of Southern River terrapins’ haplotypes, and the results supported the monophyly of  affinis . The tre affinis . The BayHaplotype network analysis was performed using the randomised Minimum Spanning Tree (MST) method. The median-joining haplotype network showed that HAP_4 is related to all the other haplotypes, indicating that it is the ancestral one, and the haplotype distribution was established on the map. In addition, a median-joining (MJ) network was created with six haplotypes based on 652 bp mtDNA D-loop sequences  to analyBatagur affinis populations from Peninsular Malaysia. The examination was based on haplotypes of the mtDNA D-loop (ca. 652 bp) from a total of 120 individuals. HAP_2 was shown be found in the greatest number of localities compared to HAP_1, HAP_4, and HAP_6. The B. a. affinis from BK, Perak, and BP, Kedah, were both found to have the greatest variety of haplotypes.The map  shows saBatagur affinis populations in Peninsular Malaysia by analysing the mitochondrial D-loop. This is the first time that the mtDNA D-loop sequences of B. a. affinis have been investigated. The use of mitochondrial D-loop DNA sequences to study the genetic variation of B. a. edwardmolli has been previously described [B. affinis, but the results were conflicting due to the small number of samples. In consequence, we investigated the genetic variation of four different B. affinis populations found on Malaysia’s east coast  and west coast . Some haplotypes were shared between the regions, indicating common sources of colonists. The six haplotypes discovered in the D-loop area of the terrapins  are attpared to , a totalpared to .Batagur species, our Hd value was 0.214 on average. Similarly, an average Hd value of 0.2 was reported for Cambodian Batagur affinis edwardmolli  [Batagur borneoensis was reported by [When compared to populations of other e = 0.2) , and, agorted by . This imorted by . HoweverBatagur affinis population in Malaysia. The π value measured was low, with an average difference in nucleotide composition (k) of 0.3%. This low value indicates that a population bottleneck might occur because of hunting for consumption and retail, habitat loss, and inbreeding in the restricted habitat. All of these could be contributing to B. affinis’ decline. Additionally, low π indicated minor haplotype differences [B. affinis affinis and three different haplotypes found in four other regions. In addition, the total number of populations was highly monomorphic (96%). A hypervariable region with high base substitution, the D-loop, allows for more accurate identification of individual diversity than the other regions [The percentage of haplotypes that were similar reached 67.8% in this study. As previously stated, it has been demonstrated that there is a critical situation for the ferences . Based o regions . Given t regions ,82.Similar biochemical processes cause polymorphisms and mutations, but the term “polymorphism” is used to avoid implying that any particular allele is normal or abnormal. Some of the consequences of inbreeding can also be explained by mutations ,84. As aBatagur affinis populations. Variations within populations were found in all the selected B. affinis populations. This could be explained by one or more factors, such as small population sizes, previous bottleneck events, or the existence of physical barriers to gene flow between populations [Batagur affinis populations have fixed haplotype differences and high STF values. This means that there was not much gene flow between the populations, which could be because rivers or large distances separated the populations [Dermochelys coriacea in the United States of America [STF values found in this study.This study discovered significant intra-population and inter-population variations in the ulations . Most Baulations . A simil America  supportsBatagur affinis are genetically and how different they are from each other. The data can be used to infer patterns of gene flow, genetic diversity, and population structure among the populations, which can be important for the conservation and management of this species.Based on DNA sequence data, Batagur affinis affinis populations from Perak and Kedah shared mainly HAP_3. As a result, this would suggest that B. a. affinis had a historically extensive natural distribution in the area.Nonetheless, a particular haplotype is distributed among several locations; for example, KE-KB, Terengganu, and BP, Kedah shared HAP_2. D value  shows an excess of low-frequency polymorphisms relative to expectation [D  indicates an excess of high-frequency variants, which can be consistent with population contraction or balancing selection [p-value is greater than 0.05, which means there is no evidence of deviation from neutrality in the population. It suggests that the observed genetic variation in the population is consistent with what would be expected under neutral evolution, and there is no reason to reject the null hypothesis of neutrality [To study the population history, neutrality tests were conducted using Tajima’s D  and Fu’sectation , indicatectation . A positelection ,90. In autrality .SF indicates an excess of rare alleles in the population. This can be an indication of recent population expansion or a selective sweep, which can result in the loss of genetic diversity in the population. In contrast, a positive value indicates the opposite [p-value for Population 4 is undetected, its significance remains unknown.Meanwhile, a negative value for Fu’s opposite . Since FD and Fu’s SF test statistics. Nonetheless, Fu’s simulations denoted that Fu’s FS was a more sensitive indicator of population expansion than