National and international efforts like the 1000 Genomes Project are leading to increasing insights in the genetic structure of populations worldwide. Variation between different populations necessitates access to population-based genetic reference datasets. These data, which are important not only in clinical settings but also to potentiate future transitions towards a more personalized public health approach, are currently not available for the Belgian population.To obtain a representative genetic dataset of the Belgian population, participants in the 2013 National Health Interview Survey (NHIS) were invited to donate saliva samples for DNA analysis. DNA was isolated and single nucleotide polymorphisms (SNPs) were determined using a genome-wide SNP array of around 300,000 sites, resulting in a high-quality dataset of 189 samples that was used for further analysis. A principal component analysis demonstrated the typical European genetic constitution of the Belgian population, as compared to other continents. Within Europe, the Belgian population could be clearly distinguished from other European populations. Furthermore, obvious signs from recent migration were found, mainly from Southern Europe and Africa, corresponding with migration trends from the past decades. Within Belgium, a small north-west to south-east gradient in genetic variability was noted, with differences between Flanders and Wallonia.This is the first study on the genetic structure of the Belgian population and its regional variation. The Belgian genetic structure mirrors its geographic location in Europe with regional differences and clear signs of recent migration.The online version of this article (10.1186/s40246-018-0136-8) contains supplementary material, which is available to authorized users. After the completion of the Human Genome Project in 2003, international efforts were initiated to map human genetic variation between populations. This variation has been described for 26 populations worldwide via the 1000 Genomes Project –7. From Therefore, a population-based cross-sectional study, called BelPHG-21 , was organized that aims at describing the genetic variability in the Belgian population and relating this variability to several indicators of health and disease to anticipate a future transition towards precision public health. To accomplish this, the BelPHG-21 study was organized in the context of the 2013 Belgian National Health Interview Survey (NHIS). The NHIS is a population-based survey that has been periodically organized since 1997. It has the purpose to assess the health status and its distribution in the population and to describe the association of this health status with its main determinants .Here, we report on the BelPHG-21 results related to the genetic structure of the Belgian population. By incorporating NHIS information about the study subject’s residence and parental country of birth, we demonstrate small but clear genetic differences between different regions and illustrate how the population’s genetic structure is shaped by recent migration waves.To study the genetic structure and variability in the Belgian population, a subset of participants from the most recent NHIS, conducted on 10,829 inhabitants in 2013 , was invDNA was extracted from saliva samples obtained from 210 consenting participants. Three of these samples contained insufficient saliva, while the DNA concentration of eight other samples was considered too low for downstream analysis  were donated by an individual with non-Belgian roots . These individuals mainly originated from neighboring countries (16), Southern Europe (7), and Africa (6). Proportionally, the highest numbers of non-Belgian samples were obtained from individuals living in Brussels , followed by Wallonia , and Flanders   analysis using continental population data  from the 1000 Genomes Project . The maiOur next goal was to examine how the Belgian population differs from other European populations. After a similar SNP filtering strategy as described above, the PC analysis was repeated using the European populations from the 1000 Genomes Project. Only the first two PCs were informative for a rather small variance of 0.76 and 0.35% respectively . Fst is a metric with values between 0 and 1 that is used to measure genetic distance between populations, in which high pairwise values indicate large population differentiation and vice versa. Fst values between the three Belgian regions were very small (between 1.8 and 2.5e−04) and smaller than with any other European population (Fst 3.2e−04 or higher) . As expIn summary, these data show minor but clear genetic differences between the different regions in Belgium with a north-west to south-east geographical correlation. However, these differences were smaller than with any other European population.The BelPHG-21 study aims at developing an approach to estimate genetic variability at the level of the Belgian population and exploring how this genetic information can be associated with information collected via NHIS questionnaires. Here, we reported on the genetic structure of the Belgian population using ± 300,000 genome-wide SNPs, derived from saliva DNA sampled from 189 participants from the latest NHIS (2013). This is the first study on the genetic structure of the Belgian population and its regional differences. The Belgian population was found to be a typical European population with signals of recent migration from the African continent. Within Europe, the Belgian population has its unique properties, which clearly mirrors the geographic orientation and again with signs of recent migration from Southern Europe. While minor genetic differences were observed between the different Belgian regions, these were smaller than with any other population studied.The linkage possibilities to health, demographic, and related data from the NHIS provide a unique opportunity to study the different factors that determine participation in a genetic study and to better understand public willingness to participate in population genetic research. Consent rates to participate in this study were rather low (14%). Related studies that recruited people with previous participation in health research reported on variable