Natural and anthropogenic disasters can have long-lasting impacts on the genetics and structure of impacted populations. The 1986 Chernobyl Nuclear Power Plant disaster led to extensive contamination of the local environment and the wildlife therein. Several ecological, environmental, and genetic studies reported various effects of this disaster on animal, insect, and plant species; however, little work has been done to investigate the genetics of the free-breeding dogs that occupy the Chernobyl Exclusion Zone (CEZ).FST-based outlier analysis, we then performed a genome-wide scan for evidence of directional selection within the dog populations. We found 391 outlier loci associated with genomic regions influenced by directional selection, from which we identified 52 candidate genes.We define the population genetic structure of two groups of dogs that reside within the CEZ, one around the reactor site itself and another living within Chernobyl City. We found little evidence of gene flow and a significant degree of genetic differentiation between the two populations dogs, suggesting that these are two distinct populations despite occupying areas located just 16 km apart. With an Our genome scan highlighted outlier loci within or near genomic regions under directional selection, possibly in response to the multi-generational exposure faced. In defining the population structure and identifying candidate genes for these dog populations, we take steps towards understanding how these types of prolonged exposures have impacted these populations.The online version contains supplementary material available at 10.1186/s40575-023-00124-1. Wildlife populations can be greatly affected by disasters, whether they are natural or man-made. Disasters that result in contamination or habitat destruction can result in population declines or influence wildlife adaptation to these adverse environmental changes. The Chernobyl nuclear power plant disaster released an enormous quantity of ionizing radiation into the surrounding environment. Abandonment of military and industrial facilities, as well as subsequent cleanup and remediation efforts, resulted in further environmental contamination by a variety of non-radioactive toxic metals, chemicals, and compounds. Earlier studies investigated local wildlife responses to some of these exposures. In this study, we address the impact of this disaster on the population structure of free-breeding dogs that live around the power plant and in the nearby city of Chernobyl. In particular, we use genetic approaches to understand how these two populations of dogs interact and their breed composition, so that we may begin to understand how these populations have adapted to over 30 years of exposure to this harsh environment. In this foundational study we determined that while the two local populations of dogs are separated by only 16 km, they have very low rates of interpopulation migration. We also detected genetic evidence that suggests that these population may have adapted to exposures faced over many generations. In future studies, we aim to determine if the genetic variation detected is indeed a biological response to enable survival after multi-generational exposures to radiation, heavy metals, organic toxins, or other environmental contaminants. In this way, we then understand how the impact of environmental catastrophes such as the Chernobyl nuclear disaster can influence animal populations.The online version contains supplementary material available at 10.1186/s40575-023-00124-1. Large-scale disasters, both natural and anthropogenic, can have multiple direct and indirect impacts on wildlife populations. Natural disasters such as tornadoes, volcanic eruptions, and hurricanes, as well as man-made disasters such as oil spills, chemical leaks, and nuclear accidents can have detrimental impacts on local species, lasting for months or even years after the incident –4. Envir2 (~ 30 km radius from the power plant) that spans regions of northeastern Ukraine and southern Belarus was established as the Chernobyl Exclusion Zone (CEZ). Heavy metals, organics, and other environmental toxins left behind following decontamination and remediation efforts, along with the abandonment of military bases and industrial complexes, contributes to ongoing contamination of the locale )Our goal in this study was to 1) understand the genetic structure of these dog populations and the degree of isolation that exists within them, and 2) scan their genomes for signatures of directional selection possibly tied to local adaptation within the contaminated area. We addressed these goals through genetic analyses of two populations of free-breeding dogs within the CEZ that face different degrees of exposure Blood samples were collected from semi-feral dogs captured around the Chernobyl Nuclear Power Plant (NPP) and 16.5 km away in Chernobyl City (CC). Blood samples were taken during sterilization and vaccination procedures conducted by the Clean Futures Fund Dogs of Chernobyl program in 2018 and 2019. In total, 116 unique dogs were included in the study, 60 from Nuclear Power Plant and 56 from Chernobyl City  and a discriminant analysis of principal components (DAPC) using the 301 K Set in ADEGENET  . We used the R package SNPRelate  to calcubreeding . The FROTo generate a measure of gene flow between the populations, we conducted analyses with MIGRATE using Bayesian inference –43. BegiWe submitted blood-derived DNA from each dog for analysis on the Wisdom Panel™ (MARS). Using the calculated percentages of breed matches, we classified predominant breeds as a breed contributing more than 10% to the genetic lineage for each dog. We compared how many breeds were present in each dog and analyzed the breed counts between the populations using an unpaired student’s t-test. We also utilized a PCA for breed composition between the populations. We compared the two populations for the number of predominant breeds per individual and the maximum percentage of a single breed found within an individual. We also looked for significant correlations between these breed-associated quantifications and ROH measures through calculation of Pearson’s correlation coefficient in the stats R package .FST was higher than the expected FST in all simulations (P(Simulated FST < sample FST) = 1).We used LOSITAN to identify outlier loci associated with directional selection . OutlierThe genome location of each significant SNP identified using LOSITAN was expanded to a 10 kb genomic interval of CanFam3 (5 kb either side of the SNP coordinates). We then surveyed each of the 10 kb regions for the presence of genes and identified corresponding gene ontology (GO) terms through the Mouse Genome Informatics Batch Query Search . GO termFST measures to allow for the best estimation of loci driving genetic differentiation.Both DAPC and PCA showed discrimination between the Nuclear Power Plant and Chernobyl City populations, and clustering highlighted a similar structure Fig. . Most ofFST corroborated the patterns found via PCA and DAPC . For our second FST analysis, where we aimed to compare our results to the Eurasian free-breeding dog populations using the 147 K set, we found a similar estimate for our populations (FST = 0.0572). The pairwise FST measures across free-breeding dog populations within the Pilot et al. dataset ranged from 0.0043 to 0.0889 . We estimated the number of migrants per generation using the modes of Θ and M values for both migration to Chernobyl City from Nuclear Power Plant and to Nuclear Power Plant from Chernobyl City and generated estimates of 0.57 and 0.75 migrants every four generations, respectively Table . Though While the WISDOM PANEL may not have a comprehensive representation of breeds from which the Nuclear Power Plant and Chernobyl City populations may have been founded, we can still use the approach to compare relative breed composition. All 61 of the Nuclear Power Plant dogs and 52/55 of the Chernobyl City dogs were identified as being at least 10% German Shepherd Dog (GSD), according to the Wisdom Panel™. None of the sampled dogs in either the Nuclear Power Plant or Chernobyl City populations were determined to be purebred, with both populations averaging 25 breed matches per dog. Further investigation of the genetic ancestry of the dogs revealed that 17 of the Nuclear Power Plant dogs and 21 of the Chernobyl City dogs had at least two predominant breeds (defined here as a single breed contributing more than 10%). While GSD was the most observed predominant breed, both populations also had moderate prevalence of Eastern European breeds, such as West Siberian Laika and Caucasian Shepherd Dog Fig. . The NucFROH, we compared the breed measures to the different ROH scores. The number of predominant breeds in each dog was not tied to the number of ROH segments, the average size of ROH fragments, nor the FROH for each individual according to Pearson’s correlation coefficient . Since the average size was not a factor considered in evaluation of the FROH, we continue to utilize the FROH to evaluate inbreeding.The two populations have a similar breed makeup on average, but dogs from the Nuclear Power Plant population tended to have a higher proportion of a single breed . xrcc4 plays a crucial role in repairing ionizing radiation-induced double stranded breaks through non-homologous end-joining in mammals [cntnap2) is an additional candidate gene and is tied to the inflammatory response [Through LOSITAN, we found 391 significant outlier loci and identified 165 genes that aligned to CanFam3.1 within 10 Kb of the outlier loci. Fifty-two of these genes, proximal to 67 outlier SNPs, have GO terms that could be associated with exposure to the contamination of the environment at the Nuclear Power Plant. These include GO terms associated with DNA repair, cell cycle signaling, response to radiation, calcium ion binding, and immune function  each with a smaller number of genotyped loci, we used a much larger number of genotyped loci (~ 3X) and approx. half the number of individuals (n = 116) and were able to confirm the population genetic structure they suggested. These results, along with the pairwise FST estimates, suggest that the two populations are maintained as separate groups and are breeding predominantly amongst themselves. This was further corroborated by the comparative estimates across different Europasian free-breeding dog populations in the Pilot et al. dataset. Pilot et al. [FST than Nuclear Power Plant and Chernobyl City, despite having much more geographic distance between them, further suggesting these populations are distinct. We suggest that this high level of differentiation among these two geographically close populations could be prompted by genetic drift, caused by small population sizes and limited migration, or by strong selection not seen in the Eurasian free-breeding dog populations.We detected a significant degree of genetic differentiation between the two populations of dogs sampled at the Nuclear Power Plant and in Chernobyl City, along with almost complete clustering at the population level through the DAPC and PCA, corroborating trends that were seen in identity by state clustering analyses in Spatola et al. (in press). While the focus of Spatola et al. (in press) was different, the studies are complementary in that while Spatola et al. assessed a larger number of individuals  terms for the molecular functions of gene products that are of interest based on a putative response to the exposures from the Chernobyl disaster. These candidate genes were of particular interest because they are involved in functions such as DNA repair and cell cycle checkpoint progression, immune response, and calcium ion binding. Our ongoing studies are designed to generate additional measures to refine the identify of outlier loci representing true signals of selection from those resulting from drift. Several of the exposures faced by the population living in the region of the Chernobyl Nuclear Power Plant are known to be mutagenic. As we explore the genomes of the dogs at this location, we aim to identify genome variants that were potentially induced by the prevailing multigenerational exposures and then subjected to ongoing selective pressures to maintain them in the population. Our long-term goal with this unique population of dogs is to establish further evidence to evaluate the degree of local adaptation and thus develop measures of the impact of the exposures experience by these dogs.xrcc4 and cntnap2. Our findings are likely to inform future studies, where we intend to search these genomic regions and candidate genes for variants, novel and previously documented, to further evaluate the degree of local adaptation within the Nuclear Power Plant and Chernobyl City populations. This approach permits us to pursue the identification of local adaptation as we continue to study the genetics of this distinctive group of dogs. This work, and future studies with these canine populations, will advance our broader understanding on the genetic effects of prolonged exposures to both radiation and non-radiation toxic exposures, and the findings potentially more broadly applicable to the adverse health effects of other environmental nuclear and non-nuclear disasters in both animal and humans.Study of the dogs living within the CEZ offers a unique opportunity to examine the long-term genetic and health consequences of multi-generational exposure to radiation, heavy metals, organic compounds, and other environmental toxicants. The present study takes steps towards understanding these consequences of these adverse environmental exposures by classifying the genetic structure of these populations. Our findings demonstrate genetic population differentiation between the two sampled dog populations at the Nuclear Power Plant and Chernobyl City. Despite having similar breed makeup and being separated by only a short geographic distance, these free-breeding dog populations are reproducing independently of each other and co-occur with little gene flow. Through our outlier analysis, we identified genomic regions that have diverse allele frequencies between the populations, including candidate genes such as Additional file 1. This file contains supplementary figures 1-3 including a plot of individual inbreeding coefficients, a breed PCA and plot of highest breed match, and a correlation plot for ROH measures compared to breed measures.Additional file 2: Supplementary table 2.