Tajima’s D [All the statistically non-significant tests were consistently compatible with the population’s being in drift-mutation equilibrium, as determined by Tajima’s jima’s D .Batagur affinis population in Malaysia. This was done to test the hypothesis that the experimental records fit the unexpected expansion model [p-values of these tests are greater than 0.05, it suggests that the observed pattern of genetic diversity is consistent with the spatial expansion model and that there is no evidence of deviation from this model. However, the p-value for Population 4 is less than 0.05, which means that the observed pattern of genetic diversity is very different from the spatial expansion model [According to , the SSDon model . The SSDon model . It is iBatagur affinis, commonly known as the Southern River terrapin, is present in the waterways within the territory between the east and west coasts of Peninsular Malaysia.Regarding population differentiation, the results of haplotype network analysis established that Population 2  and Population 3  share the most prevalent haplotypes and exhibit only minor divergence. High gene flow between populations can slow down or prevent geographic differentiation. The populations from BK, Perak and KB, Terengganu exhibited the most differentiation, as the geographical distance between the west coast of Peninsular Malaysia and the east coast of Peninsular Malaysia, populated by these terrapins, divides the two subspecies. It is possible that Accordingly, the phylogenetic interpretation of the haplotype network revealed a “glass goblet-like” topology with a large proportion of singletons. This is typically interpreted as a sign of a growing population, particularly from a small number of founders following a population bottleneck . Here, HBatagur affinis edwardmolli). On the other hand, HAP_3, HAP_5, and HAP_6 could be grouped as cluster 2 (B. a. affinis). The genetic distance between HAP_2 B. a. edwardmolli KE09 and HAP_4 B. a. edwardmolli BP31 distances were 0.2% to 0.3%. Therefore, with a significant bootstrap confidence level (>50%) in B. a. edwardmolli populations in Malaysia was revealed. This subspecies similarity was discovered in Kedah, on the west coast and north of Peninsular Malaysia . In addition, Population 2  shared a HAP_2 with Population 1 and Population 4 from Terengganu, B. a. edwardmolli. Based on the history recorded, Southern Thailand has two subspecies of B. affinis [B. a. affinis could be found in the southernmost region of Thailand. Furthermore, B. a. edwardmolli was previously recorded found in the Songkhla region of Thailand’s neighbouring, southernmost eastern peninsula. A Thailand conservation centre for B. affinis is reported to be located on the Klong Langu River in Satun Province (Southern Thailand). Therefore, the population of Southern Thailand is expected to migrate to Kedah, Malaysia. This could be due to the nature of the disaster  and the high threats faced by B. affinis in Thailand, which forced the population to migrate outside of Thailand and into Kedah, Malaysia, in search of better resources or living conditions. The author of [The topologies of our Bayesian phylogenetic tree were broadly concordant and showed that HAP_1, HAP_2, and HAP_4 could be grouped as cluster 1  among DWNP Kedah, Zoo Negara, and Zoo Melaka to reduce the potential for inbreeding in the BP Kedah population. Meanwhile, an exchange programme between the KE and KB populations is required to enhance genetic variance among the KB (Terengganu) populations. Additionally, we proposed that additional research should be conducted, such as more exploration to find new wild population sites and conducting a whole-genome sequencing study to reveal more important genetic matters crucial for conservation strategy. At this time, the ranching conservation programme should be maintained, while any sand mining should be stopped immediately. Additionally, habitat enrichment is required in at least four population regions to protect successful hatchlings from hunters and natural predators during the egg and hatching seasons.Batagur affinis edwardmolli populations in Malaysia. This information is helpful for conservation programmes aimed at sustaining the genetic stock.Our research showed low genetic diversity and significant genetic differences across the terrapin populations and revealed a novel distribution range for


Eothenomys miletus from the Hengduan Mountain (HM) region, one of the biodiversity hotspots. One hundred sixty‐one E. miletus individuals from five populations in the HM region had their reduced‐representation genome sequenced, and one additional individual from each community had their genomes resequenced. We then characterized the genetic diversity and population structure of each population and compared the phenotypic divergence in traits using neutral molecular markers. We detected significant phenotypic and genetic alterations in E. miletus from the HM region that were related to naturally occurring diverse habitats by combining morphometrics and genomic techniques. There was asymmetric gene flow among the E. miletus populations, indicating that five E. miletus populations exhibit an isolation‐by‐island model, and this was supported by the correlation between FST and geographic distance. Finally, PST estimated by phenotypic measures of most wild traits were higher than differentiation at neutral molecular markers, indicating directional natural selection favoring different phenotypes in different populations must have been involved to achieve this much differentiation. Our findings give information on the demographic history of E. miletus, new insights into