Ostrea edulis, recently begun and recommendations to preserve genetic diversity and to conduct monitoring protocols have been made. In particular, an initial step is to test for genetic differentiation against homogeneity among the oyster populations potentially involved in such programs. Therefore, we conducted a new sampling of wild populations at the European scale and a new genetic analysis with 203 markers to (1) confirm and study in more detail the pattern of genetic differentiation between Atlantic and Mediterranean populations, (2) identify potential translocations that could be due to aquaculture practices and (3) investigate the populations at the fringe of the geographical range, since they seemed related despite their geographic distance. Such information should be useful to enlighten the choice of the animals to be translocated or reproduced in hatcheries for further restocking. After the confirmation of the general geographical pattern of genetic structure and the identification of one potential case of aquaculture transfer at a large scale, we were able to detect genomic islands of differentiation mainly in the form of two groups of linked markers, which could indicate the presence of polymorphic chromosomal rearrangements. Furthermore, we observed a tendency for these two islands and the most differentiated loci to show a parallel pattern of differentiation, grouping the North Sea populations with the Eastern Mediterranean and Black Sea populations, against geography. We discussed the hypothesis that this genetic parallelism could be the sign of a shared evolutionary history of the two groups of populations despite them being at the border of the distribution nowadays.Although all marine ecosystems have experienced global‐scale losses, oyster reefs have shown the greatest. Therefore, substantial efforts have been dedicated to restoration of such ecosystems during the last two decades. In Europe, several pilot projects for the restoration of the native European flat oyster,  Although millions of oysters were regularly moved within and between countries over the last 200 years, such translocations mainly failed  whose members made several recommendations for the settlement of a European flat oyster restoration program  from Launey et al.  according to the manufacturer’s recommendations. Quality and concentration were assessed on a 1% agarose gel and by using a NanoDrop Spectrophotometer ND‐2000 (Thermo Scientific). Concentrations were equilibrated to 100 ng/μl per DNA sample.O. edulis transcriptome sequence data from eight individuals from the natural range. SNPs that were detected by sequencing a subset of individuals for candidate genes will be referred to as in vitro SNPs. Those that were detected by aligning different sequences from contig databases will be referred to as in silico SNPs. Fifty‐two in vitro SNPs were included in the array, representing 35 different gene fragments. A total of 1305 in silico SNPs fitted the proposed criteria for Illumina genotyping. Therefore, we chose the 332 in silico SNPs with the highest Illumina Functionality Score (>0.935) to complete the array.A set of 384 SNP markers was specifically developed on the European flat oyster  according to the manufacturer’s recommendations, at the Genotoul platform (Genopole Toulouse Midi Pyrénées). The clustering was realized with the GenomeStudio software (Illumina Inc.). Reliability of SNP detection and coding were the same as what is reported in Lapègue et al. . In partCrassostrea gigas  in an attempt to map the loci corresponding to our 203 SNPs. The GFF file of the genome was used to functionally annotate those loci.We used the genome assembly of 2.3https://brcwebportal.cos.ncsu.edu/zaykin/rxc  was visualized as a raincloud plot using the R code available at https://github.com/RainCloudPlots/RainCloudPlots/tree/master/tutorial_R.We obtained the estimation of linkage disequilibrium (LD) through a correlation‐based test for loci with multiple alleles using the MCLD program available at r2 value obtained were then submitted to the Tuckey’s fence method for outlier detection. This method considers as outlier a value that is higher than Q3 + k(IQR), where IQR = Q3–Q1 . Here, we set k = 3, to obtain only the strong outlier values. Loci showing r2 value being outlier in at least two pairwise comparisons were retained. Pairwise r2 values were then ordered according to the position of loci on the genetic map of O. edulis  and expected (Hnb) heterozygosity for each population. Within‐ and between‐population components of genetic diversity were decomposed using a principal component analysis (PCA), using dudi.pca function of the R package Ade4.Genetix  varying from 1 to 10, with 10 runs for each K value, to find the K value with the highest posterior probabilities and used the ΔK statistics to evaluate the change in likelihood  was performed following Excoffier et al.  with them (number of migration edges) ranging from 0 to 3.Evolutionary history of the genetic clusters was then investigated under a model of divergence and admixture events using the population graph approach implemented in Treemix  gave reliable data. Among them, 203 SNPs (52.9%) were polymorphic and used for further analyses. The high rate of genotyping failure falls within the range of values seen in other nonmodel species but are very different from those obtained in model species  between pairs of markers , showing that synteny was likely conserved for those chromosomal regions between the two species.We successfully managed to locate 50 out of our 203 loci on the me Table . Interess Figure  were alsIn order to avoid any bias due to the large number of linked loci, only one marker of each of the six LD‐groups  also gave the same result with 8.7% of the variance explained between clusters and only a small part of the variance (1.8%), still significantly different from zero, explained between populations of the clusters Table . HoweverThe population tree inferred using TREEMIX without accounting for migration Figure  is consi3.4A total of 28 SNPs was identified by both BAYESCAN and PCAdapt and 17 of those SNPs were identified in pairwise comparisons (8 between NS and Atl and 9 between MedE and MedW). If we conservatively focus on the 28 SNPs identified with the two methods, they encompassed 14 of the 15 SNPs of LDG_182 , 7 of the 9 SNPs of LDG_202 , and only SNP192 of LDG_192. Those markers were indicated in bold on Figure C. gigas genome. Functional annotation of these 13 SNPs was performed using the GFF file given alongside the genome. These SNPs are mostly located in exonic sequences of genes involved in pre‐ and posttranscriptional control and modification of RNA products, homeostasis and ER stress alleviation, and respiration . This illustrates the lineage effect of PCA axis 2. Hence populations at the edge of the distribution in the North Sea and the Eastern Mediterranean Sea have similar allele frequencies, against geography  translocation between France and Italy, particularly in the lagoon of Venice. Consequently, it would be possible that European flat oysters translocated at the same time as cupped oysters. This example illustrates the difficulty to disentangle long‐term evolutionary history and human activities as factors influencing the population genetic structure of species of interest for aquaculture , it might be of importance to further investigate the genetic status of the populations in this area.The limit between the North Sea and Atlantic clusters is likely to take place in the English Channel, maybe at the straights of Dover which coincide with a biogeographic boundary between the English Channel and the North Sea. Such a pattern has already been identified in several species, such as the common goby  and Black Sea (Crimea). In the literature, two potential biogeographical barriers are discussed. The most western is located at the Siculo‐Tunisian Strait and is particularly involved in population structure of the sea bass  and Eastern Mediterranean Sea and Black Sea (MedE)  and that of the MedE lineage with the West Mediterranean lineage (MedW) would be explained by secondary gene flow that has gradually eroded the ancestral differentiation. SNPs showing genetic parallelism between border populations would have resisted secondary intergradation and retained the genetic proximity of these clusters , so that synteny of those regions seems to have been conserved between the two species, although they do not belong to the same genus, putatively indicating the importance of the structural integrity of this region. Our study proposes hypotheses that need to be tested and deepened as we performed a preliminary indirect approach . Being able to detect human translocations due to aquaculture  might also be very useful in restoration programs.Although more work is needed to understand the origin and the factors that maintain the heterogeneous and contrasted patterns of genetic differentiation between the four genetic clusters, the identified mosaic of those clusters is genuine and they can be considered as Evolutionary Significant Units (ESUs). They should accordingly be managed with dedicated conservation strategies. Therefore, there is a need to create and conduct monitoring protocols as soon as the very beginning of a program (Bernatchez et al., Therefore, raising awareness of genetic considerations among managers of restoration programs should be continued, together with the transfer of easy to use and relatively cheap genetic molecular tools. Besides, a better characterization of the boundaries between the different genetic units in the European flat oyster needs to be conducted. This is particularly true in the English Channel and on the Atlantic coasts of Spain and Portugal where a sampling effort needs to be performed but is already somehow circumscribed. This is clearly more challenging in the Mediterranean and Black Seas where much more coasts still need to be covered. A more prospective issue could be the choice of the origin of the oysters for restocking in case one ESU becomes critically endangered. Should we choose the genetically or geographically closest population or should we take the one that potentially share barrier loci? For example, the Black Sea populations of flat oysters are declining dramatically (Todorova et al., The authors do not declare any conflict of interest.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Figure S4Click here for additional data file.Table S1Table S2Table S3Table S4Table S5Click here for additional data file.