consent rates between 21 and 85% –18. The The close mirroring of population genetic variation with geography is a well-known phenomenon that has been described at the European level previously , 9. NoveBelgium is a country of immigration, with substantial labor-related migration waves after the Second World War . This isThe NHIS contains information related to health, lifestyle, environment, etc. . TherefoThis is the first study on the genetic structure of the Belgian population and its regional variation. The Belgian population is a typical European population with minor but clear differences between the regions and clear signs of recent migration.The BelPHG-21 study is a cross-sectional population-based study, conducted in 2016. The study population was selected from the participants of the most recent (2013) NHIS. Persons from the German community or with a missing national identification number were excluded for logistic reasons. Only one member per household was included, and the participants that did not complete the auto-questionnaire part of the NHIS in 2013 , that were deceased, deregistered or with a discrepancy between self-reported and calculated age were excluded. From the resulting 5768 eligible candidates, a random subsample of 1468 individuals was invited to participate in the BelPHG-21 study. Individuals were invited via postal mail. They received an invitation letter with all the information on the BelPHG-21 study as well as informed consent forms to return in case of participation. Individuals that consented to participate in the study were subsequently sent a saliva sample collection kit ) accompanied by an information letter, a user manual for sample collection, a safety bag, and a pre-paid and UN3373-labeled bubble. Each package complied with the guidance on regulations for the transport of infectious substances. All communication with study participants  was performed by a trusted third party  that was not involved in the downstream research in any way. They submitted the received samples to the BelPHG-21 research team and provided linking information to the NHIS database.Upon receipt of saliva samples by the BelPHG-21 research team, DNA was manually extracted from the total sample following the manufacturer’s instructions . DNA samples were quantified using UV absorbance, and SNPs at ± 300,000 sites were determined using the whole genome scanning 12-sample Illumina HumanCytoSNP-12v2.1 BeadChip according to the manufacturer’s instructions. Images were captured on Cytoscan (Illumina), and data were primarily analyzed using Illumina’s GenomeStudio software.Information related to the study participants’ residence (region and province), migration background, and a set of variables that could influence the consent rate was obtained from the NHIS-2013 database. A subject was considered to have a migration background when at least one of its parents was born outside of Belgium. The variables that were considered to potentially influence consent rates were the background characteristics , health behavior and lifestyle characteristics , physical and social environment characteristics , and health and wellbeing characteristics . Selection of the variables for the predictive model was conducted by stepwise-weighted binomial regression analysis. Survey weights were calculated based on age, gender, and region of residence.http://ec.europa.eu/eurostat). Belgian geographic data were obtained using the BelgiumMaps.Statbel R package (available at http://www.datatailor.be/rcube/).Demographic data from the 2013 Belgian population were downloaded from Eurostat , and genotype information from all SNPs overlapping with the BelPHG-21 study was extracted using the R VariantAnnotation package [A principal component (PC) analysis was performed using phase 3 data from 1000 genomes . VCF and package . Belgianhttps://molgenis26.target.rug.nl/downloads/gonl_public/variants/release5/.Correlations between variant allele frequencies (VAF) were determined using Pearson’s correlation. Correlations were determined for SNPs after filtering using the higher mentioned criteria. Variant allele frequencies from the Genome of the Netherlands (GoNl) project were derived from the release 5 vcf files downloaded at Additional file 1: Table S1.SNP variant allele frequencies of the Belgian population, BelPHG-21 study, Belgium 2016. Variant allele frequencies (VAF) for all SNPs were calculated with reference to the human genome build hg19 on all samples or after exclusion of samples with a foreign origin (indicated by tab names). Columns indicate SNP ids, chromosome, position, reference allele, variant allele, VAF, and frequencies of homozygous reference (AA), heterozygous (AB), and homozygous variant (BB) alleles respectively. (XLSX 28534 kb)Additional file 2: Table S2.Population details of the samples analyzed in the BelPHG-21 study, Belgium 2016. Indication of the number of samples taken from NHIS-2013 participants in each region and province, total number of inhabitants in 2013, number of samples taken per million inhabitants, number of invited participants, and sampling percentages. (XLSX 10 kb)Additional file 3: Figure S1.Genetic variation of the Belgian population related to continental populations. Principal component (PC) analysis using 47,802 SNPs from five continental populations with mapping of the Belgian population. (a) Screeplot showing the variability captured by the first 10 PCs. (b) Pairwise plots of the first four PCs. Populations are indicated below the plot . (PDF 7654 kb)Additional file 4: Figure S2.Genetic variation of the Belgian population related to other European populations. Principal component (PC) analysis based on 41,083 SNPs from five different European populations . Panels show plots of the first two PCs with indication of the province of inhabitance as indicated. The central map of Belgium shows the geographical location of each province. Panels with Flemish provinces are shown on top while Walloon province panels are shown on the bottom. (PDF 1806 kb)Additional file 6: Figure S4.Variant allele frequency correlations between Belgian and other populations. Variant allele frequencies (VAF) for all SNPs were calculated with reference to the human genome build hg19. Plots show the VAF of Belgian versus other continental (a) and European (b) populations. Pearson correlation coefficients are shown on top of each plot. For visualization purposes, only 1000 random points are shown. (PDF 5259 kb)