their evolution and adaptability, and literature for studies of a similar nature on other wild small mammals from the HM region.An important objective of evolutionary biology has always been to grasp the evolutionary and genetic processes that contribute to speciation. The present work provides the first detailed account of the genetic and physiological adaptation to changing environmental temperatures as well as the reasons causing intraspecific divergence in the  Eothenomys miletus from the Hengduan Mountain (HM) region, one of the biodiversity hotspots. Findings provide first detailed account of genetic and physiological adaptation to changing environmental temperatures as well as the reasons causing intraspecific divergence. Some biodiversity hotspots serve as “evolutionary forewords” that spur fast divergence of tropical plant groupings and the junction of long‐distance species distribution, having a significant impact on the establishment and evolution of the world's flora and fauna.Due to Hengduan Mountain (HM) region has the characteristics with high northwest and low southeast, and its climate, which is characterized by a modest yearly temperature difference and a huge daily temperature difference, a wider range of animal species can survive there  and differentiation at neutral molecular markers (FST), commonly referred to as the QST–FST comparison, revealed that natural selection played a significant role in the cause of differentiation in quantitative traits. In several cases, putative FST and QST differentiation in various populations is compared in order to evaluate their evolutionary signatures and discover potential features implicated in local adaptation.Comparative analyses of quantitative genetic and neutral marker differentiation have given researchers a way to assess the relative contributions of stochastic genetic drift and natural selection to the explanation of among‐population divergence  with phenotypic divergence in a trait (PST), and PST counts are based on phenotypic assessments of a wild trait of several individuals across numerous populations  used in this study were caught in five sites with gradually varying altitudes: Deqin ; Xianggelila ; Lijiang ; Jianchuan ; and Ailaoshan . Detailed sampling information is provided in the Figure From November 2018 to January 2019, the voles , and the sequence of fragment lengths 464–494 was defined as a SLAF‐tag. The principles of the selective digestion plan are as follows: (1) The proportion of digestion SLAF tags located in repeat sequences should be as low as possible; (2) the SLAF tags were evenly distributed on the genome as far as possible; (3) the consistency of the fragment length should be maintained with the specific test system; and (4) the number of enzyme‐cut SLAF tags obtained met the expected number of tags. All qualified samples were digested by enzymes, respectively . Clean reads were then mapped to the reference genome of Microtus ochrogaster  software of Beijing Genomics institution Co. LTD  calling in the GATK 3.3.0  and observed heterozygosity (Ho) were calculated using PLINK 1.9  between each pair of variables in order to further quantify the impact of environmental variables, such as altitude, temperature, and latitude, on genetic diversity at five geographic populations .The expected heterozygosity  model was used to estimate changes in effective population size on the basis of heterozygous sites across the genome . The Bayesian statistical model of MIGRATE‐N software was used to estimate pairwise relative migration rates and directionality between populations based on the ancestor tracing theory  in order to test the effects of geographic distance and environmental differences on genetic differentiation. This was done using the Ecodist package in R 3.6.3  was employed as the response, the geographic distance as the predictor, and the environmental distance as the condition factor to assess isolation by distance (IBD). The geographic distance was utilized as the condition element to investigate isolation by environment, isolation by temperature (IBT), and isolation by precipitation (IBP). Moreover, the distance in altitude between paired sampling sites was calculated. Finally, we utilized Canoco 5 , torso length (T2L), chest width (CW), chest depth (CD), ear length (EL), ear width (EW), fore limb length (FLL), and hind limb length (HLL), were taken from specimen tags referring to Gao et al.  and LSD post‐hoc tests were used to assess group differences in attributes; PST measures the percentage of among‐population phenotypic variance in quantitative characteristics and is equivalent to QST  within samples is 0.5. The consequences of departure from these assumptions are considered below in the Discussion sections. Phenotypic variance components were estimated following Sokal & Rohlf, PST values for individual attributes were compared with the pairwise FST  to assess the degree of phenotypic divergence with neutral genetic divergence and independent sample t‐test was conducted for traits PST and FST using SPSS 26.0. The two‐way clustering heat map of the PST/FST value between paired populations was built using the online Heatmapper. We tested correlations between geographic distances, population pairwise altitudinal differences, pairwise FST, and pairwise PST using the Mantel test of matrix correspondence . Moreover, with K = 4, a portion of the XGLL individuals and the JC population formed one clade, and the remainder XGLL individuals and the DQ population formed one clade, in accordance with PCA, which further divided the LJ population and the DQ population , expected heterozygous (He), Nei diversity