Genomic prediction of breeding values is routinely performed in several livestock breeding programs around the world, but the size of the training populations and the genetic structure of populations evaluated have, in many instances, limited the increase in the accuracy of genomic estimated breeding values. Combining phenotypic, pedigree, and genomic data from genetically related populations can be a feasible strategy to overcome this limitation. However, the success of across-population genetic evaluations depends on the pedigree connectedness and genetic relationship among individuals from different populations. In this context, this study aimed to evaluate the genetic connectedness and population structure of Alpine and Saanen dairy goats from four countries involved in the European project SMARTER (SMAll RuminanTs Breeding for Efficiency and Resilience), including Canada, France, Italy, and Switzerland. These analyses are paramount for assessing the potential feasibility of an across-country genomic evaluation in dairy goats. Approximately, 9,855 genotyped individuals  and 6,435,189 animals included in the pedigree files were available across all four populations. The pedigree analyses indicated that the exchange of breeding animals was mainly unilateral with flows from France to the other three countries. Italy has also imported breeding animals from Switzerland. Principal component analyses (PCAs), genetic admixture analysis, and consistency of the gametic phase revealed that French and Italian populations are more genetically related than the other dairy goat population pairs. Canadian dairy goats showed the largest within-breed heterogeneity and genetic differences with the European populations. The genetic diversity and population connectedness between the studied populations indicated that an international genomic evaluation may be more feasible, especially for French and Italian goats. Further studies will investigate the accuracy of genomic breeding values when combining the datasets from these four populations. Genomic prediction of breeding values is routinely performed in several livestock species, including dairy and beef cattle, dairy sheep, and dairy goats . Genomicwww.eurogenomics.com/), which maintains a training population of ∼40,000 genotyped bulls and provides genomic estimated breeding values (GEBVs) for 11 countries. More recently, international genomic evaluations have also been implemented in beef cattle populations . Four countries  have shared 9,941 raw genotypes and pedigree information from Alpine and Saanen dairy goat populations. The animal identification (ID) was standardized in each country partner and was formed based on four components: three letters indicating the breed of the animal (ALP for Alpine and SAA for Saanen) + three letters indicating the country of origin  + one letter indicating the sex of the animal  + 16 characters with the animal identifier . For instance, the final identification of an Alpine female with local ID 5248383, born in France, and raised in Switzerland would be ALPCHEF0000000FR5248383. Imported animals may have multiple identifiers (one from the country of origin and another one in the importing country). Therefore, up to three aliases could be provided by the partners in addition to the ID of the animal. This identification is important to enable tracing the origin of the curated data but also useful for finding the connections between the different pedigrees.This study was carried out in the framework of the “practical selection tools to benefit from international harmonisation and cooperation” work package of the SMARTER project  were also present in version 2, only the SNPs from version 1 were considered for further analyses. Duplicated genotypes were filtered out based on the animal call rate, in which the genotype sample with a higher call rate was kept in the dataset. SNPs with a minor allele frequency (MAF) lower than 0.01 and a call rate lower than 0.90 were filtered out. Furthermore, animals with a sample call rate lower than 0.90 were also removed from the analyses (n = 86). Quality control was performed within the breed and country but also after merging the four datasets. The quality control analyses were performed by PLINK 1.9 software . There are currently two versions of this SNP chip panel, but 90% of the genotyping was performed based on the first version that contains 53,347 SNPs. Genotypes were exchanged in the TOP/BOT format and based on the ARS1 reference genome. As more than 90% of the genotyping was carried out based on the first version of the SNP chip panel and all SNPs included in the version 1 . In total, 12 comparisons were made to find all the connections. These analyses were performed using Python scripts prepared by the authors.https://pypi.org/project/fuzzywuzzy/) to find the matches between the two pedigrees. The verification of the proposed animal matches based on this approach was carried out manually. This approach enabled the identification of animals with typos at the time of registration.The standardization of animal identification facilitated the extraction of foreign animals present in the other pedigree files. Several strategies were developed to retrieve the pedigrees of these animals. The simplest approach was to compare the identifiers  of these animals with the source pedigree . This step was easily automated but not sufficient to find all the pedigree connections. For instance, considering the Swiss dairy goat pedigree, some French animals were registered in Switzerland with only the last digits of the French identifiers. For these animals, we used the fuzzy string-matching approaches , respectively, and pAB is the frequency of the haplotype AB, as proposed by 2 for each class. In the The extent of LD was calculated for each breed both within each country and also with merged datasets. This was determined based on the −rsoftware . The r2 2 statistic and then adding the sign of the D-value obtained with the dprime-signed option of the PLINK 1.9 software  was performed based on two methods: principal component analysis (PCA) and genetic admixture analysis. To comply with the data independence assumption for performing PCA, the genotypes were pruned using the default parameter of the option -indep implemented in the PLINK 1.9 software . A totalSince the majority of animal exchanges occurred between France and the other three countries, we have identified Canadian, Italian, and Swiss animals with French parents to estimate the importance of their descendants in the host country. 2 values stabilized around 0.10–1 Mb. The average LD for the Swiss and Italian populations was also very similar and stabilized around 0.07–1 Mb.The average LD calculated in Alpine (A) and Saanen (B), for each country separately and for multiple countries (ALP or SAA) or multiple breeds (All breeds) as a function of the SNP distance, is presented in For the Saanen breed, the range of LD values at 50 kb was wider than in the Alpine breed, with an average LD at 50 kb between 0.15  and 0.19 (Canada). Canadian populations had a higher LD than in the other countries, regardless of the distance between SNPs. For short distances, LD values for Canadian and Swiss populations were close  before differentiating for distances greater than 90 kb. The maximum difference was observed at 810 kb with an average LD of 0.09 in Swiss and 0.12 in Canadian goats.The consistency of the gametic phase according to nine classes of distances between SNPs is shown in The trends observed in Alpine were also found in the Saanen breed  but withThe breed composition for each animal calculated with the Admixture software is shown in The Canadian Saanen population seems to be largely different from the other Saanen groups. Indeed, the main ancestral cluster in Canadian Saanen . Here, some of the pedigree connections have been found, but there is still work to be carried out because some original pedigree of foreign animals is still untraceable. The importance and difficulty of exhaustive research of pedigrees have been discussed in previous studies, such as in beef cattle for Interbeef , dogs W, and racWe also have disproportional datasets with larger amounts of data in France in comparison to the other countries. This situation has also been reported in the framework of Interbeef for the Limousin cattle breed , in whicInbreeding estimated in our population was also consistent with previous estimation found in the literature. For example, in Canada, 2 metric, the bins used to group the SNPs are different. Between 10 and 100 kb, Population parameters such as LD and consistency of the gametic phase have implications for the design of across-population genomic evaluations. For a multi-population (here multi-country) genetic evaluation to be effective, there should be equivalent LD between SNPs and QTLs in each country and a relatively high consistency of gametic phases between populations from different countries . For theThe consistency of the gametic phase is a key parameter for determining the effectiveness of a multi-population genetic evaluation . This isThe consistency of the gametic phase in Alpine and Saanen breeds is similar within countries until the SNP distance of  with a decrease from about 0.92 to 0.62. After this distance, the decrease of the consistency of the gametic phase shows different trends with a higher level for Italian, a lower level for Canadian, and an intermediate level for Swiss and French populations. For French animals, these results are in accordance with those of The results of the PCA and admixture analyses contribute to determining the genetic relationship of the animals, including the breed and country of origin. The only populations with no clear distinction are French and Italian goat populations for both breeds. Italy is the country that imports most animals from France, which may explain the genetic proximity between these two populations. In contrast, the Canadian and European populations are more genetically distant. This might be explained by the little exchange of animals and the geographical distance that separates Canada and the European countries. Finally, these results are consistent with the results observed on the connections between countries based on pedigree information.Several genetic diversity studies have been conducted in goats. In France, the study of www.goatgenome.org/vargoats.html). This project sequenced goats of different breeds from all over the world including Alpine and Saanen from France, Italy, and Switzerland. They studied the phylogeny of these breeds using a neighbor-joining tree constructed with 100,000 SNPs. For the Italian, Swiss, and French populations, our results are in agreement with their study, where a close relationship between France and Italy (for both Alpine and Saanen) and a greater distance with the Swiss goat population were observed.The comparison of Alpine and Saanen genotypes within one country is well-documented more than international comparisons of these breeds. The first two components of the PCA represent less than 6% of the total variation, which is quite limited. Even if we observe two different clusters between European and Canadian populations, these populations seemed close enough in order to blend all genotypes and to analyze genotypes conjointly. Further investigations on differences along the genome between animals from different countries could be interesting to detect genomic regions specifically selected in each country.This work aimed to combine and analyze pedigree information and genomic data from four countries. Our analyses showed that an international evaluation would be most beneficial to the European populations that are genetically closer. However, it is necessary to verify the impact of Canadian data into international genomic evaluations, especially if other European dairy goat populations are added such as Yorkshire dairy goats . YorkshiThe genetic diversity and pedigree analyses performed in this study showed that the French and Italian populations are both the most genetically connected and more genomically similar. On the other hand, for the Swiss and Canadian dairy goat populations, the genetic connections are limited to the importation of a few French animals. Besides, they are genomically more distant than the other populations. The genetic diversity and population connectedness between the studied populations indicate that an international genomic evaluation might be more feasible for French and Italian goats. Further studies will investigate the accuracy of genomic breeding values when combining the datasets from these four populations.