FTO gene found in our samples. ADRB3-rs4994 and ABCC8-rs1799854 genes showed a significant association with BMI and waist circumference, and the KCNJ11-rs5219 gene with hyperglycemia. These results emphasize the importance of knowing the genetic variability underlying complex genetic diseases in indigenous peoples and the search for particular or rare variants.This study was carried out to investigate the frequency of genetic variants related to body mass index (BMI) and type 2 diabetes (T2D) and evaluating the potential impact of risk alleles on susceptibility to these disorders in six indigenous peoples from Brazilian Amazon region. The majority of Fst values for pairwise population comparisons among the indigenous groups are low or moderate. The indigenous people show high values of differentiation with Africans, Europeans and Southeast Asians and moderate values with East Asian and American populations, as expected. The allelic frequencies among indigenous indicate that the majority of associations observed with T2D in continental populations can be replicated in native Amazonians. The genetic risk scores calculated for T2D in indigenous are high and similar to those calculated for Americans and East Asians, while the estimates obtained for obesity are low, probably due to the low frequencies of the risk allele of the  Obesity and T2D are complex genetic disorders caused by a combination of genetic, epigenetic and environmental factors. They are related disorders, since obesity is a strong risk factor for T2D, and both conditions are strongly hereditary. Family-based studies clearly show that genetic factors play an important role in the susceptibility to T2D. More than 400 genetic risk variants in 250 loci for T2D have been identified, mainly through broad genome association studies (GWASs) . With reEpidemiological data have revealed that certain racial and ethnic groups are more (or less) likely to develop obesity and T2D. These data indicate a higher prevalence of obesity, T2D and dyslipidaemia in Native Americans, as well as in “Latinos” or “Mestizos” (resulting from the mixture between Native Americans and Europeans), than in Euro-Americans and that the risk for these pathologies increases with the highest proportion of indigenous ancestry . In thisBrazilian indigenous populations are potential targets for the studies of complex traits, such as obesity, since indigenous people are influenced by external factors in a more homogeneous way, which allows for more efficient identification of rare and low-frequency genetic variants. In this context, this pilot study was carried out in indigenous populations in which the degree of admixture with non-indigenous people is still low, which allows minimal influence of genes from miscegenation with other ethnicities. The aims were to analyse the prevalence of some variants of common risks associated with obesity and comorbidities in continental populations, to describe the genetic profile of indigenous populations in relation to these variants and to assess the potential impact of this genetic variability on susceptibility to these disorders.Arara: Karib-speaking individuals who live in six villages located near the Laranjal stream in the municipality of Altamira, PA, with a population of 349 individuals, and in four villages located near the Cachoeira Seca stream on the upper Iriri river, with 252 indigenous people .Araweté: The Araweté, of Tupi-Guarani language, currently inhabit 12 villages on the banks of the Xingú River and in the Ipixuna igarapé, a tributary of the right bank of the Middle Xingu, in the municipality of Altamira, PA. Population, 559 .Asurini do Xingú: Located in Pará, municipality of Altamira close to Igarapé Ipixuna, they have 260 individuals currently distributed in five villages on the banks of the Xingú River. Language of the Tupi-Guarani family .Gavião Kyikatêjê: Located in the Mãe Maria Indigenous Land in the municipality of Bom Jesus do Tocantins, southeastern Pará. Language of the Jê family, with a current population of about 400 inhabitants .Parakanã: Located in the Xingu basin in the municipalities of São Félix do Xingu, Pará. Language of the Tupi-Guarani family, with a population of 716 people. They currently live in 11 villages .Xikrin do Bacajá: At present, they live in 11 villages located on the banks of the middle Bacajá River, in the municipalities of Senador José Porfírio and Anapú, PA. Kayapó language (or Mebengokré), from the Jê language family. Current population, 1127 .The above information is presented in This was a pilot study of genetic epidemiology carried out with five indigenous peoples located in the southwest of Pará  and the Gavião Kyikatêjê people, in southeastern Pará, Brazilian Amazon. The epidemiological study was carried out with 628 indigenous people (332 women and 296 men) aged 18 or over, of both sexes, during healthcare visits carried out between 2007 and 2014, in cooperation with health teams from the National Health Foundation (Funasa) and later from the Special Indigenous Sanitary District (DSEI), of the Ministry of Health. Obtaining clinical and biological data was preceded by a meeting with the community to explain the work, benefits and risks, procedures used, and to obtain the communities’ consent through its leaders with formal collective authorization. All residents of the villages were invited to participate in the service and were instructed on the need for fasting for biochemical tests. All participants were evaluated in a medical consultation, starting the treatment of conditions that could be confirmed in the field. The adhesion of indigenous communities to health actions was high and the percentage of participants among those eligible was 55% among the Araweté, above 70% among the Asurini do Xingu, Parakanã and Gavião Kyikatêjê, and 100% among the Arara.After 9 hours of fasting, blood samples (4 mL) were collected into vacuum collection tubes containing sodium fluoride as a glycolytic inhibitor and the EDTA anticoagulant to determine blood glucose levels, and 4 ml were collected in tubes without additive to determine total cholesterol and triglycerides. These measurements were performed in the field laboratory. The fasting plasma glucose (FPG) results were classified as follows: normoglycemia, <99 mg/dL; impaired fasting glucose (IFG), 100-125 mg/dL; diabetes, ≥125 mg/dL. Dyslipidemia was defined as a serum cholesterol level ≥ 200 mg/dL or triglyceride level ≥150 mg/dL.Hypertension was defined as a systolic blood pressure (SBP) ≥ 140 mm/Hg and/or a diastolic blood pressure (DBP) ≥ 90 mm/Hg. Body mass index (BMI) classification used the WHO criteria for adults . A waistCAPN10-rs3792267, CAPN10-rs5030952, ABCC8-rs1799854, KCNJ11-rs5219 CTF7L2-rs7901695, CTF7L2-rs12255372 and PPARG-rs1801282, associated with T2D. ADRB3-rs4994 and FTO-rs8050136, associated with obesity; and ABCA1-rs9282541, associated with low levels of high-density lipoprotein (HDL-C). Genotyping was done using a TaqMan SNP genotyping assay  according to the manufacturer’s instructions. Pre-designed probes were ordered for genotyping analyses. Approximately 10-50 ng of DNA was amplified with 5 μl of 2X TaqMan Universal PCR master mix, 0.5 μl of 40X primer and TaqMan probe dye mix. Cycling conditions consisted of 10 min at 95 °C, followed by 40 cycles of 15 s at 92 °C and 1 min at 60 °C. Allelic discrimination was performed on an Applied BioSystem RT-PCR system - Real-time PCR. These genes were selected for this pilot study using as a criterion previous studies demonstrating the association of variants with obesity and/or type 2 diabetes in continental populations, and for which there were results described for Native American people, from Brazil, in particular, or for the Brazilian population in general.Genomic DNA was extracted from 300 mL of EDTA-treated blood using the NeoIsoColumn kit  according to the manufacturer’s instructions. DNA was eluted into 200 μL of elution buffer (provided with the kit). The following single nucleotide polymorphisms (SNPs) were analysed: a priori genotypic models  was performed to calculate risk allele-specific odds ratios (ORs), 95% confidential intervals, and corresponding p-values, after adjusting for sex and age. These analyses were performed with the Statistical Package for the Social Sciences (SPSS) for Windows, version 20.0 . An overall significance level of 0.05 was set for statistical analyses, considering that the tests were applied to 10 genetic markers, the critical p-value to be considered after Bonferroni correction is 0.005. Composite genetic scores (CGS) for obesity and type 2 diabetes, considering the cumulative effect of all variants on the basis of the number of risk SNPs and the number of copies of risk alleles, were estimated at the population level according to Allele frequencies, Hardy-Weinberg equilibrium (HWE) for each locus, mean heterozygosities, interpopulation variability (Fst) and analysis of molecular variance (AMOVA) were determined using the Arlequin v.3.5 program . EffectiThis study was approved by the National Research Ethics Committee (CONEP) Report no. 1,062/2.006 and Report no. 961,451/2,015).Overall, 628 individuals were studied (52.9% women and 47.1% men), with ages ranging from 18 to 88 years and from six indigenous groups in the Brazilian Amazon: Arara, Araweté, Asurini do Xingú, Parakanã, Gavião Kyikatêjê and Xikrin do Bacajá. The mean age was 38.7 ± 16.6 years, and there was no difference between men (39.6 ± 17.3 years) and women (37.8 ± 15.9 years) (p = 0.089). Anthropometric and metabolic variables are presented by sex in High levels of fasting blood glucose were found in 8.6% of the investigated sample (52/607), of which 1.2% were diagnosed as type 2 diabetes. Men and women had similar prevalence.The overall prevalence of dyslipidemia was 30.4% , and the highest proportion was observed among the Asurini do Xingú (30.0%). The prevalence of arterial hypertension was 1.8% in total, and there was no difference between genders.The allele frequencies observed for 10 variants in eight genes related to T2D and obesity in the six indigenous populations are shown in ARL, Arara; ARW, Araweté; ASX, Asurini do Xingu; XKB, Xikrin do Bacajá; PRK, Parakanã; GAV, Gavião Kyikatejê; BRA, Brazilian Amerindians; EUR, European AFR, African; AMR, American; EAS, East Asian; SAS, Southeast Asian. ABCA1 (rs9282541) in the Araweté people, FTO (rs8050136) in Gavião Kyikatêjê, and CAPN1063 (rs5030952) and CTF7L2B (rs12255372) in Parakanã. The effective population sizes Ne obtained for all polymorphisms studies were presented in The intrapopulation variability observed in indigenous