index, as well as PIC. The genetic diversity of the five E. miletus populations from the HM regions was highest in the ALS population, followed by JC population, and least in the XGLL population.Table D, and observed heterozygosity, there was no link between altitude and genetic diversity indices (p > .05), however, there were substantial relationships between ambient temperature, latitude, and diversity indices (p < .05).According to Table 3.4Nm) patterns. According to the FST technique, there were 0 to 62.52 migrants on average per generation between all populations. There were asymmetric patterns of gene flow between the DQ and XGLL populations and the XGLL and LJ populations, with the Nm between the DQ and XGLL populations having the highest mean of 62.92. The next Nm was from the XGLL population to the LJ population. Furthermore, there were no Nm between the JC and ALS populations as well as the LJ and JC populations. Additionally, the maximum likelihood tree of Nm between five populations was constructed using Treemix  over time were evaluated with the PSMC model for each five populations  ranged from 0.019 to 0.188  in this study. Moreover, the heat map of the pairwise FST showed that JC population and ALS population have high genetic differentiation with the other three populations, and there were medium score genetic differentiation between the remainder populations  and temperature distance  = 0.766; PFST/(1−ST) < 0.01, Figure rFST = 0.618; PFST > 0.05; Mantel rFST/(1−FST) = 0.627; PFST/(1−FST) > 0.05, Figure rFST = 0.182; PFST > 0.05; Mantel rFST/(1−FST) = 0.166; PFST/(1−FST) > 0.05, Figure rFst = −0.528; PFST > 0.05; Mantel rFst/(1−FST) = −0.520; PFST/(1−FST) > 0.05, Figure rFST = −0.443; PFST > 0.05; Mantel rFST/(1−FST) = −0.442; PFST/(1−FST) > 0.05, Figure FST and FST/(1 − FST). Moreover, RDA analysis showed that there was a highest contribution of temperature distance on genetic diversity . From the two‐way clustering heat map of PST/FST value, several interesting findings have emerged. First, most of pairwise PST of phenotypic traits were higher than the pairwise FST , based on the assumption that there is no environmental variance. The graphs in Figure PST–FST ratio would take for different values of h2. The majority of PST values were greater than the pairwise FST value, even though the pairwise FST value was at its minimum when the h2 was assumed to be one. However, it is well understood that the h2 cannot be one, and must be less than one. With our original assumptions, we concluded that most traits are the consequence of natural selection. Except for a few exceptions, the only conditions under which PST would be much lower than FST are if environmental variance is close to zero, and the critical value of c when the h2 is one is shown in Table No data were available to estimate the genetic variances of traits in this study due to the fact that the animals in this study are wild, but we can determine the effect on 5E. miletus in the HM region and used population genomic techniques to provide insights on its differentiation, adaptation, and history. In conclusion, our data show that E. miletus from the HM region exhibits phenotypic and genetic alterations related to naturally occurring diverse environments. It is interesting to note that there are differences between phenotypic clusters and genetic change patterns. Furthermore, phenotypic and genetic changes are linked to environmental factors, such as latitude, altitude, and average annual temperature, and phenotypic traits are more influenced by environmental factors; however, it is still unknown whether other environmental factors may also have an impact on phenotypic and genetic changes. Additionally, the significant biological stratification brought on by the tectonic uplift of the HM region during the late Pliocene results in spectacular topography, which has an impact on the asymmetric gene flow patterns found in E. miletus. Five E. miletus populations demonstrate an isolation‐by‐island model, which is supported by gene flow and a link between FST and geographic distance. Last but not least, PST estimates for the majority of wild traits are higher than differentiation at neutral molecular markers, indicating that directional natural selection favoring various phenotypes in various populations was likely involved in achieving this much divergence. Our findings provide as a foundation for studies on other HM region wild small animals.In this study, we investigated the widely dispersed Yue Ren: Data curation (lead); formal analysis (lead); investigation ; validation (lead); visualization (lead); writing – original draft (lead); writing – review and editing (lead). Ting Jia: Resources . Yanfei Cai: Investigation ; resources . Lin Zhang: Resources ; software . Hao Zhang: Investigation ; resources . Zhengkun Wang: Conceptualization ; supervision . Wanlong Zhu: Conceptualization (lead); data curation ; funding acquisition (lead); supervision ; writing – review and editing .The Yunnan Ten Thousand Talents Plan Young & Elite Talents Project (YNWR‐QNRC‐2019‐047), National Natural Scientific Foundation of China (Grant No. 32160254), National Natural Scientific Foundation of China (Grant No. 31760118), and Yunnan Provincial Middle‐Young Academic and Technical Leader (2019HB013) candidate provided financial and Excellent Doctoral Award of Shanxi Province for Scientific Research (SXBYKY2022123) support for this work.The authors declare no competing financial interests.https://doi.org/10.5061/dryad.kkwh70s8b.This article has earned an Open Data badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at Data S1Click here for additional data file.