Thunnus atlanticus, is a small tropical tuna exploited by recreational and commercial fisheries in various parts of its range. Information on stock structure is needed to develop management plans for this species but is currently lacking. In this work, 470 blackfin tuna from nine geographic populations were assayed at 13 homologous microsatellite markers to provide a first assessment of stock structure across the species range. The overall divergence among locality samples was very low  indicating high connectivity of blackfin tuna across their range. No clear grouping of localities in differentiated units was inferred but structuring followed a weak isolation by distance pattern . Pairwise exact tests and spatial analysis of molecular variance suggested divergence of the sample collected offshore Baía Formosa (Brazil) possibly reflecting reproductive isolation of Brazilian populations from those in the Caribbean region and further north. Further study of the status of Brazilian populations and the transition between this region and the Caribbean is warranted. Cryptic subdivision within the Northern Hemisphere part of the range is possible and should be evaluated using increased marker density and a more comprehensive geographic coverage.The blackfin tuna,  However, detecting subdivision and delineating units is often challenging in marine species due the lack of physical barriers to movement across wide geographic areas3 and the high dispersal potential of many species, as well as to the large sizes of marine populations, which leads to slow accumulation of genetic differences among units3. Pelagic species such as tunas combine life history characteristics that are likely to promote high connectivity. Most studies of tunas and other large pelagics have often shown weak or lack of structure across large geographic areas in some cases spanning entire oceanic basins6 highlighting the importance of studying connectivity at the scale of a species range to identify units when they exist.Understanding the structure and connectivity of populations is essential to design efficient conservation and management strategiesThunnus atlanticus, Lesson, 1830) is a small tuna found in tropical and subtropical waters of the Western Atlantic Ocean, ranging from the mid-Atlantic region of the United States east coast to northern Brazil8. Blackfin tuna are exploited commercially using purse seines, and various hook and line techniques  in Brazil and in several parts of the Caribbean Sea, including Cuba, the Dominican Republic, the Lesser Antilles, and Venezuela12. The species is not commercially fished in the U.S. However, yearly recreational landings have increased in the past few decades from an average of 87,295 specimens for the period 1981–1985 to 294,256 specimens for the period 2015–2019 .Blackfin Tuna . Interestingly, conventional tagging studies in the areas of St Vincent and the Grenadines and Bermuda islands revealed that tagged fish were recaptured in close proximity of the tagging location22 even when recaptures occurred after multiple years (up to more than 3 years in the study of Singh Renton and Renton22), but individuals tagged off Bermuda were recaptured during the summer months only, suggesting the occurrence of migrations to southern portions of the range during the colder months21. Hypotheses proposed by Lukhurst et al.21 to explain these results were that (i) blackfin tuna may observe seasonal migrations from feeding areas to spawning areas and would show fidelity to specific feeding and spawning grounds, respectively, and (ii) some populations of blackfin tuna may tend to be more sedentary than others, as observed in other tunas23. In this context, the analysis of genetic stock structure in combination with approaches to study movement such as physical tagging and otolith microchemistry, is essential to reveal occurrences of differentiated genetics stocks and their habitat use, and to avoid overexploitation and depletion of some of them24.Despite the commercial and recreational exploitation of blackfin tuna stocks, information on population biology and stock structure is still very limited and insufficient to develop reliable assessment and management of this species. Blackfin tunas, like other tunas, have a strong potential for dispersal at all life stages. They can disperse by pelagic passive transport of eggs and larvae after spawning25 who surveyed mitochondrial DNA and 6 heterologous microsatellites in the Gulf of Mexico and northwest Atlantic regions. The study revealed significant albeit low divergence between the two populations. The present study employs 13 of the microsatellite markers developed by Antoni et al.26 to investigate the genetic variation in blackfin tuna across 9 localities spanning most of the species range. The dataset was used to assess whether blackfin tuna are subdivided within their range and to test whether genetic heterogeneity reflected discrete units potentially separated by barriers or a continuous model driven by isolation by distance. The occurrence of cryptic structure was also assessed by testing temporal stability of spatial patterns and clustering approaches.To date, information on the population genetic structure and connectivity of blackfin tuna is limited to one study by SaxtonTissue samples were obtained from a total of 470 blackfin tuna from nine offshore localities throughout the western Atlantic Ocean Fig. , Table 1Tissue samples were collected postmortem from fish carcasses during fisheries-dependent sampling. Tissue collections in La Martinique was conducted by IFREMER scientists during sampling activities part of the MAGDELESA project and authorized in the context of that project. The data used in this research, from Brazil, were obtained with the approval of the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) of the Brazilian Ministry of the Environment (permit no. 53702) to Dr. Fabio Hazin. Tissue sample collections in US and Venezuelan waters were conducted on carcasses of fish of legal size caught during commercial fishing or recreational tournaments.https://aquila.usm.edu/datasets/6/).Sampling targeted adult specimens of reproductive size (over 50 cm Fork Length) that were captured by hook and line. Samples were obtained in two different years for four of the localities. Capture coordinates were not known for most samples, which were obtained from recreational or charter boats at landing. Capture was assumed to have occurred within a 100 miles radius of the landing port in those cases. The sampling was not stratified. Collection information specific to each individual sample is available along with genetic data in the University of Southern Mississippi public repository Aquila (2O) and returned to USM for analysis.Tissue samples, primarily fin clips, were stored in 95% ethanol or in DMSO salt-saturated fixative , and 1X of reaction buffer. The amplification cycle consisted in an initial denaturation at 95 °C for 5 min, 35 cycles at 95 °C for 30 s, annealing temperature for 30 s, 72 °C for 45 s, and a final extension at 72 °C for 10 min. The composition of each multiplex panel and annealing temperatures used during amplification are available on the Aquila public repository (https://aquila.usm.edu/datasets/6/). Microsatellite amplification products were electrophoresed using an ABI 377 automated sequencer , following the manufacturer’s instructions. Resulting chromatograms were analyzed in GENESCAN v. 3.1.2 (Applied Biosystems) and alleles were scored using GENOTYPER v. 2.5 (Applied Biosystems).All samples were assayed at 13 of the microsatellite loci  developed by Antoni et al.A), allelic richness (AR), expected heterozygosity (HE) (unbiased gene diversity), and the inbreeding coefficient FIS (measured as Weir and Cockerham27f) were generated for each sample locality and locus, using FSTAT v. 2.9.3.2 (available at http://www2.unil.ch/popgen/softwares/fstat.htm)28.Summary statistics for microsatellite data, including number of alleles  expectations were carried out for each microsatellite, using an exact probability test as implemented in GENEPOP v. 4.332 accounting for variation among localities and among years within locality was conducted in ARLEQUIN v. 3.533. Samples were aggregated by sampling year within locality for this analysis. Significance of molecular variance components  were assessed using 10,000 permutations of haplotypes.Analysis of molecular varianceST) was estimated as Weir and Cockerham’s27θ, as implemented in FSTAT v. 2.9.3.228. Homogeneity among samples (the probability that θ = 0) was tested using exact homogeneity tests computed in GENEPOP and using the same Monte Carlo Markov Chain as above. The Benjamini–Hochberg34 correction was applied for all multiple tests performed simultaneously.The degree of divergence among samples (Fixation index Fhttp://pritch.bsd.uchicago.edu/structure.html)36. Multiple Monte Carlo searches were run varying the number of putative clusters (K) from 2 to 9 in order to identify the most likely value for K. The correlated allele frequency setting was used in computations along with information on collection location used as prior to assist during clustering. The latter option improves recovery of structure within the data when genetic divergence among populations is weak37. Monte Carlo searches were run for 1.1 108 steps with the first 107 steps discarded as burnin.Occurrence of cryptic structure within the dataset was further explored using Bayesian clustering implemented in STRUCTURE v. 2.3.4 (available at 38 in ADEGENET v. 2.1.338. The number of clusters (K) was determined by applying the find.cluster procedure with increasing values of K and minimizing the obtained Bayesian Information Criterion and the number of Principal Components retained for the analysis was determined using cross validation.Cryptic structure was also evaluated using discriminant analysis of principal components39. SAMOVA employs a simulated annealing algorithm to optimize allocation of N geographic populations into K groups (2 ≤ K < N) in order to maximize the proportion of total genetic variance due to genetic variation among the inferred groups. Computations were conducted in SAMOVA v. 2.0 (available at http://cmpg.unibe.ch/software/samova2/). One hundred simulated annealing processes were used to determine optimal allocation of the nine geographic samples into two, three, four, five, six, seven, or eight groups.Spatial structure was examined using spatial analysis of molecular variance (SAMOVA)40 was used to examine whether there was a significant correlation between genetic divergence and geographic distance. Isolation by distance was tested between geographic samples and using individual models. Genetic distance was estimated as FST/(1-FST) for Mantel tests implemented on samples grouped by localities and using the distance of Smouse and Peakall41 for individual models. Geographic distance was log transformed as recommended by Rousset42 to account for dispersal in a two dimensional habitat. Significance of the correlation was tested using 10,000 permutations of the distance matrices.A Mantel test implemented in GENALEX v. 6.52 = 3.47, df = 8, P = 0.902; gene diversity: Χ2 = 6.46, df = 8, P = 0.596). Departures from HWE were significant in15 out of 195 tests before correction for multiple tests controlling for a 5% false discovery rate but only one test remained significant after correction . None of the possible null allele cases was associated with significant departure from HWE. Based on these results, all the loci were kept for further analysis.Summary statistics for the 13 microsatellite loci in each sample locality are given in Supplemental file CT = − 0.0017, P = 0.903) or among years within locality . Pairwise exact tests comparing samples collected in different years within the same locality also were non-significant (data not shown). Samples were pooled across sampling years for further analyses considering the non-significant variance between years and the relatively short interval between temporal samples . Exact tests of population differentiation  across loci indicated significant heterogeneity among geographic locations . The corresponding overall estimate of FST was very low (0.0004). Pairwise FST estimates averaged -0.0015 and were all less than 0.0039 . The optimized partition of geographic samples separated the Brazil sample from Baía Formosa (BR-BF) in one group and all the rest of the samples in the second group. The