populations based on the expected average heterozygosity under the Hardy-Weinberg hypothesis for all loci in the set of indigenous populations was 0.320 ± 0.183, ranging from 22.9% in Araweté to 35.3% in Xikrin do Bacajá . Hardy-WThe levels of genetic differentiation observed among indigenous groups based on Fst values were classified as low (≤ 0.05) or moderate (> 0.05 and ≤ 0.15) in most of the paired comparisons. However, the Araweté exhibited somewhat higher differentiation coefficients (0.168) in relation to the Asurini do Xingú and Parakanã people, which in turn also exhibited a somewhat higher coefficient in relation to the Arara. Regarding the continental populations, the indigenous people presented high values of differentiation in comparison with Africans, Europeans and Southeast Asians, in decreasing order. Fst values indicative of moderate genetic differentiation (<0.15) were observed in relation to East Asian and American populations .ARA, Arara; ARW, Araweté; ASX, Asurini do Xingu; XKB, Xikrin do Bacajá; PRK, Parakanã; GAV, Gavião Kyikatejê; BRA, Brazilian Amerindians; EUR, European AFR, African; AMR, American; EAS, East Asian;; SAS, South Asian. Fst = <0.05: low genetic differentiation; Fst = 0.05-0.15: moderate genetic differentiation; Fst => 0.15 high genetic differentiation;The analysis of molecular variance (AMOVA) revealed that most of the genetic variation among indigenous people occurs within populations (~90%) and that ~10% occurs among populations. When the AMOVA includes samples from the 26 populations of 1000 Genomes Project grouped according to their continental regions and our six populations grouped in an Amazonian cluster, most of the genetic variation also occurs within populations (85.5%), while the variation that occurs among groups is 12% and the variation among populations within groups is 2.5%.ADRB3 (rs4994) gene showed a significant association with waist circumference measurements and BMI. The ABCC8 (rs1799854) gene and the KCNJ11 gene (rs5219) showed lower p-values (<0.05) in association analysis with BMI and hyperglycemia, respectively, under dominant and co-dominant models  and anthropometric (BMI and waist circumference) changes in the indigenous populations studied are still low to allow robust analyzes of the association with genetic variants, the t models , althougCAPN1043, CAPN1063, ABCC8, KCNJ11, CTF7L2A, CTF7L2B and PPARG), revealed mean risk scores for ranging from 0.484 in the Arara group to 0.620 in the Xikrin do Bacajá group. For obesity (ADRB3 and FTO genes) the lowest average risk score was calculated for the Parakanã (0.188) and the highest for the Araweté group (0.413)   and 3.  and 3.COverall, the prevalence of excess weight (31.7%) among the natives of the Brazilian Amazon analysed in this study was lower than that described for urban Brazilian adults (63.1%), as was the prevalence of obesity (6.4% vs 22.9%) . HoweverThe prevalence of metabolic changes among the Amazonian indigenous peoples studied here  was still very low, being much lower than that observed in non-indigenous Brazilian adults: 52.6% IFG and 20.0% T2D . They arThe whole indigenous population investigated here exhibited low prevalence of nutritional and metabolic changes, with the exception of the Gavião Kyikatêjê and Asurini do Xingu, in which high frequencies of obesity and abdominal obesity were observed, certainly as a reflection of accentuated cultural and environmental changes experienced by these groups. The available data reveal that the process of “westernization” of customs is ongoing, to varying degrees, and that the nutritional and epidemiological transitions are at early stages. ADRB3) was found with slightly higher frequencies (average of 39.3%) in indigenous people than in continental populations (frequencies ranging from 8.2% in Europeans to 15.7% in Southeast Asians) and can be considered a good marker for association studies in indigenous. However, in the FTO gene, the risk allele (A) of the rs8050136 variant was absent or found at very low frequencies among indigenous people. So, we could infer that this estimate of genetic risk was weak because of the low allelic frequency found for FTO in associations with changes in BMI in indigenous people of the Amazon. FTO has been recognized as a major genetic factor for obesity risk in different human groups. In addition to showing signs of balancing selection in Europeans , found with frequencies ranging from 0.4% to 22% (average of 10.7%) in natives of the Amazon, was found only in Americans (4.2%) in ABCA1 cholesterol transporter gene (ATP-binding cassette transporter A1) is associated with low levels of high-density lipoprotein (HDL-C), constituting a functional variant that may have contributed to the adaptive evolution of Native American populations  of rs9282541  compared to Africans, Europeans and Southeast Asians, but a genetic differentiation classified as moderate in relation to American populations and East Asian populations. This finding is in line with expectations considering that available data confirm that current Native Americans are descended from human expansions that entered North America from East Asia. However, issues such as number, mode and time of secondary migrations to the Americas are still the subject of intense debate , the most commonly investigated ADRB3 variant, with abdominal obesity and excess weight among indigenous people corroborates results already described in different populations. The ABCC8 and KCNJ11 genes are responsible for the coding of ATP-dependent potassium channels (KATP) expressed in pancreatic beta-cells, which play a central role in the glucose-stimulated insulin secretion of pancreatic beta cells. These two genes (ABCC8 and KCNJ11) are adjacent and located on chromosome 11p15.1. Studies have shown that variants in KCNJ11 are associated with T2D in Europeans in the United Kingdom (ABCC8 have been correlated with T2D in Japanese populations (ABCC8 gene (rs1799854) with abdominal obesity found among the indigenous people studied here is probably a spurious association and unrelated to biological processes. The low p-values (<0.05) from association of KCNJ11 (rs5219) with hyperglycemia, on the other hand, is in line with that observed in other populations.The β3 adrenergic receptor, encoded by the isorders . Thus, t Kingdom , and varulations , Caucasiulations  and Chinulations . Thus, tAlthough our samples are representative of the populations studied and the care taken with regard to phenotypic data collection, our study has some intrinsic limitations, especially with regard to sample size and to complex traits. Association studies are more robust when applied to large samples , especially when considering the application of correction of multiple tests (such as Bonferroni), something difficult to be circumvented when considering the small sizes of the Amazonian isolates. Also, it is difficult to find association of genetic markers with complex traits, as multiple genetic factors contribute to the phenotype. Additional studies in Amazonian populations may give us more clues about variants related to the important phenotypes considered in this study.FTO) variant among indigenous, unlike that observed in other ethnic groups.The comparison of the average genetic risk scores estimated for T2D in indigenous peoples and continental populations showed that the values observed in indigenous (0.592) are high and more similar to those calculated for East Asians (0.503) and Americans (0.481), and slightly higher than those calculated for Africans (0.438), Southeast Asians (0.395) and Europeans (0.414). These results are in line with expectations considering the ancestry shared by indigenous, American and East Asian peoples and deserve to be confirmed with additional case-control studies. On the other hand, the average genetic risk scores for obesity in indigenous and continental populations showed that mean value in indigenous (0.312) is smaller than those calculated for all continental groups that vary from 0.517 in East Asians to 0.666 in Africans and Europeans. This is certainly due to the very low frequency of the risk allele (A) of the rs8050136 (FTO gene. Despite the low prevalence of nutritional and metabolic alterations in most of the analyzed indigenous groups, a significant association was found between the ADRB3 gene and abdominal obesity and excess weight, and the KCNJ11 gene with hyperglycemia for indigenous people in general. The results obtained in this pilot study also underscore the importance of knowing the frequencies of risk alleles related to complex genetic diseases in indigenous people in order to assess the potential of these variants as biological risk factors and to identify those that may constitute a panel of mutations to be used in the prevention of diseases, such as obesity and comorbidities among indigenous and admixed populations, such as those in Brazil. Moreover, the results suggest that a further analysis of the underlying genetic profile to obesity and comorbidities has good prospects to contribute important information on the genetic basis of these diseases in indigenous, especially in the context of the current epidemiological and nutritional profile of indigenous peoples in the middle Xingu after the compensation policies used for the construction of the Belo Monte Hydroelectric.In summary, the epidemiological data obtained in this study reveal that the studied indigenous populations are in an initial stage of transition, which would allow health education actions to be used to contain or minimize their progression. These data are very important from an epidemiological point of view because they reflect a peculiar period in which the indigenous peoples of the Xingu had not yet been socially impacted by the construction of the Belo Monte Hydroelectric, mainly due to the abundant distribution of financial resources and subsistence items for the indigenous people, mainly from 2012. The genetic profile of indigenous people analyzed in this study was characterized by differentiation indices classified as moderate or high in relation to continental populations, but the distribution of allele frequencies among indigenous pe indicates that the majority of associations observed with T2D in continental populations can be replicated in indigenous people of the Amazon. The genetic risk scores calculated for diabetes in indigenous people are high and similar to those calculated for Americans and East Asians, while the risk scores for obesity are low, probably due to the low frequencies of the risk allele studied in the