Understanding genetic diversity is a core issue in conservation genetics. However, previous genetic diversity evaluations of narrowly distributed species have rarely used closely related widespread species as a reference. Furthermore, identifying natural hybridization signals between narrowly and widely distributed sympatric species is of great importance for the development of species conservation programs.Geodorum eulophioides (endemic and endangered in Southwest China), and a widespread species, G. densiflorum. A total of 18,490 high-quality single nucleotide polymorphisms (SNPs) were identified at the whole-genome level.In this study, population genotyping by sequencing (GBS) was performed for a narrowly distributed species, G. eulophioides were significantly higher than those of G. densiflorum, confirming that narrowly distributed species can still preserve high genetic diversity. Consistent with taxonomic boundaries, all sampled individuals from the two species were divided into two genetic clusters and showed high genetic differentiation between species. However, in a sympatric population, a few G. eulophioides individuals were detected with genetic components from G. densiflorum, suggesting potential interspecific natural hybridization. This hypothesis was supported by Treemix analysis and hand-hybridization trials. Invasion of the habitat of G. eulophioides invasion by G. densiflorum under anthropogenic disturbance may be the main factor causing interspecific hybridization.The results showed that the nucleotide diversity and heterozygosity of G. eulophioides populations. This study provides valuable information for future conservation programs for narrowly distributed species.Therefore, reducing or avoiding habitat disturbance is a key measure to protect the The online version contains supplementary material available at 10.1186/s12870-023-04285-w. Geodorum: a narrowly distributed species and a widespread species.Genetic diversity is an important component of biodiversity and is the sum of all genetic information carried by different individuals within a species or a population . UnderstGeodorum is a ground-growing type of herb in the Orchidaceae family with medicinal and ornamental values, comprising approximately ten species [Geodorum eulophioides is an orchid endemic to Southwest China. The species was first discovered and named by a German plant taxonomist Schlechter in 1921 in Luodian, Guizhou province. However, this species has not been found in the wild for more than 80 years. In 2004, G. eulophioides was rediscovered in Yachang, Guangxi Zhuang Autonomous Region. Due to the extremely narrow range of this species in the wild, G. eulophioides has been classified as endangered on the Threatened Species List of China’s Higher Plants [G. eulophioides is only narrowly distributed in parts of Guangxi, Guizhou, and Yunnan provinces in China. This species prefers shade and grows primarily in canyons, broad-leaved forests, and thickets at about 600 m elevation. The number of extant plants of G. eulophioides is very low. The total number of individual plants found in the Yachang Orchid Nature Reserve in Guangxi does not exceed 400, while several field populations with a small population distribution of 104 plants in total have been found in Guizhou [Geodorum densiflorum is a typical widespread species with distribution in Guangxi, Guangdong, Taiwan, Hainan, and other provinces in China, as well as India, Vietnam, Malaysia, Japan, and other countries in Asia. It is sun-loving and grows primarily in sparse forests, streamsides, and grassy slopes below 1500 m elevation. The flower structure and color of G. densiflorum differ markedly from those of G. eulophioides  improved population fitness via hybridization. Nevertheless, natural hybridization between widespread and narrowly distributed species may result in some negative effects. For example, hybridization may cause genetic and demographic swamping and accelerate the extinction of narrowly distributed species [Melastoma penicillatum) and a widespread species (M. candidum), based on a few low-copy nuclear genes and chloroplast DNA intergenic spacers. They hypothesized that the result may be detrimental to the conservation of the narrowly distributed species. In fact, a precedent for natural hybridization has been found in orchids [G. densiflorum and G. eulophioides species through genomic evidence is of great important for the development of conservation programs.Natural hybridization between species may contribute to increasing a population’s genetic diversity, promoting environmental adaptability, and potentially creating new species . Based o species . Zhou et species  identifi orchids . TherefoGeodorum, a previous study used SNPs generated by double digest restriction site-associated DNA sequencing (RAD-seq) data to assess the genetic diversity of G. densiflorum [G. eulophioides. To date, genome-wide SNP data have not been previously used to assess the genetic diversity and population structure of G. eulophioides.Single nucleotide polymorphisms (SNPs) are the most abundant variant form in the genome and are highly stable and easily detected . SNPs arsiflorum . In contsiflorum  to assesG. eulophioides and the widespread G. densiflorum, (2) to investigate the genetic differentiation and population structure between the two species, and (3) to detect the possibility of natural hybridization between the two species, and to provide meaningful conservation strategies and suggestions for narrowly distributed species.The main objectives of this study were to use genome-wide SNP data (1) to compare the levels of genetic diversity between the narrowly distributed G. eulophioides individuals from five populations and 52 G. densiflorum individuals from six populations, as well as three Eulophia graminea individuals, using GBS  was somewhat higher than that of G. densiflorum (45.89%). A total of 4,911,676 raw SNPs were called using Stacks’s de novo pipeline. After linkage disequilibrium (LD) pruning and filtering, 18,490 high-quality SNPs were obtained for further downstream analysis.After filtering, a total of about 334 G clean bases were obtained in 38 GBS Fig. . The aveGBS Fig. . The aveG. eulophioides and G. densiflorum. At the species level, the expected heterozygosity (HE) (0.0247) and observed heterozygosity (HO) (0.0117) of G. densiflorum were both significantly lower than that of G. eulophioides  (Table G. eulophioides (0.1580) was about six times higher than that of G. densiflorum (0.0250). These results may be related to the higher number of private alleles in G. eulophioides. Similar results were observed at the population level, with the highest π in G. eulophioides in the GE-DS population (0.2063) and G. densiflorum in the GD-ZX population (0.0262). At the individual level, we found that the heterozygosity of two individuals  was significantly higher than that of other individuals  while, in comparison, only one G. densiflorum population showed a negative FIS