significance of the among-group component of molecular variance could not be assessed because there were only nine different configurations of two groups comparable to the optimal configuration during permutations 44, such that the lowest possible P-value was P = 0.114 .The highest variance among groups was obtained with K = 2 groups during spatial analysis of molecular variance . When the Baía Formosa sample was removed, the slopes of the population and individual models dropped to r = 0.103 (P = 0.404) and r = 0.040 (P = 0.039), respectively.The slope of the isolation by distance model was moderate but positive and the Mantel test indicated a significant association between genetic and geographic distances . In addition, adults migrating from one of the two stocks to the other may show limited reproductive activity post migration due to the shifting of reproductive periods. Additional sampling in South America and at the transition between Brazil and the Caribbean Sea is therefore warranted to confirm the demographic independence of the two regional stocks, describe stock boundaries, and identify factors restricting gene flow between the two regions. The St Peter and St Paul archipelago population did not diverge significantly from either the northern localities or the Baía Formosa population sample. This location is further north from Baía Formosa and may have received migrants from both ‘Northern Hemisphere’ and Brazilian populations, which would contribute to maintaining the St Peter and St Paul population similar to both groups. Considering the small sample size in the St Peter and St Paul area  and the very low divergence between the ‘Northern Hemisphere’ and Brazilian stocks, the divergence of St Peter and St Paul from either of the two other stocks may be too small to be detected with this dataset. The archipelago is very close to the equator line and, while spawning in this area seems to be more active in the first semester as in Brazil, actively spawning individuals are found almost all year round43, suggesting that adult migrants of both northern and southern origin would find conditions conducive to spawning and that their progeny would also find favorable conditions for development leading to successful incorporation of migrant genotypes in the local gene pool. Finally, pairwise comparisons also revealed marginal differences between La Martinique Island and the Gulf of Mexico, Puerto Rico the Keys and East Venezuela (and Baía Formosa). La Martinique did not differ significantly from the larger (West) Venezuela sample, St Peter & St Paul or the US East coast; thus, these small differences in allele frequencies are more difficult to interpret but could possibly signal some degree of isolation of blackfin tuna in the lesser Antilles region.None of the pairwise exact tests were significant after correction for multiple tests but four of the eight tests comparing the Brazil Baía Formosa population to others were significant before correction and another two tests were close to significance (P < 0.078). Only tests comparing Baía Formosa to two of its closest geographic neighbor populations (St Peter and St Paul and West Venezuela) were insignificant. The divergence of the Baía Formosa geographic population was also suggested by spatial analysis of molecular variance, during which the most supported configuration separated Baía Formosa in one group from all the rest of the samples in a second group. The sample size of Baía Formosa was small ; thus, the divergent status of this population will need to be confirmed with additional sampling, but collectively, the above results suggest that this population shows some degree of demographic independence from the other parts of the sampled range. Populations off the coast of Brazil, including the Baía Formosa area, are separated from the Caribbean region and the rest of the species range by the outflows of the Amazon River in northern Brazil and of the Orinoco River in eastern Venezuela. These barriers have been shown to effectively restrict gene flow for several reef fishes that disperse pelagic larvae55, a scenario that may apply to blackfin tuna as well. Variation due to selection and adaptation may not be strictly geographic but could also be cryptic related to parameters of the landscape or habitat characteristics such as temperature and habitat use. This study only employed 13 markers which is insufficient to provide information on the effects of selection. Examination of regions of the genome influenced by selection through the implementation of high-density genome scans is therefore warranted for future studies. We note that the among-years component of molecular variance was close to significant and larger than the spatial  component which suggests that some form of cryptic structure might exist. The occurrence of cryptic structure was examined using model-based Bayesian clustering in STRUCTURE and discriminant analysis of principal components. Model-based Bayesian clustering failed to identify subunits within the dataset. While the levels of divergence between such units, if they exist, are unknown, the divergence between geographic samples was very weak (below 0.005), an order of magnitude below those that can be detected by STRUCTURE56. Discriminant analysis of principal components supported the occurrence of four clusters. While these clusters might indicate the occurrence of some form of cryptic structure, the distribution of cluster membership was uncorrelated to the spatial origin of the samples in this study and this analysis may reflect artifacts considering the weak divergence across the sampling surface. However, cryptic structure within the dataset is possible and could be related to groups featuring different habitat use and movement patterns. This study sampled adults of reproductive size but could not confirm whether specimens were collected on or close to their spawning grounds. Sampling nursery sites in future studies would be useful to better assess the breeding structure of blackfin tuna as was successfully done in other tunas57.Population structuring related to natural selection has been detected in a number of marine species even where neutral variation was very low25 who detected statistically significant, yet low, divergence between the Gulf of Mexico and the NW Atlantic populations. Saxton25 used only 6 heterologous microsatellites and one mitochondrial gene and samples were taken in 1994–1995 for the East US coast and 2001–2007 for the Gulf of Mexico such that spatial variations between the two regions were potentially confounded by temporal variations. This study addressed these limitations by employing a larger panel of homologous loci and more comprehensively sampling the species range. Fine-scale structuring within US waters as suggested by Saxton25 may occur but is likely very weak considering the results of the present study.The lack of significant subdivision in US waters in this study contrasts with the findings of SaxtonIn conclusion, this study indicates the occurrence of weak spatial structuring of blackfin tuna populations, which warrants further investigation to determine if management of the resource needs to account for multiple stocks. Isolation by distance throughout the range was suggested by the outcome of the Mantel tests and will need further description with finer geographic sampling to determine the spatial scale of dispersal and the distance at which stocks are expected to be demographically independent. While the results suggest divergence of the Brazilian and Northern Hemisphere populations, further research is needed to confirm the occurrence of distinct stocks, their geographic delineation, and their relationships, in particular their degree and pattern of connectivity and the mechanisms involved in isolation. The analysis of high-density genome scans in future studies may prove useful to improve inference power for the detection of differentiated stocks and assess variation under selection.Supplementary Information 1.Supplementary Information 2.


Sus scrofa), have experienced severe climatic and size fluctuations that have shaped their genetic variation. Habitat fragmentation and human‐mediated translocations have further contributed to the complex demographic history of European wild boar. Danish wild boars represent an extreme case of a small and isolated population founded by four wild boars from Germany. Here, we explore the genetic composition of the Danish wild boar population in Klelund. We genotyped all 21 Danish wild boars that were recently transferred from the source population in Lille Vildmose into the Klelund Plantation to establish a novel wild boar population. We compared the Danish wild boars with high‐density single‐nucleotide polymorphism genotypes from a comprehensive reference set of 1263 wild and domesticated pigs, including 11 individuals from Ulm, one of two presumed founder locations in Germany. Our findings support the European wild background of the Danish population, and no traces of gene flow with wild or domesticated pigs were found. The narrow genetic origin of the Danish wild boars is illustrated by extremely long and frequent runs of homozygous stretches in their genomes, indicative of recent inbreeding. This study provides the first insights into one of the most inbred wild boar populations globally established a century ago from a narrow base of only four founders.Demographic events such as series of bottlenecks impact the genetic variation and adaptive potential of populations. European megafauna, such as wild boars ( This population was established with the introduction of 12 individuals in 2016, with additional supplementation of nine individuals in 2017. All 21 individuals originated from the source population in Lille Vildmose, Jutland, Denmark, which was founded in 1926 by four founders, two of each sex. There is no information on the population size in Lille Vildmose at the time of the two translocations of 12 and nine individuals in 2016 and 2017, respectively. After the yearly spring population regulation in 2020, the source population size, that is Lille Vildmose, was assumed to be stable around 150 individuals, whereas the population size of boars in Klelund Plantation was around 65 pigs.Our study population is wild boars  in PLINK 1.9  and total ROH length  were significantly different between the two populations. Moreover, the genomic autozygosity (FROH) in the Ulm population was consistently lower compared with Danish wild boars (Table p < 0.001) in Klelund Plantation, Denmark compared to Ulm, Germany . Chromosome 15 showed almost half of the SNPs (1050 out of 2581) to have a frequency above 90%, while the majority of the SNPs (1782 out of 2965) on chromosome 7 had a frequency below 50%  and other gene families that have important functions in immunity . In European wild boar populations, the mean number of piglets and the survival % of piglets ranges from 3.5–6.9 to ~65%–93.7%, (see Table Oryx dammah), which was previously declared extinct in the wild, is an example of a successful use of genomics in conservation management to generate a large‐scale reintroduction project by a careful selection of founders from captive populations (Humble et al., Demographic histories shape the genetic structure of differentiation between populations (Auton et al., This study provides the first insights into the demographic history and genetic structure of the Danish wild boars. Wild boars from Klelund Plantation do not show any apparent fitness costs from an extremely small founder base and living in isolation. However, there may be hidden costs related to their high level of inbreeding and having the longest ROHs found in pigs and wild boars globally. Such hidden costs of inbreeding may only become apparent when these inbred animals have to compete directly with less inbred conspecifics. With the use of genomic analysis, those hidden costs can be unravelled, for instance by inferring if deleterious high‐frequency alleles are present. In the current situation, the Klelund population seems to thrive. However, with changing environments low diversity may result in lower potential to adapt. All in all, the Klelund population has strong potential to untangle how small populations with high levels of inbreeding succeed to persist.The authors have no conflict of interest to declare.Supplementary MaterialClick here for additional data file.Table S4Click here for additional data file.