Liocarcinus depurator  located along this transition were analysed in six consecutive years (2014–2019) using a fragment of the COI (Cytochrome Oxidase subunit I) gene. All sequences (966) belonged to two well defined haplogroups: ATL (most abundant in Atlantic waters) and MED (predominant in Mediterranean waters). Following a geographic variation, the frequency of ATL decreased significantly from Cadiz to North Catalonia. However, this variation presented steps due to the effect of oceanographic restrictions/fronts. Significant effects were recorded for GS , AOF  and IC (2016). The intensity and precise location of these fronts changed over time. Multivariate analyses distinguished three main population groups: Cadiz, Alboran Sea and the remaining Mediterranean populations. These findings could be relevant to properly define Marine Protected Areas and for conservation and fisheries policies.An interesting evolutionary question that still remains open is the connectivity between marine populations. Marine currents can favour the dispersal of larvae or adults, but they can also produce eddies and gyres generating oceanographic fronts, thus limiting gene flow. To address this subject, we selected the Atlantic-Mediterranean transition, where several fronts are located: Gibraltar Strait (GS), Almeria-Oran Front (AOF) and Ibiza Channel (IC). Seven populations of the marine crab  Additionally, marine currents and counter currents, eddies and other displacements in the three-dimensional marine environment can largely affect not only larval dispersion, but also the distribution of juveniles or adults, thus transporting them at great distances from where parental spawning took place. These processes would thus generate appreciable gene flows7. Accordingly, connectivity among populations would highly depend on a balance between the gene flow generated by dispersing currents, and marine fronts preventing it. Furthermore, these oceanographic processes are temporally dynamic and generate high levels of temporal variability in connectivity patterns. Also, behavioural factors, especially in the pelagic larval phase, may be relevant in connectivity.A superficial/preliminary appraisal would tend to suppose that many marine species  could freely move, even far away, in the water mass. These movements would generate a high gene flow, the consequence of which would be a low population differentiation. Under this assumption, distance would be the only factor generating isolation/genetic differentiation between marine populations. However, this is not the actual situation, since available, relatively recent information on the occurrence of oceanographic barriers has been accumulating. These fronts are the product of physical movements of the water masses and can significantly restrict the movement and spread of marine species3, the Mediterranean Sea is especially adequate to study these processes due to its specific physical and biological processes at play in this area. The present Mediterranean oceanographic conditions are the result of both geological events (historic factors) and contemporary processes, which together shape the biodiversity patterns that are responsible of the present marine species distributions and of the genetic variability observed in their populations8. All these processes have contributed to produce and shape the rich biodiversity currently present in the Mediterranean Sea, which is considered a biodiversity hotspot12.Although oceanographic fronts have been described in all world oceans and seas13. It is also connected with the Red Sea via the Suez Canal. The Mediterranean Sea receives a large amount of solar radiation, low input of freshwater, and the winter northerly winds  are characteristically very dry and cold15. All these factors combine to produce a substantial evaporation of surface water in both winter and summer, which thus implies a remarkable increase in salinity, in addition to a remarkable decrease in temperature in winter. These processes very often imply that the large increase in density  contributes to the winter advection of these waters towards the bottom in some areas16. It is worth remarking that in the latter case, these processes have also the important biological implication of transporting oxygen to the deep sea. As a consequence of both winter evaporation (due to the above-mentioned processes), and summer evaporation due to solar irradiation, coupled with a remarkable decrease of river inputs due to industrialization and damming, the Mediterranean basin suffers from an important water deficit, which equilibrium is mainly supported by the epipelagic influx of Atlantic water (due to its lower density) through the Strait of Gibraltar. It then circulates along the northern shores of the Alboran Sea until Almeria/Cape Gata, where it heads then southeast towards the North African coasts17. This movement generates two anticyclonic gyres18, the western (WAG) being usually stronger and more stable than the eastern (EAG)21. The current follows along the African coast until Sicily (Algerian current), where one of its branches is deviated to the North of Italy, South of France and North of the Spanish coasts  until the Ibiza Channel22. There it usually continues southwest, but, due to particular winter conditions in some years, it may recirculate northwards along the western coasts of the Balearic Islands, thus reinforcing the Balearic current and creating a temporal cyclonic gyre in the basin23. The other branch of the Atlantic jet flows to the Eastern Mediterranean14. Thus, the dynamics of all these water masses and their circulation generate several oceanic barriers in the Western basin, such as the Gibraltar Strait (GS), the Almeria-Oran Front (AOF), the Ibiza Channel (IC) and the Balearic Front (BF)5.This sea is a semi-enclosed basin connected with the Atlantic Ocean by the Gibraltar Strait and divided in two main sub-basins (Western and Eastern) by the Sicily StraitLiocarcinus depurator   are epipelagic and freely disperse until megalopas (recruitment phase) settle on the continental shelf bottom32. Planktonic larval duration, although highly dependent on temperature, is estimated to be of at least five weeks34. Juvenile crabs grow rapidly, and sexual maturity is reached during their first year of life28. For these reasons, the adult individuals observed in a particular population and year are mainly dependent on the recruitment from the previous year.The main aim of this research was to study the connectivity between populations of benthic animals as well as to analyse how gene flow is affected by the oceanographic processes. Furthermore, and principally, we wanted to study the possible temporal variation in the connectivity patterns due to fluctuations of the oceanographic discontinuities. To this purpose, we focused on the Atlanto-Mediterranean transition along the Iberian Peninsula coasts to study the effect of GS, AOF and IC in the gene flow among populations of the marine crab L. depurator as an appropriate species to perform this study is also a consequence of the specific characteristics of a useful genetic marker, a fragment of the mitochondrial COI (Cytochrome Oxidase subunit I) gene. Previous observations detected that L. depurator haplotypes could be clearly classified in two well-differentiated groups: one predominant in Atlantic waters, whereas the other is mainly detected in Mediterranean waters35. Accordingly, this species was chosen as an adequate organism to test whether gene flow between populations is affected by oceanographic fronts, since it is also commonly distributed along the whole region of interest (the Atlanto-Mediterranean transition). Furthermore, it presents a very valuable genetic marker to study the connectivity processes and interannual variability between their populations.The selection of L. depurator populations. Using the COI gene fragment, the two main aims of our research were to estimate the genetic structure and the connectivity of seven populations of L. depurator distributed along the Atlanto-Mediterranean transition . Accordingly, we intended to assess the effect of the marine barriers located in this area  on the gene flow between populations. For this objective, the selected populations were located on each side of these barriers. Thus, Gibraltar Strait is placed between Cadiz and West Alboran, the Almeria-Oran Front is flanked by East Alboran and Alacant, and the Ibiza Channel is located between Alacant and Valencia. Acting as controls, two populations not affected by any barrier, Ebro Delta and North Catalonia, were also analysed. Finally, since these oceanographic fronts are temporally semi-permanent and their precise placement is relatively dynamic over time37, it was assumed that their intensity and precise placement may be useful to elucidate to what extent their variations could influence the connectivity levels between populations estimated from crab populations surveys along six consecutive years (2014–2019).The hypothesis of our research was based on whether oceanographic fronts condition the connectivity among L. depurator sequences obtained according to population and year are presented in Table The parameters estimating the molecular diversity of the 966 35, thus confirming the occurrence of two stable haplogroups. In the present research, 64 out of 118 different described haplotypes belonged to the ATL haplogroup, whilst 54 to the MED haplogroup. All four sequences reported from IALB (Alboran Island) belonged to the ATL group.Concerning the networks computed with the haplotypes, either studying all sequences together Fig.  or separST and Snn values along with their respective P values  were computed, and the corresponding results presented in Table L. depurator . Moreover, a variation pattern of both haplogroups was observed along the Atlantic-Mediterranean study area, the ATL haplogroup being the most frequent in CADI, while decreasing its proportion towards the populations located further inside the Mediterranean , which is the stage that performs the final settlement to the benthos. Most crab species are coastal or continental shelf dwellers25, but during larval development, the first zoeal stages are usually located in epipelagic waters40. Subsequent zoeal stages are usually found in deeper waters, but megalopae are again usually associated with surface or near-surface waters, as is the case of the present species32.In the marine benthic and epibenthic environment, species population dispersion takes place mainly during the pelagic larval stages, since larvae may travel larger distances when associated to oceanic currentsCOI mitochondrial gene of L. depurator, which