value. In addition, Tajima’s D values were positive for all populations except for the GE-DS population  0.047 and obG. eulophioides and G. densiflorum were divided into two different clades, while GE-DS-6 and GE-DS-2 in G. eulophioides were the two individuals most closely related to G. densiflorum  in the GE-DS population of G. eulophioides, suggesting the possible presence of interspecific gene flow.The maximum likelihood (ML) phylogenetic tree of the individuals showed that FST) (0.061) between the G. eulophioides populations was lower than that of G. densiflorum (0.263)  within the populations of G. eulophioides and G. densiflorum were 4.064 and 1.695, respectively   Fig. a. CorresFST) 0.06 between florum 0.63  distributed in India and between three other orchids , suggesting that the genetic diversity of G. densiflorum may generally be at a low to moderate level. In G. eulophioides, Ying [HE and HO values of 0.6968 and 0.7797, respectively. These values were higher than in this study based on SNP data . Due to the differences in the estimation of genetic diversity using different molecular markers, it was difficult to make substantial comparisons across studies. However, many previous studies have shown that the use of only a few SSR markers may overestimate the heterozygosity of the population, compared to the use of large-scale genome-wide SNP loci [G. eulophioides, was significantly higher than that of the widespread species, G. densiflorum.For biallelic SNP markers, nucleotide diversity (π) is an overall indicator of population genetic diversity, with higher π values indicating higher genetic diversity , 32. They et al. . The resy et al.  study wees, Ying  used 18 SNP loci , 34. UsiG. eulophioides and G. densiflorum populations were positive  was detected in all G. eulophioides populations  in the DS population were more likely to be the result of interspecific hybridization [G. densiflorum to the GE-DS population was observed. The Admixture results could also be due to other factors, such as ancient gene flows [G. densiflorum. If the habitat preference of G. densiflorum and G. eulophioides is followed, they rarely have the opportunity to have overlapping habitats. Anthropogenic habitat disturbance may be an important factor for the occurrence of actual sympatric distribution of the two orchid species. We hypothesized that during the initial natural forest-dominated period of the DS population, which was a more suitable habitat for G. eulophioides, and during the subsequent plantation period, G. densiflorum gradually invaded the G. eulophioides habitat, and during their sympatric period, natural hybridization between the two species may have occurred, resulting in interspecific gene flow. However, in the other two sympatric populations (ZX and XY), no evidence of interspecific gene flow was detected  and the other is external ecological factors  , 49. Bot changes , 51. Alt changes . There a species . With thG. eulophioides and the widespread species G. densiflorum were investigated using 18,490 SNP markers. The results showed that the genetic diversity of G. eulophioides was higher and this species showed significant genetic differentiation from G. densiflorum. However, interspecific gene flow was detected in a sympatric population (DS) from G. densiflorum to G. eulophioides, which may natural hybridization signal. Invasion of the G. eulophioides habitat by G. densiflorum under anthropogenic disturbance may be the main factor causing interspecific hybridization. Therefore, reducing or avoiding habitat disturbance is a key measure to protect the G. eulophioides populations. This study provides valuable information for future conservation programs for the endangered orchid.In this study, the genetic variation and differentiation between the narrowly distributed species G. eulophioides individuals from five populations and 52 G. densiflorum individuals from six populations . The final pooled libraries were sequenced on an Illumina Novaseq 6000 platform with a paired-end 150-bp read length. Quality assessment of reads was performed with FastQC [ustacks, cstacks, sstacks, tsv2bam, and gstacks modules, for SNP calling. Next, LD pruning of the raw SNP dataset was performed using populations module of Stacks with the parameter “-write-random-snp”. The LD-pruned SNPs were then filtered using VCFtools v0.1.16 [Genomic DNA of 93 samples was extracted using the E.Z.N.A. Tissue DNA kit . The quantified DNA  was used for ddGBS library construction using the h FastQC . Adapterh FastQC . We usedh FastQC , includi v0.1.16  using thpopulations module of Stacks was used to calculate the π, HO, HE, and FIS. The Tajima’s D values of each population were estimated using VCFtools, with a sliding window size of 3000 bp. Since the calculation of Tajima’s D value requires at least four individuals, the GD-EG population was excluded.The E. graminea individuals were used as outgroups. The phylogenetic tree was visualized using the Phylogenetic tree view function of ImageGP [K) were set from 1 to 15. The optimal K value was determined based on the minimum CV error.A phylogenetic tree was constructed using the ML method in IQtree v2.0 , using t ImageGP . The Pli ImageGP  was used ImageGP  and the FST between populations was calculated using VCFtools. Based on the geographic coordinate information of the population, the actual geographic distance was calculated using the R package geosphere. The Mantel test was then performed using the R package vegan for FST and geographical distance with Spearman’s correlation coefficient and 9999 permutations to test significance. The interpopulation Nm was calculated according to the equation: E. graminea as an outgroup, and the “-noss -k 500” parameters were added to prevent over-correction of sample sizes, and reconstruct the ML tree by resampling blocks of 500 SNPs. Residuals were used to choose the best-fit migration model. According to the Treemix recommendation [The ix v1.12 . The assendation , when thG. eulophioides and G. densiflorum, we randomly selected five G. eulophioides and five G. densiflorum individuals in the wild to conduct hand-hybridization trials. Flowers that were about to bloom were sequentially emasculated, bagged, and pollinated according to the method described by Dafni [G. eulophioides and ten G. densiflorum individuals were marked, and their fruit set under natural conditions was recorded as controls. The fruit-set rate was recorded three months after the trial was conducted. Since an individual may produce more than one flower, the fruit set was counted by the number of flowers.To verify the possibility of hybridization between by Dafni . Eight wAdditional file 1: Fig. S1. The corresponding residuals when using all individuals to simulate 0–5 migration events (m). SE, standard errors.Fig. S2. The corresponding residuals when using individuals after removing admixtures to simulate 0–5 migration events (m). SE, standard errors. Table S1. Sampling and sequencing information.