Aedes albopictus originates from Southeast Asia and is considered one of the most invasive species globally. This mosquito is a nuisance and a disease vector of significant public health relevance. In Europe, Ae. albopictus is firmly established and widespread south of the Alps, a mountain range that forms a formidable biogeographic barrier to many organisms. Recent reports of Ae. albopictus north of the Alps raise questions of (1) the origins of its recent invasion, and (2) if this mosquito has established overwintering populations north of the Alps. To answer these questions, we analyzed population genomic data from >4000 genome‐wide SNPs obtained through double‐digest restriction site‐associated DNA sequencing. We collected SNP data from specimens from six sites in Switzerland, north and south of the Alps, and analyzed them together with specimens from other 33 European sites, five from the Americas, and five from its Asian native range. At a global level, we detected four genetic clusters with specimens from Indonesia, Brazil, and Japan as the most differentiated, whereas specimens from Europe, Hong Kong, and USA largely overlapped. Across the Alps, we detected a weak genetic structure and high levels of genetic admixture, supporting a scenario of rapid and human‐aided dispersal along transportation routes. While the genetic pattern suggests frequent re‐introductions into Switzerland from Italian sources, the recovery of a pair of full siblings in two consecutive years in Strasbourg, France, suggests the presence of an overwintering population north of the Alps. The suggestion of overwintering populations of Ae. albopictus north of the Alps and the expansion patterns identified points to an increased risk of further northward expansion and the need for increased surveillance of mosquito populations in Northern Europe. Aedes albopictus in Europe across the Alps. While we detected a weak genetic structure and high levels of genetic admixture, the recovery of a pair of full sibling consecutive years in Strasbourg, France, suggests the presence of an overwintering population north of the Alps. The expansion patterns points to an increased risk of further spread and the need for increased surveillance of mosquito population in Europe.Population genomics study to investigate the recent invasion of  It is considered one of the most invasive species worldwide . Second, we assess dispersal at the European scale and genetic structuring across the Alps using a dataset consisting of a subset of 137 individuals, which included only European samples from 39 sites , plus additional samples collected outside of the target study area to facilitate detection of potential long‐range introductions and their origins. The dataset comprises a total of 208 specimens, including 5 populations from the USA and Brazil, as they are considered to be a bridgehead for the European invasion  and were collected during summer months in 2006, 2016, 2017, and 2018. This dataset includes samples from across the Alps in Switzerland, neighboring countries , and from Albania and Greece. Samples from across the Alps included collections made along national highways around and in the city of Basel in Northern Switzerland , as well as 23 specimens in the global dataset (1.native_invasive) that did not have published ddRAD data available  at the Department of Biosystems Science and Engineering (D‐BSSE), ETH Basel, Switzerland, using paired‐end, HiSeq Flow Cell v4, HiSeq SBS Kit v4, and with a 10% PHiX spike.2.3process_radtags function in STACKS v2.2  available on NCBI GenBank  with VCFtools. The window size corresponded to our maximum fragment size, thus each SNP belongs to a single DNA fragment. After conducting a relatedness analysis, we excluded one individual per sibling pair from the analyses (see section below). The reduced dataset was split into two cleaned datasets: 1.native_invasive_cleaned, including 153 samples and 4714 loci and SNPs, and 2.europe_cleaned, including 93 samples and 6308 loci and SNPs . The second approach was to choose the most likely value for K using the ADMIXTURE's cross‐validation procedure. Genetic differentiation was further investigated with fineRADstructure v1.7.20  and Discriminant Analysis of Principal Components (DAPC)  by assessing their statistical significance with 1000 bootstrap samplings and estimated allelic richness (AR). We calculated the observed heterozygosity (HO) using VCFtools (−het). Individuals were grouped by country and differences in mean HO between groups were tested with a non‐parametric Kruskal–Wallis (KW) test for statistical significances in R. Expected heterozygosity (HE) per country was computed using the R package adegenet v2.0.To evaluate the degree of genetic differentiation, we estimated pairwise  Goudet,  and esti2.europe_cleaned dataset), we estimated FST, FIS, AR, HO, and HE using the R package adegenet v2.0, FIS and pairwise FST using the R package HierFstat  using the R package adegenet (propShared function). We visualized pairwise Dps with neighbor‐net networks with the software SplitsTree v5.0  per individual aligned to the reference genome. Table We sequenced 160 individuals obtaining a total of 828 million reads, with 5 million reads per sample on average, ranging from 7 thousand to 17 million. After filtering and removal of duplicate siblings, the dataset 3.21.native_invasive_cleaned and 2.europe_cleaned). Although we had only 15 full siblings in our dataset, we found two siblings from the same location in two consecutive years. These were detected at a distance of 330 m in 2017 and 2018 in Strasbourg, France. The first sibling was collected from an egg sample in fall 2017, while the second one was caught as an adult during the second half of July 2018.We identified 15 full‐ and 10 half‐sibling pairs from the same collection sites Table . One sib3.31.native_invasive_cleaned dataset Figure 1.native_invasive_cleaned dataset with individuals collected in Indonesia forming one cluster genetically distinct from all the other individuals . However, as the cross‐validation errors were very similar for a number of K's , we further explored K = 2, 3, 4, and 5. K = 4 appears to be the most likely scenario with two clearly distinct genetic groups, including specimens collected in Indonesia and Hong Kong, and two genetic groups with a high level of genetic admixture, including specimens collected in Europe, Brazil, the USA, and Japan  are reported in Figure K = 1; see Figure At the European level, different analysis suggests a weak substructuring. In the ADMIXTURE analysis, the individuals collected in Europe, except for some individuals from Greece and Albania, shared high levels of genetic ancestry and showed the highest proportion of their genome being assigned to either cluster 2 or 4  and expected (HE) heterozygosity ranged from 0.041 to 0.055 and from 0.048 to 0.075, respectively. HO, HE, and FIS within country for the dataset 1.native_invasive_cleaned are reported in Table H0 differed among countries  and between the native and the invasive range . The highest heterozygosity measured within a country was among the Indonesian specimens  and “recently‐established” (after 2003) populations. With this approach, we identified three groups of populations from Italy  than the long‐established Italian populations   did not show lower genetic diversity  are relatively low, whereas those collected in France (pop_fr) are higher Figure . Simulat4Ae. albopictus into Switzerland and across the Alps, and to estimate if the current populations are self‐sustaining. As a point of reference, we compared the genetic variability in mosquitoes from Switzerland to populations from Italy that have been established for over 25 years, to recently established populations in neighboring France, Germany, and Liechtenstein, and to populations from the mosquito's native range in Japan, Indonesia, and China. We found that populations from Switzerland had similar genetic variability to those from well‐established populations in Italy , we detected the presence of four genetic clusters ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); project administration (lead); resources (lead); writing – original draft (lead); writing – review and editing (lead). Ann‐Christin Honnen: Data curation ; formal analysis ; methodology ; supervision ; validation (supporting); visualization (supporting); writing – review and editing . Norah Saarman: Data curation ; formal analysis ; methodology ; resources ; software (lead); writing – review and editing . Adalgisa Caccone: Formal analysis ; investigation ; methodology ; resources ; software ; supervision ; writing – review and editing . Pie Müller: Conceptualization (lead); funding acquisition (lead); investigation ; methodology ; supervision (lead); visualization ; writing – review and editing .The authors declare no competing interests.Appendix S1Click here for additional data file.


Petrous bones and teeth are the skeletal elements most often targeted by researchers for ancient DNA (aDNA) extraction, and the sources of the majority of previously published ancient African genomes. However, the high temperature environments that characterise much of Africa often lead to poor preservation of skeletal remains. Here, we successfully reconstruct and analyse genome-wide data from the naturally mummified hair of a 4000-year-old individual from Sudan in northeastern Africa, after failed attempts at DNA extraction from teeth, petrous, and cranium of this and other individuals from the Kadruka cemeteries. We find that hair DNA extracted with an established single-stranded library protocol is unusually enriched in ultra-short DNA molecules and exhibits substantial interior molecular damage. The aDNA was nonetheless amenable to genetic analyses, which revealed that the genome is genetically indistinguishable from that of early Neolithic eastern African pastoralists located 2500 kms away. Our findings are consistent with established models for the southward dispersal of Middle Nile Valley pastoral populations to the Rift Valley of eastern Africa, and provide a possible genetic source population for this dispersal. Our study highlights the value of mummified hair as an alternate source of aDNA from regions with poor bone preservation. Since then, the field of ancient DNA (aDNA) research has grown rapidly, with over 6000 ancient genomes published to date. This extraordinary growth has largely been based on analyses of ancient human bones and teeth, with their preservation widely regarded as superior to that of soft tissue and hair2. Meanwhile, very few studies have attempted to extract aDNA from hair or soft tissue3, especially from samples of significant age.The first complete ancient human mitochondrial genome, published in 2008, was generated from the frozen hair of an individual of the Saqqaq archaeological culture from Greenland, dated to 4750 years ago5, in particular drawing on samples from present-day Kenya and Tanzania. aDNA studies have revealed multiple waves of human migrations associated with the spread of pastoralism, and complex subsequent admixture events with local hunter-gatherers in eastern Africa5. Although there is broad consensus that early pastoralists in eastern Africa originated from population dispersals from the north, from regions such as Sudan and Ethiopia7, no aDNA data related to early pastoralists from these regions has yet been published.While only accounting for 1.7% of all published genomes, Africa is no exception to this trend. So far, aDNA studies focused on Africa have reported nearly 100 ancient whole genomes, reconstructed from aDNA preserved in petrous bones and teeth from sub-Saharan Africa. Over half of these studies focused on understanding the spread of pastoralism in ancient eastern Africa8. As we show in our analysis, sequence data generated from hair aDNA using this single-stranded library protocol features unique characteristics that pose challenges in downstream genetic analyses.Here, we extract DNA from five specimens  deriving from four individuals from middle Holocene cemeteries in the Dongola Reach of Upper Nubia, northern Sudan. Selected samples for this study represent three individuals from the early 7th millennium BP Neolithic site of Kadruka 21, and one Kerma period individual, directly dated to 4033 cal. BP, from Kadruka 1. Of all the tissues and samples tested, only one sample of preserved hair, from the Kerma period individual, yielded a sufficient amount of authentic ancient human DNA to study. We argue that a critical prerequisite in this extraction was the first application of a state-of-the-art single-stranded library laboratory protocol on hair material, which is able to turn single-stranded DNA molecules into a sequencing libraryOur population genetic analysis of this hair sample from northern Sudan reveals a close genetic affinity of the Kerma period individual to early pastoralists from the Rift Valley in eastern Africa. Drawing on this evidence, as well as previously published proteomic evidence of dietary proteins extracted from the dental calculus of this individual, we conclude that our sample (“Sudan_Kadruka1_4000BP”) represents one of the earliest and most northerly ancient pastoralist populations to have been genetically characterised in Africa to date, as well as potentially a genetic