shows a large molecular variability35, is an excellent model to try to understand the connectivity patterns among its populations along the Atlantic-Mediterranean transition, where currents and three potential oceanographic fronts condition gene flow. Likely, the larger WALB diversity could be due to the mixture of Atlantic and Mediterranean waters in the Alboran Sea region. Over the study area, the smallest diversity estimates corresponded to VALE, probably as a consequence of the engulfed waters in the collecting area, which may act as a cul-de-sac, thus generating higher levels of population isolation. Also, this population is the last of the studied areas potentially receiving direct gene flow via the Northern current. However, and as in previous studies35, all haplotypes could be classified in just two haplogroups, ATL and MED. The haplotype coalescence time was estimated at between 113–138 kya and 34–46 kya8. This last estimate would correspond to a period before the Last Glacial Maximum, when both haplogroups likely diverged. This period, which was characterized by a sea level decrease of around 120 m41, was coupled to a drastic descent of sea temperatures in the North Atlantic, which also produced an intensive cooling of the Alboran Sea42. Therefore, both haplogroups may be the result of adaptation to these different environmental conditions, which still exist presently, due to the marked differences in temperature and salinity between Atlantic and Mediterranean waters. Currently, the gene flow consists mainly in the gene content of individuals from the ATL haplogroup entering the Mediterranean across GS.In this context, the 527 pb fragment of the 43, or to the possible occurrence of advanced zoeal stages in the deeper Mediterranean outflux, which both imply moving a fraction of Mediterranean larvae into the Atlantic. The proportion of ATL frequencies in the Alboran Sea region changed over time . Between VALE and ALAC the IC usually allows the current to reach the ALAC areaL. depurator COI haplotypes, taking into account all populations and years, the general overview depicted by the multivariate analysis identified three main areas: Gulf of Cadiz, Alboran Sea, and the remaining Mediterranean populations along the Iberian Peninsula , characterized by their low proportion of the ATL haplogroup  in Merluccius merluccius. The possibility of selective differences between ATL and MED haplogroups deserves to be further studied in the future.From these results an interesting question arises: if the ATL haplogroup is constantly penetrating into the Mediterranean Sea, why does it not increase in frequency until it reaches fixation? This would be the expected consequence under a migration model. However, in the studied region, two main evolutionary factors would coexist: migration and selection. Although haplotypes from the ATL group are continuously reaching the Mediterranean populations, they are not so well adapted to the present environmental conditions as those from the MED area. The higher temperature and salinity of Mediterranean waters would be some of the most important environmental conditions to which 78 an increase in temperature, and therefore, in changes in other environmental factors, should be expected, generating adverse conditions for ATL L. depurator crabs in the Mediterranean. Although population genetics models of migration-selection are complex81, they deserve to be explored and applied to L. depurator in the Atlantic-Mediterranean transition. Moreover, it would be necessary to follow studies of temporal series to better understand the evolutionary dynamics of these populations.Furthermore, under the marine global warming scenarioL. depurator in the Atlantic-Mediterranean transition could be used as a model species with high genetic richness that could contribute to understand the evolutionary dynamics of marine species with similar overall patterns of dynamic biogeography. Furthermore, the information obtained can be useful for properly describing Marine Protected Areas in species with high intraspecific genetic diversity, as well as to develop efficient conservation strategies.In summary, L. depurator samples , and the Gulf of Cadiz in the Eastern Atlantic (ARSA surveys). All samplings were performed on board the research vessel R/V Miguel Oliver. The sampling gear used in the MEDITS surveys was the experimental bottom trawl gear GOC-7385, with a vertical opening of 3 m and a codend stretched mesh size of 20 mm. The ARSA surveys used a Baka 40/60 trawl gear as used in Atlantic ICES fisheries research surveys83. Once on board, the total catch was separated by species, counted, and weighed before biological sampling by species taking place. Occurrence and abundance data were then standardized by swept area to obtain information on density and biomass as well as additional fisheries research information.The sampled area included the continental shelf and upper/middle slope from depths around 40 m down to 800 m from the Gulf of Cadiz  to Cape Creus (Northwest Mediterranean Sea). les Fig.  were obt35. Thus, the Gulf of Cadiz (CADI) and the Western Alboran Sea (WALB) are separated by the Gibraltar Strait. No apparent major discontinuity is however present between WALB and Eastern Alboran Sea (EALB). The Almeria-Oran Front (AOF) is present between EALB and Alacant (ALAC) sector i.e. between Cape Palos and Cape La Nao. No apparent oceanographic barriers are located between ALAC and the Valencia sector (VALE), located between Cape La Nao and the Columbretes Islands. The exception would be the occasional winter “closure” of the Ibiza Channel, when a frontal oceanographic barrier that occasionally restricts the north–south of the Northern Current that runs along the continental shelf break, forcing it to in a north–south way, and then forces it to return northwards along western Mallorca65. The Ebro Delta sector (DELT) expands from the Columbretes islands to north of the Ebro River, on a wide continental shelf area, and is widely affected by the outflow of the Ebro River, which implies a concomitant decrease in salinity throughout the whole area. North of the Ebro Delta, the Catalonia sector (NCAT) constitutes a geomorphologically diverse region between the Ebro Delta and the Pyrenees, with a markedly indented continental shelf with steep submarine canyons in the north, along which the Northern Current flows.The seven target populations were selected so that they were located at either side of putative oceanographic barriers Fig. , or by dEach population was sampled consecutively each year from 2014 to 2019, except for EALB and NCAT, which could only be sampled in the period 2016–2019, although with usually low sample sizes. In 2019 it was also possible to collect a small sample from the Alboran Island (IALB), located in the middle of the Alboran Sea, between the European and northern African coasts.28. Therefore, only those crabs presenting a carapace width of 41 ± 6 mm were selected, since they would correspond to one year old mature individuals35. Accordingly, and for each year, the main phase of dispersion (planktonic larvae) that gave rise to our sampled crabs would have taken place during the previous year winter. Therefore, the oceanographic conditions found on the previous year would be those affecting larval dispersal35.Given that crabs of different age classes can usually be observed in natural populations and trying to avoid using individuals from different cohorts within the same year, all crabs used for genetic analyses were adult crabs, according to the growth characteristics of this species, suggesting that adult Mediterranean populations strongly depend on the previous year spring recruitment86 (2014–2017 samples) or QIAmp® DNA Mini Kit (Qiagen Inc.) following the provider’s instructions (2018 and 2019 collections). A partial fragment of the COI (Cytochrome Oxidase subunit I) mitochondrial gene was amplified by PCR using the universal primers LCO1490 and HCO219887. The total volume of the reaction was 20 µl which contained: 1 µl of DNA, 12.7 µl of water (ECOLAV), 4 µl of buffer (× 5) (Promega), 1 µl of MgCl2 (25 mM) (Promega), 0.5 µl of dNTPs (1 mM), 0.4 µl of primer Forward , 0.4 µl of primer Reverse  and 0.2 µl of GoTaq (5U/µl) (Promega). The amplification protocol was: 4 min at 94 °C, 30 cycles of 1 min at 94 °C, 1 min at 48 °C, 1 min at 72 °C and a final extension of 7 min at 72 °C. PCRs were cleaned with ExoSAP, which is a mixture of 1.2 U of Exonucleasa (Epicentre), and 1.2 U of “Thermosensitive Alkaline Phosphatase” (Promega), and were incubated 15 min at 37 °C and 15 min at 80 °C. DNA sequencing were carried out by Serveis Científics i Tècnics de la Universitat de Barcelona .One section of the third or fourth pair of legs was immediately extracted on board, dehydrated, and preserved with 96% ethanol in a labelled vial and stored at 4 °C. Once in the laboratory, DNA was extracted using Chelex resin 10% (Fluka)88 to obtain a final fragment of 527 bp. The number of haplotipes (Nh), haplotype diversity (Hd) and nucleotide diversity (π) was calculated for each population and year using DnaSP v5.10.1 89. In order to follow the same haplotype nomenclature, in the present work we continued the haplotype series described in35. Haplotype network was constructed using the Median Joining network algorithm90 implemented in the NETWORK v5 software (Fluxus Technology) and phylogenetic tree using Neighbour Joining method of MEGA X91. With DnaSP v5.10.1 software89, GammaST genetic distance values were computed, and their significance (P < 0.05) was calculated using Snn statistic92. In all cases of multiple comparisons, the FDR correction was applied93, and significance was reported for P < 0.05. Geographic distances, to explore the possible correlation between genetic and geographic distances, were obtained with Karto V5.294, following the 200 m depth isobathic line. To obtain this correlation for each year, Mantel tests were calculated. Furthermore, to analyse the distribution of all population samples a PCoA  was computed. For this study, the only NCAT population used was that of 2019, given the scarceness of collected individuals in the rest of the years. Finally, the dependence of the haplogroup frequency on the geographic distance and the year of capture was analysed using regression models (ANCOVA and linear regression). All these computations were carried out using R language functions95.All sequences were visually inspected, aligned and trimmed using BioEdit v7.2.6.1Supplementary Figure S1.Supplementary Figure S2.Supplementary Legends.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.