Identifying DNA markers such as Short Tandem Repeats (STR) can be used to investigate genetic diversity based on levels of heterozygosity within and between populations. Allele frequencies and forensic data for STRs were obtained from a sample of 384 unrelated individuals living in Bahia, Northeastern Brazil. Thus, the present study aimed to identify the allele frequency distribution, in addition to the forensic and genetic data, of 25 STR loci in the population of Bahia. Buccal swabs or fingertip punctures were utilized to amplify and detect 25 DNA markers. The most polymorphic loci were SE33 (43), D21S11, and FGA (21). The least polymorphic were TH01 (6), TPOX, and D3S1358 (7). Forensic and statistical data were obtained through data analysis, which revealed a large genetic diversity, with an average value of 0.813 for the analyzed population. The present study was more robust than previous STR marker studies and will contribute to future research on population genetics in Brazil and worldwide. The results of this study allowed the establishment of haplotypes found in the forensic samples of Bahia State to serve as a reference in the elucidation of criminal cases and paternity tests, as well as population and evolutionary investigations. Advances in molecular biology have provided powerful tools for reconstructing genetic history. DNA contains valuable information that includes sequences from the evolutionary past that can be extracted from any type of biological material ,2. HumanThe identification of DNA markers such as short tandem repeats (STR) or microsatellites, which constitute highly variable genetic loci, has become the test of choice for genetic linkage analysis . The appMoreover, the identification of polymorphisms is of great importance for the reconstruction of historical human migrations . Since ip-value) [The use of bioinformatics tools provided statistical data with regard to population genetics through forensic parameters such as genetic diversity (GD), polymorphic information content (PIC), heterozygosity (H), and the probability of the Hardy-Weinberg equilibrium  ,5,7. TheForensic analyses and studies of the distribution of allele frequencies, GD, and PIC in heterozygous populations are necessary to create databases for reference populations and to obtain information on the genetics of the population under study. To determine the genetic diversity and gene flow in this population, the present study determined the distribution of allele frequencies in addition to forensic and genetic data from 25 STR loci in the Bahia population.® cards  according to the manufacturer’s protocol [A total of 384 samples from unrelated individuals living in Bahia, northeastern Brazil, were obtained as secondary data from paternity cases performed between 2016 and 2017 at the Laboratório de Investigação de Vínculo Genético do Centro de Diagnóstico do Grupo de Apoio à Criança com Câncer (CDG). Patients had previously given consent to the paternity tests. DNA was extracted from buccal swabs or directly from fingertip puncture using FTADNA extract (5 µL) was used for amplification of the 25 STRs examined in this study: . Electrophoresis and genotyping were performed using an ABI PRISM 3500 Genetic Analyzer . Data collection was performed using ABI PRISM 3500-Data Collection v1.0 software (Applied Biosystems), and for profile analysis we used GeneMapper ID -X v1.2 software (Applied Biosystems).p-value).Samples were calculated to obtain statistical data and forensic parameters from 384 individuals from the state of Bahia. After obtaining the allele profiles, the allele frequencies of the 25 markers were determined. The allele frequencies were determined using the relative frequency method and the number of allele repeats observed in the samples. The data were analyzed using GenAlEx version 6.5 software  to calcuThe allele frequencies for the 25 STR loci studied in Bahia’s population are presented in For each analyzed locus, p-values obtained ranged from below 0.001 (PENTA D) to 0.9998 (FGA). No deviations from the Hardy-Weinberg equilibrium were observed. The high values obtained for these forensic parameters in the present study confirms the high genetic variability of Bahia’s population.The forensic parameters obtained for the 25 loci are presented in This is the first study in Brazil reporting on 25 markers that were analyzed to evaluate the genetic variability of a specific human population. The allelic frequencies were compared to other regions of Brazil. No relevant differences were observed when comparing the frequencies of other studies performed in Rio Grande do Norte, Paraíba, Pernambuco, Santa Catarina, Mato Grosso do Sul, Rio Grande do Sul, Rio de Janeiro, Amazonas, or Paraná, with one other Brazilian study (Brazil) ,18,19,20Among observed studies, the most polymorphic loci were D2S1338, D18S51, D21S11, PENTA E, FGA, and SE33. In all studies, the least polymorphic locus was TPOX ,18,19,20The least polymorphic loci in the study with the Bahia population were TH01 and TPOX, similar to the other studies ,18,19,20We suggest that the movement of individuals from one population to another (immigration and emigration) implies the identified gene flow. In Northeastern