source population for one of the earliest prehistoric pastoralist dispersals into eastern Africa.9. In accordance with broader archaeological sequences for this region of the Middle Nile Valley12, cultural deposits were primarily linked to the Neolithic (spanning the 7th millennium BP), as well as the more recent Kerma period (3450–4450 BP). Excavation programs in the Kadruka district focused on several cemeteries from these cultural periods, including Kadruka 1 and Kadruka 21. Reflecting local environmental fluctuations between dry and wet conditions associated with more intensive Nile floods during the Middle Holocene, Neolithic skeletal remains at these cemeteries are typically highly degraded14. In contrast, progressive local aridification and floodplain contraction15, has facilitated enhanced preservation of organics, including hair and leather items, in the more recent Kerma period burials at Kadruka 116.Archaeological fieldwork in the Kadruka district of northern Sudan has revealed widespread past occupation of the alluvial plain and paleochannel systems to the east of the current Nile River channelBovinae) or sheep (Ovis) in the dental calculus of individual SK129 from Kadruka 21 and to goat (Carpa) in the dental calculus of individual SK68 from Kadruka 117. Furthermore, isotopic analyses of hair from individual SK68 (named “Sudan_Kadruka1_4000BP” in this study) broadly indicate a diet primarily composed of C3-based resources .A recent study exploring the diet of individuals from Kadruka 21 and Kadruka 1 detected milk proteins attributable to domesticated cow  Table . The hai20 which uses simulations to estimate fractions of spurious and authentic alignments given certain read length cut-offs. For a cut-off length of 25 bp, SpAI estimated a spurious alignment fraction of 10%  and 34 bp in the raw data processing steps, and summarised EAGER statistics and respective contamination estimates in Table We found that the average read length was relatively short , we further explored alignment statistics. We find two unusual characteristics in the aDNA library generated from the Kadruka hair sample. The first is that the sample is enriched in unusually short DNA molecules, giving a median read length of 25 bp, compared to 44 bp for typical bone-derived shotgun aDNA in a previous African aDNA study using the same laboratory pipeline4. We investigated whether there is a notable difference in terms of aDNA preservation in nuclear DNA and mitochondria from the hair material. Specifically, we examined if the two idiosyncratic features of hair aDNA we describe above apply to both nuclear and mitochondrial DNA from hair. In Fig. We find the ratio of mitochondrial to nuclear DNA in the hair-derived DNA library to be relatively high , likely due to capture preferentially targeting molecules without damage (due to more effective hybridization) than with damage. In addition, longer molecules are preferentially captured over short molecules.Given the success of SNP capture techniques for poorly preserved human DNAata Fig. , corrobof3 . We projected ancient Africans and ancient Near Easterners on the background of modern African groups25. Although the number of available populations in SGDP and HGDP is limited, we observe clear separations of African populations from different regions, with eastern/northern, southern and western African populations falling into the right, left and top corner of PC1/PC2 space, respectively.We performed Principal Components Analysis (PCA) and Outgroup-f3 Figs. , 3 to inf3 Figs. . To maxi5, such as Kenya_EarlyPastoral_N  and Kenya_Pastoral_Neolithic . Kenya_EarlyPastoral_N is a group of two pastoralist individuals dated to the early stage of the eastern African Pastoral Neolithic, both of whom are genetically derived from admixture between two early northeastern African-related ancestries from Sudan and Northern Africa/Levant5. To estimate the level of noise resulting from the sparsity of our SNP data, we computed a standard error for the projected PCs of Sudan_Kadruka1_4000BP using a block jackknife approach. Specifically, we computed pseudo-values by deleting each chromosome of the genotype data in turn and then used the resulting estimates from the remaining data as input for the weighted jackknife calculation27. We find the standard errors of Sudan_Kadruka1_4000BP to be relatively small compared to overall genetic variation within Africa, which gives us confidence that the location calculated from the full data . Figure f4  to validate the close PC location between Sudan_Kadruka1_4000BP and Kenya_EarlyNeolithic_N. Consistent with PCA location, f4-statistic result confirms the genetic cladality between Sudan_Kadruka1_4000BP and Kenya_EarlyPastoral_N given that none of the tested populations breaks cladality significantly, suggesting that these two individuals are indistinguishable in terms of allele frequencies  denaturation of double-stranded DNA molecules to single-stranded DNA, and ii) more frequent breakage of DNA molecules, resulting in shorter DNA fragments. Hence the predominance of short, single-stranded DNA in hair explains the excess C- > T substitution rate in the interior of sequencing reads. Meanwhile, the arid preservation environment and the specific properties of hair potentially reduce enzymatic activity, resulting in reduced hydrolysis damageGiven these properties, the single-stranded library protocol we employed is uniquely well-suited as a sequencing tool in this case, maximising library complexity from heavily degraded and in large parts single-stranded source DNA fragments by building library molecules from both forward and backward strand DNA fragments regardless of the DNA strand orientation. In such cases, both short and long DNA fragments are sequenced effectively. Hence, our shotgun sequencing on the single-stranded library accumulates the short DNA fragments, as reflected in the read length distribution with a peak around short read lengths, and keeps the long fragments with interior damage, as reflected in the average high interior damage with C->T substitution rate over 10% throughout the sequence read Fig. .To our surprise, only the DNA from hair, rather than from the tooth of the same individual, showed signatures of authentic human aDNA preservation, as seen from the pattern of C- > T substitutions, regardless of rather similar endogenous DNA content in tooth and hair Table . From thf3-statistics that this individual, from a rural agro-pastoral population linked with the Kerma culture of Upper Nubia16, shares close genetic affinity with Levantine groups. Moreover, we could show that this individual is genetically indistinguishable from early Pastoral Neolithic individuals dated to 4000BP living over 2500 km away in Kenya and Tanzania, even when correcting for relatively large standard errors in population genetic estimates due to the low coverage. This close relatedness to early pastoral populations in eastern Africa is consistent with archaeological evidence for the dispersal of herding populations southwards along the Nile River Valley following their establishment in the Kadruka region from the early 7th millennium BP30, although we caution that inference from a single sample can at best be tentative.The very low coverage data resulting from this unique sample posed a challenge for downstream population genetic analyses. Nevertheless, we see from 31. These early eastern African pastoralists at sites like Lothagam in the Turkana Basin32, while featuring regional forms of cultural expression, also broadly maintained traditions of monumental mortuary expression, as well as traditions of burial with elaborate grave goods for which the Neolithic and Kerma period cemeteries of the Middle Nile Valley, including Kadruka 1, are recognised9.The high affinity of the Kerma period individual from Kadruka 1 with Neolithic pastoral groups far to the south, and genetic indistinguishability of their sequenced DNA, would be consistent with this sample representing a possible genetic source population for the earliest eastern African pastoralists who settled in the Rift Valley. This in turn would point to a high degree of mobility of pastoralists between the Middle Nile Valley and present-day Kenya, potentially before or around the Pastoral Neolithic. It would imply that the southward dispersal of pastoralists from the Middle Nile Valley did not involve genetic exchange with pre-existing human groups along the migration route, particularly local foragers, and may therefore have been relatively rapid. Indeed, archaeological research suggests that dispersals were likely driven in part by increasing population pressure and regional aridification, with a marked period of aridity in Upper Nubia preceding the first appearance of pastoral groups in the Turkana Basin of eastern Africa at 5000 BPWhile much archaeogenetic research in Africa has focused on the Neolithic populations of eastern Africa, their source populations have, from a genetic standpoint, remained elusive. Highlighting the need for additional aDNA from ancient northeastern African populations, the aDNA recovered from ancient human hair in this study provides important new genetic data relevant to the detailed reconstruction of early pastoral dispersals from northeastern to eastern Africa. Our findings also point to the value of preserved ancient hair as a source of aDNA, particularly in regions where more typical aDNA archives are unavailable or poorly preserved, with exciting implications for future aDNA research.33, and performed both whole genome shotgun and 1240 k-hybridization capture23 to maximise the amount of aDNA.We sequenced five specimens  in total from Kadruka 21 and Kadruka 1 for aDNA in dedicated clean rooms at the Max Planck Institute for the Science of Human History in Jena, Germany. We constructed both single-stranded and double-stranded DNA libraries for skeleton remains and single-stranded library for the hair sample following previously published protocols2O and dried before combustion. Combustion and graphitization of a subsample of the pre-treated hair (5.35 mg) was carried out according to the methods outlined in Dee et al. (2020)34. The 14C measurements were carried out using an Ionplus AMS-MICADAS36. C14 ages are calibrated to calendar years with OxCal version 4.337, using calibration curve IntCal1319.We sampled from a mixed composition of hair for aDNA analyses and report the direct C14 date for the hair sample from separate additional sampling. A bulk hair sample was sampled for dating at the Centre for Isotope Research (CIO) Groningen in 2019. A sample of 78.5 mg of hair was chemically treated with 4% HCl, rinsed with decarbonized H38, with adaptors removed by AdapterRemoval v239 using flags AdapterRemoval –threads 4 –trimns –trimqualities –preserve5p –minlength 25 –minquality 20 –minadapteroverlap 1, reads mapped to hs37d5 by BWA v0.7.1240 using flags bwa aln -t 4 -n 0.01 -l 32 , and polymerase chain reaction duplicates removed by Dedup software v0.12.238. mapDamage v2.0.941 was used to calculate substitution rate in read termini of sequenced DNA fragments.We processed DNA sequences using the EAGER v1.92.5621, which is designed for single-stranded library DNA sequences, to examine the contamination level. Finally, we applied a minimum read length filter of 25, and a minimum base quality of 30 and a minimum mapping quality of 30 for genotype calls. We call pseudo diploid genotypes by randomly sampling an allele for each site in the 1240 k panel using Samtools v1.342 and pileupCaller (github.com/stschiff/sequenceTools).We estimated the amounts of spurious alignments at various read length cut-off using SpAI (bioinf.eva.mpg.de/SpAl/). We tested three different read length filters  to yield the maximum efficiency for endogenous aDNA while assuring contamination free. We used AuthentiCT50, together with 30 high-coverage modern African populations from the Simons Genome Diversity Project (SGDP)24 and Human Genome Diversity Project (HGDP) datasets25.For downstream population genetic analyses, we combined our new data with previously published ancient African and Near Eastern genomes51 with “numthreads: 8” and “lsqproject: YES” option in the parameter file, and projected ancient individuals into the PC space calculated from modern African populations in SGDP  of the PC location of Sudan_Kadruka1_4000BP52. We dropped one chromosome (i.e. block) each time and iterated for 22 chromosomes, to get an estimate of SE.We performed PCA using smartpca v16000DP Table 24. No shf3 and f4 statistics using qp3pop v435 from the AdmixTools v5.1 package26 using options “outgroupmode: YES”, and qpDstat v755 from the same package26 using options “f4mode: YES” and “printsd: YES”. We performed the Outgroup f3  test using Chimpanzee as the outgroup population. Population X represents published ancient African and Near Eastern populations and high-coverage modern populations from SGDP and HGDP datasets documented in Table f3 test results using more than 1000 overlapping SNPs. Given that Sudan_Kadruka1_4000BP shares high genetic affinity with early eastern African pastoral groups, we further tested the same list of population X using f4  and f4  for the genetic cladility between Sudan_Kadruka1_4000BP and Kenya_EarlyPastoral_N.We calculated Supplementary Information 1.Supplementary Information 2.