Amphibalanus eburneus is a marine crustacean, which presents near-cosmopolitan distribution due to extensive introduction and exhibits a wide spectrum of phenotypic variation. To elucidate geographical differentiation among populations through invasion, we investigated variation in genetic structure, shell morphology, and recruitment pattern for A. eburneus, from the southern Korean Peninsula where it has been established since the late 1980s. We selected samples from four populations in corresponding ecologically-relevant regions representing all surrounding South Korean waters. From these we amplified the mitochondrial genetic marker cytochrome oxidase subunit I (COI) from 57 individuals and performed a populational genetic analyses with 11 additional GenBank sequences to evaluate population structure. To examine morphological variation, we applied two-dimensional landmark-based geometric morphometrics to the scutum and tergum for 148 and 151 individuals, respectively. Furthermore, we estimated the density of year-old individuals in the field to compare recruitment responses among localities. We detected 33 haplotypes among the four locations belonging to three distinct clades based on moderate intraspecific pairwise genetic distance (≥3.5%). The haplotypes in these clades were not locality-specific in their distribution. In contrast, we did detect interpopulation variation in opercular shape and morphospace structure, and one population could be separated from the rest based on its distinct tergum morphotype alone. This morphologically distinct population was also differentiated by displaying the lowest mean recruitment density. Our results indicate that although there is no relationship between molecular variation in the COI gene and geographic regions in South Korea, association with locality for operculum morphology, and recruitment response suggest ecological adaptation by this barnacle in a new habitat.The ivory barnacle  These regions provide distinct habitat conditions for marine life due to their unique ocean dynamics and physical environments, leading to biogeographic division of three ecoregions characterized by different biota . The wesA. eburneus emergence was noted in an ecological monitoring project in the Korean harbors (“Improvement of management strategies on marine ecosystem disturbing and harmful organisms”). Underway since 2013, the project is aimed at monitoring various alien marine organisms invading the Korean harbors through ship fouling or ocean currents. During a survey in April 2021, extensive A. eburneus spread was documented in four major harbors, namely Incheon, Tongyeong, Sokcho, and Hanlim, representing all surrounding Korean waters, including the Yellow Sea, East China Sea, Korea Strait, and East Sea (Sea of Japan). Considering the substantial geographic distances and heterogeneous habitat conditions among these regions, we speculated that A. eburneus might exhibit adaptive genetic and morphological variation across geographies and possible routes of invasion.Recently, A. eburneus populations using a mitochondrial genetic marker. Then, we examined variations in the shape and left-right symmetry of the opercular plates of A. eburneus, which are of evolutionary and taxonomic significance , Tongyeong , Sokcho , and Hanlim  harbors . We acceeburneus  remains eburneus . We did eburneus .We randomly selected 30 individuals from each locality sample and isolated genomic DNA with the aid of the LaboPassTM Kit  following the manufacturer’s protocols. We amplified partial sequence of the mitochondrial gene cytochrome C oxidase subunit I (COI) with polymerase chain reaction (PCR) using PCR premix  in an AllInOneCycler™ PCR system . We used the universal primer pair, jgLCO1490 (5`‒TIT CIA CIA AYC AYA ARG AYA TTG G‒3`) and jgHCO2198 (5`‒TAI ACY TCI GGR TGI CCR AAR AAY CA‒3`)  with thehttps://digitalworldbiology.com/FinchTV) to check the quality of signal and sites with possible low resolution. We deposited all obtained sequences in GenBank  and performed a population genetic analysis at the continental scale, comparing the level of pairwise genetic differentiation between all obtained sequences. We performed sequence alignment using the MAFFT v7.313 , four from Mastic beach , and three from Gomishan Wetland . We estimated uncorrected pairwise distances using Geneious prime (https://www.geneious.com/prime/) and used the TCS algorithm implemented in PopART . For each individual, we first separated the left and right sides of the opercular plates from the body and subsequently divided them into scutum and tergum. We used 5% sodium hypochlorite (NaClO) to clean the surface of dissected parts. We placed the dissected parts on a petri dish covered by black paper and included a scale to take calibrated microscope images. To make a dorsal perspective angle perpendicular to the microscope objective, we manipulated the specimen using forceps. After each specimen was adequately positioned consistently, we took images on ventral view of scutum and tergum in multiple foci using a camera DP22  implemented in the dissecting microscope. To illustrate the three-dimensional depth of field more fully, we used stacking software Helicon Focus 7.7.5  to combiWe generated two TPS files for the scutum  and the tergum  separately to evaluate the geometric variation in size and shape, including the asymmetry in side-by-side pairs (matching symmetry between left and right parts) . We geneWe employed algorithms implemented in Morpho J package software ver. 1.07d  for all A. eburneus per attachment plate across sampling areas as the number of barnacles found in each plate photograph by its area (9 dm2). We counted the individual number of A. eburneus based on pixel analysis of photographs using ImageJ software . The average nucleotide frequency of the aligned sequences was 28.8%, 37.4%, 16.1%, and 17.7% for each A, T, G, and C, respectively. In other words, the sequences were AT-rich (66.2%). The uncorrected pairwise distances between the four populations ranged between 0 and 3.5% , with thThe parsimony network generated with TCS  detectedA. eburneus, the evidence of population genetic structuring in Korea was not statistically significantly different as revealed by pairwise Fst analysis  and tergum . However, neither population nor asymmetry side-by-side contributed to the observed variations in the size of the scutum  and tergum . The effect of asymmetry side-by-side was significant, contributing to the variations in the shape of the scutum  and tergum  and exceeding that of individual variability. The population most significantly contributed to the variations in the shape of the scutum  and tergum , and its contribution exceeded that of the asymmetry by the side.The ANOVA results  yielded p = 0.011) and the right  scutum datasets, thus the null hypothesis regarding isometric shape development was rejected. PCA based on residuals revealed major shape variations of the left  and right  tergum datasets. The first two PC axes of the left . CVA of the left and right tergum datasets showed that the first two axes explained respectively 81.4%  and 88.5%  of the total variance.Based on the Mahalanobis distances , the TonA. eburneus recruitment in the surveyed areas. The Incheon population showed the most significant mean density at 65.27 indi/dm2, followed by Hanlim, Sokcho, and Tongyeong populations .A. eburneus. Based on the pairwise distance of the COI sequences, A. eburneus populations showed a low diversification rate. The values ranged between 0% and 3.5%, falling within the level of intra-specific variability for this species, compared to the much higher inter-specific values of COI sequences in other balanomorph barnacles  has been found to exhibit a complex haplotype admixture with global distribution , showed a distribution confined to eastern and southern parts of the Korean Peninsula owing to its weak capability to vary physically (The present study investigated geographical differentiation in the genetics, morphology, and recruitment of Korean populations of alien barnacle arnacles . Parsimoeratures , compararibution  based onribution . It showribution . Contrarysically . Indeed,ysically .A. eburneus. Indeed, fouling organisms including barnacles often form dense populations on ship hulls as large founding populations and/or potential admixture, which is necessary for the invaders to overcome the founder effect and demographic bottlenecks (A. eburneus populations are more frequently found in ports than in exposed habitats. It is also likely that shipping along the coastline is more responsible for the gene flow among Korean populations than internal shipping. Because internal shipping across the Korean Peninsula is impossible due to the mountain range existing along the eastern half, disturbing direct linkage between the east and west coast.Parsimony network analysis revealed that Korean populations shared haplotypes with North Atlantic and the Caspian Sea populations. The genetic structure indicated by Fst value showed that there was genetic differentiation only between Korean and North Atlantic populations. This corresponded to the AMOVA results, where within-population variations accounted for the majority of the total variation. Our results might indicate a possible genetic relationship between the Korean and Caspian populations, although the latter region is geographically isolated at a large distance from the Far East. However, it is difficult to confirm such a hypothesis clearly due to extremely small numbers of sequence in the Caspian Sea population, which did not allow for establishing the overall genetic structure of both regions. Consequently, it is also challenging to speculate on the actual invasion pathway into the Korean Peninsula from our results owing to insufficient genetic information. Shipping and other anthropogenic activities might have played an important role in shaping the current population genetic structure of tlenecks . Previoutlenecks  support A. eburneus, which are reported for the first time in the study of barnacles. We identified two different aspects of morphological variations in the datasets: allometry and shape variations between sides and among populations. Allometry is a known factor contributing to morphological integration (A. eburneus have evolved in size independently, despite being connected to each other to form the opercular plate.Our LBGM analysis revealed substantial geometric variations in the opercular plates of egration , 2016, aegration . The exaegration . In thisFurthermore, similar trends were noted in the size-corrected shape variations between the sides. As such, the left and right sides of the plates were not described by the identical direction of LM shift see  and 5D. A. eburneus. Based on linear measurement results, they reported that A. eburneus in cold regions differed markedly from in the warm regions by having the basal margin of the carinal side of the tergum deeply hollowed out. Chthamalus anisopoma Pilsbry, 1916 exposed to the predatory snail Mexacanthina lugubris angelica Oldroyd, 1918; as such, under predation pressure, some juveniles developed a bent morphology. Chthamalus fissus Darwin, 1854, which possesses an oval operculum, manages predation risk by changing the shape of the plate to become narrower, which is advantageous in escaping the predatory gastropod, Mexacanthina lugubris lugubris Sowerby, 1821. In the present study, we were unable to determine possible correlations of the observed shape variations in Tongyeong populations with annual record of water temperature and salinity due to a lack of significant differences in these parameters with other localities (A. eburneus communities in the monitoring plates. Compared with direct observations in natural habitats, surveys using monitoring plates offer limited opportunities to witness natural phenomena representing the relationships among organisms due to limited space and resource accessibility. Nonetheless, based on our genetic analyses, the genetic diversity in the Korean A. eburneus populations imply a great plasticity for adaptation, which can drive variations in shape of the tergum in response to certain external stimuli. Therefore, further studies are warranted on A. eburneus in the adjacent natural habitats using additional information on their community structure and trophic relationships.Regarding population differentiation, the Tongyeong population could be clearly differentiated from the rest based on variations in the shape of the tergum on both sides of the opercular plate. Several external factors that may cause this phenomenon. High recruitment density can influence barnacle shape, often resulting in columnar shells in response to intensive competition for the space and foraging . Our anacalities . In addiA. eburneus in Korea. Genetic comparisons among four regions confirmed three clades based on 3.5% pairwise genetic distances and 33 haplotypes, albeit without regional specificity. In contrast, the quantitatively described variations in opercular plate size and shape did identify a unique convergence in narrow elongated tergum in the population from Tongyeong. This population was also differentiated from all others by reduced recruitment density. Despite the inability to predict major factors driving dispersal and morphological variation in A. eburneus in South Korea, we demonstrated an unique phenotypic form of A. eburneus among diverse genotypes at a single location.Using mitochondrial gene sequence and LBGM analyses, the present study successfully unveiled genetic structure, shell morphology, and recruitment pattern in the alien barnacle 10.7717/peerj.14002/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14002/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14002/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.14002/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.14002/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.14002/supp-6Supplemental Information 6Click here for additional data file.