Brazil, migrants reproduced and, via their genes, contributed to the genetic total of the receiver population , with thFew studies have contemplated the 25 markers presented in this study in Northeastern Brazil. When we compared the most frequent alleles for each locus of a study of the Brazilian population and that of Bahia, we noticed that among 13 loci analyzed , six presented similarities . In another study, we noticed that of 21 loci studied , 17 presented similarities . According to Moyses et al. [In addition to allelic analysis, the present study provides statistical and forensic data for the 25 analyzed STR loci in Bahia’s population. Other Brazilian studies realized in this category present an average of 8 to 13 analyzed loci ,18,19,20The analysis of Genetic Diversity (GD) values ranged from 0.7316 (TPOX) to 0.9339 (SE33) . These hThe expected heterozygosity (He) was higher than or equal to the observed heterozygosity (Ho) in 14 of the 25 loci analyzed . The highest discrepancy between parameters was 0.046 (TPOX). He values ranged from 0.731 (TPOX) to 0.934 (SE33), while Ho values ranged from 0.685 (TPOX) to 0.971 (SE33). The higher Ho as compared to He in the other 11 loci  is indicative of external breeding [Previous small-scale forensic studies in other northeastern states, such as Rio Grande do Norte, Paraíba, and Pernambuco have shown similar values for the discrepancy between observed and expected heterozygosity ,11,12. DHowever, when comparing studies in other states such as Santa Catarina, Rio Grande do Sul, Rio de Janeiro, Mato Grosso do Sul, and Amazonas, it is possible to identify a preliminary average discrepancy between Ho and He which is lower than 0.05 for the Brazilian population ,15,16,17The same type of study done on a small scale with other populations and regions worldwide shows that populations with a history of miscegenation, such as Brazil, Africa, and northern Europe, present substantial genetic diversity and low discrepancies between He and Ho. Populations with low miscegenation present low heterozygosity ,24. A stThe values for PIC obtained in this study were higher than 0.6 for every locus analyzed, ranging from 0.6905 (TPOX) to 0.93 (SE33), which means that each of the 25 loci analyzed were highly polymorphic, and would contribute to the genetic variation of the analyzed population. Previous studies in other populations realized with PIC have shown similar results within some of the analyzed loci: TPOX, CSF1PO, PENTA E, FGA, and TH01 ,12,13. DValues for probability of identity (PI) within the studied loci ranged from 0.008 (SE33) to 0.113 (TH01). TH01, DS2441, TPOX, and CSF1PO presented the highest PI values in our analysis, ranging from 0.1046 (CSF1PO) to 0.113 (TH01). Previously available studies on TH01, TPOX, and CSF1PO in northeastern Brazil have shown similarities in values for PI in these same loci . The PI For the analyzed group, the PE ranged from 0.771 (TH01) to 0.989 (SE33) for the loci studied. The PE values were higher than 0.7 for every locus, confirming a great genetic variability in the genetic profile of Bahia’s population. Previous studies undertaken in northeastern Brazil with PE analysis demonstrate only three loci in common with the present study  ,11,12. Tp-value obtained in this study ranged from below 0.001 (PENTA D) to 0.9998 (FGA). A total of 18 of the 25 analyzed loci  presented non-significant p-values (p > 0.05). Seven of them  presented significant p-values (p < 0.05). Thus, the vast majority of the loci in the analyzed population either reached or were likely to reach the Hardy-Weinberg equilibrium [The p-values approximating p > 0.05, except for the TPOX locus, which presented a p-value of < 0.05 in Bahia’s population. This scenario reveals the genetic proximity of the studied populations, which can be explained by their geography and history.Similar results for the CSF1PO, TH01, TPOX, VWA31, D16S539, D13S317, D21S11, D8S1179, and FGA loci have been found in studies conducted in the Rio Grande do Norte, Paraíba, and Pernambuco ,11,12. TWhen compared to other populations, an analysis of the data confirms that Bahia’s population is genetically diverse. Furthermore, the present study provides statistical forensic data that may help guide future research on STR markers.When comparing the allele frequency of the 25 STR markers in Bahia’s population with other studies done in different populations in northeastern Brazil, no significant differences were found, revealing great similarities between populations. When forensically analyzed, Bahia’s population reveals great genetic variety, a sign of significant miscegenation in its genetic formation. The allelic frequencies of the studied population revealed contributions from Native Americans, Africans, and the Portuguese. The results also show that studies in specific populations are more likely to produce reliable results. To better understand the genetic diversity of this particular Brazilian population, more studies on the populations that contributed to its formation are needed.Finally, the present study provided forensic and statistical data that may help guide future research on STR markers and forensic studies in miscegenated populations, such as that of Brazil.