Pseudemys alabamensis is one of the most endangered freshwater turtle species in the United States due to its restricted geographic distribution in coastal Alabama and Mississippi. Populations of P. alabamensis are geographically isolated from one another by land and saltwater, which could act as barriers to gene flow. It is currently unknown how differentiated these populations are from one another and whether they have experienced reductions in population size. Previous work found morphological differences between Alabama and Mississippi populations, suggesting that they may be evolutionarily distinct. Other Pseudemys turtles such as P. concinna and P. floridana occur naturally within the same geographic area as P. alabamensis and are known to hybridize with each other. These more abundant species could threaten the unique genetic identity of P. alabamensis through introgression. In order to evaluate the endangered status of P. alabamensis and the level of hybridization with other species, we used mitochondrial and nuclear microsatellite markers to assess genetic variation within and among populations of P. alabamensis throughout its range and estimate admixture with co‐occurring Pseudemys species. In P. alabamensis, we found no variation in mitochondrial DNA and an excess of homozygosity in microsatellite data. Our results show genetic differentiation between Alabama and Mississippi populations of P. alabamensis, and low estimated breeding sizes and signs of inbreeding for two populations . We also found evidence of admixture between P. alabamensis and P. concinna/P. floridana. Based on our results, P. alabamensis is highly endangered throughout its range and threatened by both low population sizes and hybridization. In order to improve the species’ chances of survival, focus should be placed on habitat preservation, maintenance of genetic diversity within both the Mississippi and Alabama populations, and routine population‐monitoring activities such as nest surveillance and estimates of recruitment. Pseudemys alabamensis is one of the most endangered freshwater turtle species in the United States. Populations of P. alabamensis are geographically isolated from one another by land and saltwater, which could act as barriers to gene flow, resulting in the evolution of unique populations. Other Pseudemys turtles such as P. concinna and P. floridana occur naturally within the same geographic area as P. alabamensis and are known to hybridize with each other. These more abundant species could threaten the unique genetic identity of P. alabamensis through introgression. We used mitochondrial and nuclear microsatellite markers to assess genetic variation within and among populations of P. alabamensis throughout its range and estimate admixture with co‐occurring Pseudemys species. We found that P. alabamensis is highly endangered throughout its range and threatened by both low population sizes and hybridization. Conservation measures must be developed to prevent further reduction and extinction of this endangered species. Introgression with native P. concinna and P. floridana, or non‐native species that may have been introduced to the area, would have major conservation implications for P. alabamensis, as it would threaten the unique genetic identity of an already highly geographically restricted species with a likely low population size.Although hybridization has been observed within the a Mount, . In addiP. alabamensis, (2) measure intraspecific genetic diversity, (3) investigate the possibility of recent reductions in population sizes, and (4) assess potential hybridization with sympatric species. Our results can serve as a necessary basis to further develop conservation and management activities in collaboration with local conservation organizations and authorities and to raise awareness of the current status of imperilment of this species. As climate change increasingly impacts coastal populations, understanding the current distribution and genetic diversity of P. alabamensis will be critical for determining its long‐term survival potential, especially for small and isolated populations.Here, we utilize mitochondrial DNA (mtDNA) and microsatellite markers to (1) identify the genetic structuring of populations of 22.1This research was conducted under US Fish and Wildlife Service permit #TE40523A‐2, Mississippi Department of Wildlife, Fisheries, and Parks permit #0614181, and Alabama Fish and Wildlife permits #2018063278468680 and #2019097050868680. Trapping and handling methods were approved by the University of South Alabama Institutional Animal Care and Use Committee (IACUC Protocol No. 921991‐3).2.2P. alabamensis . Hoop nets were 1.2 m in diameter and 4.6 m in length, while lead nets were 1.2 m in height and 9–12 m in length. Floats were added to hoop nets to maintain flotation and ensure access to air. Nets were anchored to the substrate with PVC tubing. Traps were left un‐baited and checked once every 36 h. Specific trap site selection was based on multiple factors: water depth, substrate, disturbance, basking logs, observed boat traffic, and submerged aquatic vegetation. In addition to trapping turtles, samples were also collected from roadkill individuals on the Mobile Bay Causeway , males have elongated foreclaws for titillation, a cloaca that is located outside the edge of the carapace, and thicker tails; females have a cloaca located closer to the edge of the carapace. Individuals that could not be confidently sexed were considered as juveniles. Sampled animals were also weighed with a scale and measured with a caliper for carapace and plastron width and length and shell height. The geographic locations of sampling sites were recorded with a handheld GPS. To prevent re‐sampling, turtles were marked for identification by notching the marginal scutes. Due to admixture between individuals of the cooter complex in the area (P. concinna and P. floridana), many individuals captured in this study presented mixed morphological characteristics; therefore, individuals were identified to the most similar species following morphological descriptions of the species in Alabama as in Mount  was collected from each animal using a 23‐gauge needle and a 3‐ml syringe. All animals were released at the point of capture after blood sampling was performed and after ensuring that the puncture site was not bleeding and the animal was well. One ml of sampled blood was stored in 2‐ml microcentrifuge tubes with 1 ml of prepared blood preservative that consisted of 100 mM Tris–HCL, 100 mM EDTA, 10 mM NaCl, and 0.5% SDS. Samples were stored on ice until returned to the lab where they were then placed at −20°C for long‐term storage until DNA extractions were performed. DNA extractions were carried out using the Qiagen DNeasy Blood and Tissue kit  following the manufacturer's instructions for nucleated blood.2.3Chrysemys picta). Twenty‐five μl reactions were prepared using 12.5 μl GoTaq G2 Green Master Mix (Promega), 0.5 μl 10 mg/ml bovine serum albumin, 1.2 μl each of 10 μM forward and reverse primers, 6.8 μl H2O, and 2.8 μl DNA extract. PCR conditions were as follows: 95°C for 3 min, 35 cycles of 95°C for 1 min, 55°C for 30 s, 72°C for 1 min; and a final 10‐min extension at 72°C. PCR products were checked on a 1% agarose gel to ensure proper amplification and then purified using ExoSAP‐IT (Applied Biosystems) according to the manufacturer's instructions. Sequencing was carried out by the DNA Analysis Facility at Yale University. Sequences were checked and manually edited using FinchTV . Only a subset (N = 27) of P. floridana samples from Weeks Bay were amplified due to the high number of turtles caught (N = 68); all other P. floridana individuals sampled elsewhere were amplified . Allelic diversity, presence of private alleles, observed (HO) and expected (HE) heterozygosities, and inbreeding coefficient (FIS) were assessed with the software Genetix v4.05  with a burn‐in period of 100,000 followed by an additional 100,000 repetitions. In order to determine the best value of K (number of clusters) for each species, the ΔK statistic  was used to further assess the level of introgression based on microsatellite data using the software Genetix v4.05  were low . For the Mobile‐Tensaw Delta, our sampling included more females than males, as a large portion of our samples for this area came from road‐kill individuals, which affects female turtles more than males  was common among all sampled populations, while the other (Pen) was present only in a single individual from Dog River , a species that is normally only found on the Florida peninsula. Out of 200 samples of P. concinna and P. floridana, 19 variable nucleotide positions, including one insertion found in two individuals from Biloxi, Mississippi (haplotype = MissCon7), were identified, defining 22 haplotypes. The ARBT haplotype of P. alabamensis was found in four individuals of P. floridana and two of P. concinna. Twelve haplotypes were unique to individuals morphologically identified as P. concinna , three haplotypes were unique to individuals morphologically identified as P. floridana , and six haplotypes were shared between P. concinna and P. floridana   for this species. Haplotype diversity for P. concinna averaged 0.76 (range 0.53–0.86 among populations) and was 0.64 in the P. floridana Weeks Bay population (Table N > 5. Haplotype sequences have been deposited in NCBI GenBank (see Data Availability section for accession numbers).A 587 bp fragment of the mtDNA control region was amplified from all 296 er Table . BLAST s3.2P. alabamensis, and two loci (B91 in Pascagoula and D55 in Pascagoula and Fowl River) for P. concinna. The eight loci ranged between 4‐10 alleles each for P. alabamensis, between 4‐17 alleles each for P. concinna, and between 3‐12 alleles each for P. floridana. In P. alabamensis, private alleles were found exclusively in the Mobile Bay populations, with the Mobile‐Tensaw Delta possessing four private alleles, Weeks Bay three private alleles, and Dog River two private alleles   in one population (Fowl River) for P. concinna. Overall, null alleles were identified in 23 of the 96 combinations of loci × populations × species . F‐tests run on corrected and uncorrected FST values obtained using FreeNA indicated that the presence of null alleles does not affect FST estimates (p > .05 for each species comparison). Therefore, all microsatellite loci were used in subsequent analyses. No loci showed significant linkage disequilibrium (p < .01) across populations providing evidence of independent segregation of loci used. All populations of P. alabamensis and all but one P. concinna population showed departure from Hardy‐Weinberg Equilibrium at the locus D79, most likely as a result of the null allele and higher homozygosity levels. Among all populations, P. concinna from Biloxi possessed the most significant departures at five of the eight loci, with lower‐than‐expected heterozygosity. Similarly, the single P. floridana population with N > 5 displayed significant departure from Hardy‐Weinberg