Daphnia magna. For comparison, we characterized diversity in a single, large, and stable D. magna population. We found reduced (synonymous) genomic diversity, a proxy for effective population size, weak purifying selection, and low rates of adaptive evolution in the metapopulation compared with the large, stable population. These differences suggest that genetic bottlenecks during colonization reduce effective population sizes, which leads to strong genetic drift and reduced selection efficacy in the metapopulation. Consistent with the propagule model, we found lower diversity and increased differentiation in younger and also in more isolated subpopulations. Our study sheds light on the genomic consequences of extinction–(re)colonization dynamics to an unprecedented degree, giving strong support for the propagule model. We demonstrate that the metapopulation evolves differently from a large, stable population and that evolution is largely driven by genetic drift.The dynamics of extinction and (re)colonization in habitat patches are characterizing features of dynamic metapopulations, causing them to evolve differently than large, stable populations. The propagule model, which assumes genetic bottlenecks during colonization, posits that newly founded subpopulations have low genetic diversity and are genetically highly differentiated from each other. Immigration may then increase diversity and decrease differentiation between subpopulations. Thus, older and/or less isolated subpopulations are expected to have higher genetic diversity and less genetic differentiation. We tested this theory using whole-genome pool-sequencing to characterize nucleotide diversity and differentiation in 60 subpopulations of a natural metapopulation of the cyclical parthenogen  Metapopulations, i.e., subpopulations interconnected by extinction–(re)colonization dynamics, are ubiquitous . They dieN, of subpopulations and the degree of population structure adaptive nonsynonymous substitutions, NAω and Aω. Thus, population genetics can help to determine the ecological setting of a particular metapopulation and give insight into how evolutionary mechanisms differ among metapopulations  shows this pattern of metapopulation with small effective population sizes, low gene flow, and genomic bottlenecks colonization dynamics, and strong colonization bottlenecks  by single colonizers that then undergo clonal expansion  genomic diversity, (2) population differentiation, and (3) (non)adaptive evolution. Our objective is to understand how this metapopulation evolves and, more generally, how evolution in metapopulations differs from the evolution in larger, stable populations. Considering D. magna subpopulations than in older ones. Regarding population differentiation, recurrent genomic bottlenecks might lead to high differentiation between founder populations, which might, however, erode over time due to gene flow (D. magna (N and πS and (non)adaptive substitutions like NAω and Aω. Because of recurring bottlenecks, eN should be low, which might weaken the efficiency of natural selection and strengthen the effect of genetic drift. Under this scenario, we predict a genomic signature of weak purifying selection, i.e., an excess of nonsynonymous polymorphisms (high ratio of πN/πS), and few polymorphisms fixed by adaptation (low Aω). By understanding the variation in genomic diversity, genomic differentiation, and (non)adaptive evolution in this metapopulation, we try to unravel the general principles of evolution in metapopulations and how they evolve differently from large, stable, panmictic populations using empirical and simulated data.In this ene flow . This hiD. magna , across Daphnia magna metapopulation located on the Tvaerminne archipelago in southwestern Finland . Daphnia magna is a Holarctic-distributed, freshwater planktonic crustacean that inhabits small rock pools on the islands of this archipelago. The rock pools (mean volume about 300 l) are depressions in the bare rock of the islands that fill with rainwater but also sometimes collect seawater. We call them rock pools or ponds to avoid confusing them with “pools” from our genomic pool-seq samples. The shallow rock pools are mostly frozen solid in winter; many dry up during the summer as well. Environmental variables including pond geometry, water salinity, humic acid content, pH, calcium concentration, distance to the sea, and height above sea level are available for all ponds . Daphnia magna is a cyclic parthenogen; sexual reproduction results in resting eggs that allow it to survive the winter freezes and the drying out of ponds during the summer. These resting eggs disperse passively by wind, water, or birds and are, therefore, crucial for the migration and colonization of vacant habitat patches. Daphnia magna also reproduces asexually, which allows clonal expansion after colonization.This study uses samples from a natural ll ponds . Since 1D. magna from all occupied ponds (subpopulations) in the core sampling area in late May/early June of 2014  to the sample tube and ground the sample using a plastic pestle. Then, we added 20 μl Proteinase K for overnight incubation at 55°C, after which we added 20 μl RNAse for RNA digestion for one hour at 37°C. For protein removal and DNA precipitation, we followed the instructions of Qiagen GenePure DNA Isolation Kit, with the addition of 2 μl glycogen (Sigma-Aldrich) to aid DNA precipitation. We then suspended the purified DNA in 80 μl of Qiagen DNA hydration solution and measured DNA concentration using a Qubit 2.0 (Invitrogen). Libraries were prepared using Kapa PCR-free kits and sequenced by the Quantitative Genomics Facility service platform at the Department of Biosystem Science and Engineering  Basel, Switzerland, on an Illumina HiSeq 2500 sequencer. Two samples failed this sequencing step, leaving a total of 60 samples for subsequent analyses  of the variance and were ecologically meaningful. The first principal component described the impact of the sea or “marineness” , and represented a gradient from marine to terrestrial ponds. PC2 described geophysical properties independent of the sea  and represented a gradient from small to large ponds. The age of the subpopulation was assessed using biannual sampling data, with the maximum observed age being 31.5 years, as sampling started in 1982 (D. magna is 0.74 in this survey (10(age + 1)-transformed for statistical analyses. The geographical distances between ponds were calculated using the R package geodist v.0.0.7  using a principal component analysis (PCA) in R v.4.0.3   and subsequently trimmed to remove low-quality sequence and adapter contamination using the default setting on Trimmomatic v.0.39 (https://github.com/lh3/seqtk). The D. magna XINB3 individual genome  was used as the reference genome when mapping interleaved reads with bwa-mem2 v.2.2.1 (http://scicore.unibas.ch/) scientific computing center at the University of Basel using a snakemake (Ad) of the minor allele equal to 1 using VcfFilterJdk. By applying this minor allele read count filter for each individual sample, we chose a filter that is DP-aware, and thus, more conservative than the more commonly used minor allele frequency (MAF) filters in pool-seq studies to avoid sequencing errors  and the number of mitochondrial haplotypes (a rough proxy for the number of founders and immigrants). We estimated pond volume as a pyramid based on depth and surface area. We estimated the number of mitochondrial haplotypes by reconstructing them from the trimmed sequencing data, starting with mapping interleaved reads to the mitochondrial reference sequence  using bwa-mem2. We then used the resulting BAM files as input for RegressHaplo v.0.1 , we performed a multiple regression analysis using a type two ANOVA from the car v.3.0–10 , v.3.0–10  R packagv.3.0–10  online. v.0.3–14  and deco v.2.5–7 , with siD. sinensis genome  from NCBI and RNA-seq reads  from EMBL. Daphnia sinensis is closely related to D. magna  on the transcriptomes, we used diamond v.2.0.11.149 (https://github.com/brentp/pyfasta/) was used to split intermediate FASTA file outputs. To obtain transcript hints, the individual transcript files were concatenated and mapped using minimap2 v.2.22-r1105 (https://github.com/Magdoll/cDNA_Cupcake).To estimate (non)synonymous divergence and the rate of (non)adaptive nonsynonymous substitutions, we downloaded the available D. magna . We remoD. magna  and checD. magna . Specifiv.2.7.4a . The datv.2.7.4a . We checv.2.7.4a  and the 0.11.149  as well 22-r1105  before cD. sinensis genome as a reference. The rate of adaptive substitution, α, was calculated for each sample separately based on the total counts of (non)synonymous polymorphisms, Pn and Ps, and substitutions, Dn and Ds, as 1—(Ds*Pn)/(Dn*Ps) synonymous substitutions per site, dN and dS, using SNPGenie. Afterward, we calculated Aω as α(dN/dS) and NAω as (1-α)(dN/dS). We tested whether Aω and NAω were correlated with eN using Spearman correlations. eN was approximated with genomic diversity at synonymous sites, πS, using SNPGenie.We repeated the methodology for read mapping and VCF file preparation using the /(Dn*Ps)  using SND. magna population, we estimated overall genomic diversity, π, genomic diversity at (non)synonymous positions, πN, and πS, (non)synonymous