equilibrium at three loci. Within species, FST values among populations ranged between 0 and 0.28 and between 0 and 0.19 for P. alabamensis and P. concinna, respectively. Populations from Pascagoula (Mississippi) for both P. alabamensis and P. concinna were the most distinct  from their counterpart populations in Alabama , likely as a result of admixture between the two species.Two loci, B21 and D79, showed evidence of null alleles in all three species for half or more of the sampled populations, with the Biloxi population especially affected by the presence of null alleles in N ≥ 5 ranged from low (2.88) to moderate (4.75) in P. alabamensis (mean 3.94), from 3 to 5 (mean 4.27) in P. concinna, and was relatively higher (5.75) in the single P. floridana population found in Weeks Bay (Table HO) ranged from 0.35 to 0.48 in P. alabamensis, from 0.38 to 0.45 in P. concinna, and was 0.41 in P. floridana. Expected heterozygosity (HE) ranged from 0.45 to 0.55 in P. alabamensis, from 0.45 to 0.57 in P. concinna, and was 0.51 in P. floridana. All populations for all species, except for P. concinna in Pascagoula, showed an excess of homozygosity with HO having much lower values than HE. In P. alabamensis, bottleneck analysis identified one significant occurrence (p < .05) for the Pascagoula population (N = 17) under the Stepwise Mutation Model. Ne estimates showed support for low breeding population sizes (Ne < 30) in the Biloxi River and Fowl River populations of P. alabamensis  in populations with sis mean .94, fromsis mean .94, fromPseudemys species considered in this work identified an optimum clustering of K = 2 with evidence of some admixture  Biloxi River, (2) Fowl River, Dog River, and Mobile Tensaw Delta, and (3) Pascagoula River and Weeks Bay populations. FST of P. concinna clusters was generally low, and follows the cluster numbers listed above: cluster 1 vs. 2 = 0.043, cluster 1 vs. 3 = 0.078, cluster 2 vs. 3 = 0.072. All microsatellite allele scoring information is provided in Supplementary Data 1 on Dryad.STRUCTURE analysis of all e Figure . The two3.3P. alabamensis, two individuals were considered to be potential hybrids based on mixed morphological characteristics and the presence of reduced P. alabamensis identifying characteristics. One of these individuals from Dog River possessed a P. peninsularis mtDNA haplotype, seven microsatellite loci with alleles matching P. alabamensis, and one microsatellite locus possessing an allele not found in any other individual or species studied here. The other individual (from Bayou La Batre), despite having the P. alabamensis mtDNA haplotype (ARBT), clustered with the cooter species (P. concinna and P. floridana) in the STRUCTURE analysis based on microsatellite loci. Of the 102 P. concinna and 30 P. floridana individuals with both mtDNA data and microsatellite data, 27 (26.5%) P. concinna and 6 (20%) P. floridana individuals possessed conflicting species assignments between the two marker types. Two morphologically identified P. concinna and four P. floridana individuals possessed the P. alabamensis haplotype (ARBT), although the P. floridana individuals did not group with P. alabamensis in the STRUCTURE analysis based on microsatellites. One of these four individuals possessed a bright red plastron, a characteristic not present in P. floridana, which typically possess plain yellow plastrons; however no other potential P. alabamensis morphological characteristics were seen in these six individuals. Five of the six cooters that displayed the P. alabamensis haplotype were found in the rivers of Weeks Bay, while the sixth was found in the Mobile Tensaw Delta.Of the 96 samples morphologically identified as FST values between P. alabamensis and each of the other two sympatric Pseudemys species were lower  than FST values observed between P. alabamensis from Pascagoula versus the populations in Alabama . One private allele from locus D121 that was only found in the Weeks Bay population of P. alabamensis was also found to be a common allele in P. concinna . One allele from locus D28 that was a private allele in the Biloxi P. concinna population was also found in the neighboring Pascagoula population of P. alabamensis .Based on microsatellite data, P. concinna and P. floridana. FST between the two species was 0.065, much lower than between P. alabamensis and either of these two species and even within P. alabamensis. The P. floridana population with >5 individuals possessed 6 of the alleles that were private alleles within P. concinna populations and 1 allele that was considered a private allele within a P. alabamensis population.Hybridization appears to occur at a higher rate between P. alabamensis were assigned to P. concinna . Another individual from Pascagoula morphologically identified as P. alabamensis showed admixture with mixed assignment between P. alabamensis and P. concinna (Q = 0.62). Signs of hybridization with P. alabamensis were also found in individuals morphologically identified as P. concinna. Of the 102 individuals morphologically identified as P. concinna, 12 individuals (12%) were assigned to P. alabamensis with Q ≥ 0.7, and another 7 (23%) showed mixed assignments (0.5 < Q < 0.7) between the two species. Across all the populations, the Biloxi river was the locality where many individuals (8 out of 38 with Q ≥ 0.7) morphologically identified as P. concinna were assigned to P. alabamensis on the basis of microsatellite data. We also found that two individuals out of 30 (7%) that were morphologically identified as P. floridana showed evidence of admixture (Q ≥ 0.7). Finally, of all individuals of P. concinna and P. floridana, 33 out of 103 (32%) P. concinna were either assigned to P. floridana (Q > 0.7) or showed admixture (0.5 < Q < 0.7), and 5 out of 30 (17%) P. floridana were also either assigned to P. concinna (Q ≥ 0.7) or showed admixture . Finally, PCA run on microsatellite and mtDNA data indicates a clear distinction of P. alabamensis individuals  and P. floridana (white) along PCA1. The individuals showing admixture between P. alabamensis and P. concinna/P. floridana (pink) were placed in between the two main groups and grouping more towards P. concinna/P. floridana, suggesting the presence of both F1 hybrids and backcrosses of F1 hybrids with P. concinna/P. floridana individuals  that were morphologically identified as 4P. alabamensis and co‐occurring congeneric species. While previous studies have also addressed some of these questions . Despite the overall low genetic diversity observed in P. alabamensis, microsatellite data support genetic differentiation between Mississippi and Alabama populations of this species, in agreement with slight morphological differences previously observed between these areas  between P. alabamensis and P. concinna/P. floridana individuals and was confirmed by microsatellite data . To our knowledge, these data represent the first published evidence of hybridization between P. alabamensis and sympatric Pseudemys species. In all of these cases of haplotype sharing, animals were morphologically identified as P. concinna or P. floridana, but had the P. alabamensis mtDNA haplotype, suggesting that hybridization in P. alabamensis may be largely driven by males of P. concinna and P. floridana breeding with female P. alabamensis. In Weeks Bay and in the Mobile‐Tensaw Delta, where we found instances of haplotype sharing among species, we sampled an excess of female versus male P. alabamensis ; this individual grouped with P. concinna and P. floridana in STRUCTURE, but had the mtDNA haplotype of P. alabamensis, suggesting a possible P. concinna × P. alabamensis hybrid origin. Furthermore, our data point to the presence of both F1 hybrids between P. alabamensis and P. concinna/P. floridana and backcrosses of F1 hybrids with P. concinna/P. floridana individuals. This indicates not only that the hybrids are viable and able to reproduce, but that backcrosses of F1 hybrids occur only with individuals of P. concinna/P. floridana, further supporting the hypothesis that hybridization may be driven by lower population sizes in P. alabamensis. Future studies using more loci can better assess the level of genomic introgression among these co‐occurring species. Finally, based on mtDNA data, we found one female in Dog River that was morphologically identified as P. alabamensis that possessed a P. peninsularis haplotype. The home range of P. peninsularis is isolated to the Florida peninsula and is not native to the range of P. alabamensis. It is possible that this individual represents a P. peninsularis × P. alabamensis hybrid offspring of a female P. peninsularis that was released into Dog River and bred with native P. alabamensis.We observed admixture among the three species. Individuals of o Mount, . We founis Table . HybridiP. alabamensis is experiencing significant admixture with congeneric co‐occurring species across its entire restricted distribution range. Hybridization is a well‐known phenomenon in species of conservation concern with limited population sizes  levels in P. alabamensis populations of higher conservation concern due to their low population sizes and consequent inbreeding and hybridization: Bayou La Batre, Biloxi, Weeks Bay, and Fowl River. Furthermore, considering the observed genetic distinction of populations from Alabama and Mississippi, specific management actions should be developed to preserve their uniqueness. This includes searching for additional unknown branches of these main riverine systems where the species could occur. Finally, climate change is predicted to strongly affect coastal areas and wetlands in the Gulf of Mexico , but never funded. Local monitoring activities to ensure habitat protection of the few sites where the species occurs, the potential development of a head‐start program for genetically pure P. alabamensis individuals, education of local citizens on the consequences of translocating and moving turtles to different water bodies , maintenance of nesting sites, assessment of recruitment throughout the species’ range, and monitoring of population sizes should therefore be developed for this species.Our results also identify several Nickolas Moreno: Data curation (lead); Formal analysis (lead); Resources ; Writing – original draft ; Writing – review & editing . Andrew Heaton: Data curation (supporting); Resources (supporting); Writing – review & editing (supporting). Kaylin Bruening: Resources (supporting). Emma Milligan: Resources (supporting). David Nelson: Conceptualization (supporting); Resources (supporting); Writing – review & editing (supporting). Scott Glaberman: Conceptualization (lead); Formal analysis (supporting); Investigation ; Methodology ; Resources (supporting); Supervision ; Writing – review & editing . Ylenia Chiari: Conceptualization (lead); Formal analysis (supporting); Funding acquisition (lead); Investigation ; Methodology ; Project administration (lead); Resources (lead); Supervision ; Writing – original draft ; Writing – review & editing .None of the authors have competing interests.