divergence, dN and dS, and the rate of (non)adaptive nonsynonymous substitutions, NAω and Aω, for the D. magna population from the large Aegelsee lake near Frauenfeld, Switzerland . This population is at least 60 years old and has an estimated minimum population size of over 10 million individuals. The Aegelsee does not entirely freeze in winter nor dry up in summer. However, fall and winter conditions result in little to no overwintering of D. magna synonymous genomic diversity and effective population size in this metapopulation, we simulated different-sized populations and compared their variation in (non)synonymous genomic diversity with estimates from our collected natural subpopulations. Using a nonWright–Fisher model in SLiM v.3.6.0 , we simupulation . We simupulation . We mades = 0.0001), as reported for Drosophila (Aω and NAω more meaningful.To compare the observed (non)synonymous substitution rates in the metapopulation to in silico data, we performed a second set of simulations with substitution tracking enabled. We increased the number of generations per simulation to 100,000 and the simulated sequence length to one Mbp to get a significant number of polymorphisms that would reach fixation. Moreover, 1.5% of the nonsynonymous mutations were beneficial . As expected, most samples originating from the same island clustered together. The PCs reflected geographic distribution to some degree, with longitude being positively correlated with PC1 (Spearman's r(58) = 0.71, P < 0.001) and latitude negatively correlated with PC3 (Spearman's r(58) = −0.50, P < 0.001). The clustering by island became more defined when samples were pinpointed on a 2D map based on the first five PCs using t-distributed stochastic neighbor embedding . Interestingly, in some cases, samples from some islands fell into multiple subclusters , reflecting distinct regions on the islands (B).A total of 60 equenced . Betweene genome  online. yond 20×  online. yond 20× A. As expg t-SNE; B. Intere islands  online.  islands B.S, can be used to approximate the theoretical quantity eN, acting as a stand-in to predict how populations will behave evolutionarily. This is because πS is determined by mutation rate and eN. Since the mutation rate is similar across subpopulations, the observed differences in their πS are largely due to their variation in eN. The smaller the eN, the stronger the genetic drift, and thus, the faster the loss of mutations (S with pond volume (assuming larger ponds have larger populations and are more likely to receive immigrants) and with the number of mitochondrial haplotypes (assuming the number of haplotypes is representative of the number of colonists and immigrants). While both variables showed a positive association, as expected, it was not a strong correlation: the correlation of πS with pond volume was Pearson's r(58) = 0.25, P = 0.05, whereas the correlation of πS with mitochondrial haplotypes was F = 4.502, P = 0.038, and R2 = 0.07. The weak correlations are not entirely unexpected because the degree to which eN correlates with population census size, N, is highly variable and depends on a range of ecological and evolutionary details in an individual biological system  and sexual (fig. 3B). Therefore, observed and simulated results coincided with expectations from population genetic theory, that purifying selection is more efficient in removing deleterious nonsynonymous polymorphisms from populations with higher synonymous diversity (populations with higher eN)  (Spearman's r(58) = 0.36, P = 0.005; Aω, was higher in populations with higher synonymous diversity, a pattern also seen in the data from the metapopulation estimated with the outgroup D. sinensis . This supports that higher synonymous diversity is an approximation for higher effective population size allowing for more efficient selection. Because natural selection and genetic drift are nonindependent, positive selection may be more efficient, as it is less affected by genetic drift.  . Furtherulations  online).D. magna subpopulation. It ranged by nearly an order of magnitude from 3 × 10−4 to 1 × 10−3 with a mean of 6 × 10−4. We tested whether genomic diversity was associated with subpopulation age since colonization, NN2, ecological variables, and infection status. Using multiple regression analysis, we found that young, isolated subpopulations were less diverse than older, less isolated subpopulations  = 0.30, P = 0.02), when testing the ecological variables underlying the habitat PCA separately. This became nonsignificant when correcting for multiple testing, which was necessary because several ecological variables underlying the habitat PCA were tested.Genomic diversity, π, was estimated separately for each ulations  online. ulations  online. ll model . HoweverF-model-based FST using GESTE, had a mean of 0.06 and a range from 0.02 to 0.21. GESTE's test for association with subpopulation age, environment, and isolation suggested that genomic differentiation was best explained by a model with only the measure of isolation, NN2, i.e., the model with the highest posterior probability  = 67.961, P < 0.001, and R2 = 0.75). Furthermore, as estimated using poolfstat, pairwise genomic differentiation correlated with mean NN2 , meaning that subpopulations separated by greater geographical distance were more differentiated than geographically closer subpopulations, i.e., IBD. We also found IBD when looking specifically at combinations of subpopulations within islands (mean R2 = 0.38). Correlations between pairwise genomic differentiation and the remaining candidate variables (mean of each PC1 (marineness), PC2 (pond geometry), and subpopulation age) were nonsignificant. This pattern corresponds with results from the GESTE, pointing to IBD as the dominant pattern. On the other hand, the immediate effect of genetic bottlenecks during population founding on higher population differentiation among young subpopulations is not as strong in the overall analysis. However, the finding by FST = 0.49, CI95 = 0.47–0.51; >2 and ≤15 years old: mean FST = 0.35, CI95 = 0.33–0.37; >15 years old: mean FST = 0.36, CI95 = 0.35–0.38; Wilcoxon's W = 21,774, P < 0.001 and W = 22,103, P < 0.001 for old and intermediate age classes vs. young age class, respectively). The finding that intermediate and old age classes did not differ in mean pairwise FST  reflects the nonsignificant correlation between pairwise population differentiation and mean age and agrees with Genomic differentiation between subpopulations, as estimated with D. magna population in Switzerland. Based on the analysis of 1,056,626 SNPs, we estimated a π value of 1.5 × 10−3, about 2.5 times higher than that estimated for the entire metapopulation. The estimates of πN (1.0 × 10−3) and πS (2.0 × 10−3) corresponded with our expectations for a larger population, as they showed higher πS and more deviation from the one-to-one line than the metapopulation samples (A). Also, Aω (−0.18) and NAω (0.32) were higher and lower than in the metapopulation samples, respectively, as expected for a larger population colonization dynamics are a key aspect in distinguishing dynamic metapopulations from larger, more stable populations with gene flow . UnderstD. magna metapopulation has found high turnover dynamics, small numbers of subpopulation founders, and the accumulation of deleterious mutations in the mitochondrial genome, all consistent with the propagule model synonymous diversity and (non)adaptive evolution in insular versus continentally distributed populations of different bird species (Fringilla coelebs). eN was positively associated with Aω. We undertook a similar approach within a metapopulation of a single species with subpopulations of different ages and isolation levels and found the same relationship between πN and πS as well as πS and Aω. This suggests that πS explains variation in evolution within a species just as it does across species, as previous studies have noted deleterious nonsynonymous variants, a pattern we describe in the present work. This same pattern has been described for D. magna in We contrast our findings with previous studies using whole-genome data to explore the relationship between π species . Their cve noted . By simuN and πS, is that in all studies the datapoints fall very close to a nearly straight line. Testing this relationship for just one contig of the genome revealed a large amount of variation around this line (D. magna (sub)populations relative to the other species is unclear. It might be due to the partial asexual reproduction, although we did not observe large differences in our simulations  leads to an elevation in genetic load colonization of vacant habitat patches, whereby the expectation of s is 0.5 . We estius sizes . Founderus sizes . Under t favored . In popuulations  may, theulations .FST estimated with the F-model approach and could be shown to correlate with pairwise FST colonization dynamics. The genomic consequences of metapopulation ecology include recurrent bottlenecks during population founding, which can lead to high genomic differentiation between subpopulations and low genomic diversity within subpopulations. Even though pulation , it woulpulation . Our stupulation  and provmsac264_Supplementary_DataClick here for additional data file.