Echinaster sepositus. The species is relatively abundant in some areas of the Mediterranean Sea, but some populations have dramatically decreased over recent years due to direct extraction for ornamental aquariums and souvenir industries. Analyses across most of the distribution range of the species based on the mitochondrial cytochrome c oxidase subunit I gene and eight microsatellite loci revealed very low intraspecific genetic diversity. The species showed a weak genetic structure within marine basins despite the a priori low dispersal potential of its lecithotrophic larva. Our results also revealed a very recent demographic expansion across the distribution range of the species. The genetic data presented here indicate that the species might be highly vulnerable, due to its low intraspecific genetic diversity.Understanding the phylogeography and genetic structure of populations and the processes responsible of patterns therein is crucial for evaluating the vulnerability of marine species and developing management strategies. In this study, we explore how past climatic events and ongoing oceanographic and demographic processes have shaped the genetic structure and diversity of the Atlanto-Mediterranean red starfish  In marine benthic species with limited mobility during adulthood, larval dispersal plays an important role in the intraspecific distribution of genetic diversity, and in both the genetic structure and connectivity of populations234556789111314151718Along the Atlanto-Mediterranean arch, the Almeria-Oran Front is considered the real boundary between the Mediterranean Sea and the Atlantic Ocean, acting as an important barrier to gene flow in a number of marine species20212222242528203132Historia Animalium: the red starfish Echinaster sepositus (Retzius 1783). The species is distributed across the Mediterranean Sea and the temperate waters of the eastern Atlantic, from the south-eastern limit of the English Channel to Cape VerdeE. sepositus in the north-western Mediterranean have dramatically decreasedIn this paper, we analyse one of the most emblematic echinoderms found in the Mediterranean Sea, and the first starfish mentioned in Science, by Aristotle 2,300 years ago in E. sepositus releases lecithotropic larvae that remain as free swimmers for no longer than 5–6 days42544It has been reported that E. sepositus from which to infer the effective dispersal and levels of gene flow. In comparison, genetic patterns of other starfish species have been widely studied in recent years4647484950E. sepositus, at both the evolutionary and geographical scales. We focus on the potential effect of major marine corridors, the Strait of Gibraltar and the Siculo-Tunisian Strait, on the genetic structure and connectivity; and also on the relevance of geographical distance for the divergence of populations in a species with a short-lived larval stage. This represents a twofold contribution to marine biology: a) it increases the knowledge of population genetics and phylogeography of benthic invertebrates across the Atlanto-Mediterranean transition, and between the two Mediterranean sub-basins, which is currently patchy and controversial; and b) it generates the knowledge required to understand the potential vulnerability of this species to exploitation, according to its genetic features. It is well known that the intraspecific genetic diversity of species plays a crucial role in the long-term survival of populations because it is the raw material on which natural selection actsVery limited published genetic information is available for E. sepositus. As a mitochondrial marker we chose the cytochrome c oxidase subunit I (COI) gene because of its high resolution in phylogeographic analysis17322224264555To address our objectives, we used both mitochondrial and nuclear markers to understand past and present events shaping the geographical distribution of the intraspecific genetic diversity in −6 SD) and 0.619 (±0.021 SD), respectively. Values of genetic diversity for COI were similar for the Atlantic and the Mediterranean basins, , although within the Mediterranean basin, the eastern Mediterranean seemed to have lower values of diversity . The population from Livorno, located in the western Mediterranean, presented only two haplotypes, and was thus the population with the fewest haplotypes and the lowest Hr (0.522).From a total of 325 sequences (657 bp) of the COI fragment, we obtained 23 different haplotypes. Most individuals (92%) had one of three main haplotypes , with only one mutation step between each other see . We deteThe haplotype network obtained from COI  showed as: 2.994; all the other populations Rs: from 3.882 to 4.340); in agreement with the occurrence of only two mitochondrial haplotypes in this population (see o) in most populations, and populations from north-western Mediterranean and the Atlantic showed significant FIS values, and Hardy-Weinberg disequilibrium .The eight microsatellite loci showed between 6 and 32 different alleles per locus in the populations analysed. We observed differences in allelic richness among populations, but the population from Livorno displayed much lower values of allelic richness than any other population  for both COI and microsatellites supported the patterns obtained from STRUCTURE. The analysis revealed that most of the genetic diversity was retained, both within populations and individuals . Significant differences were observed between groups , and among populations within geographical groups for microsatellites, but not for COI. Taormina, a population on the limit between eastern and western Mediterranean, seemed to be more closely related to the western Mediterranean group. This was observed through an increase in the variation between groups for the two sets . The othST-FST and Jost’s D values revealed significant genetic differences between populations from the Atlantic and Mediterranean basins, with only a few exceptions  mean values, followed by the eastern Mediterranean and the Atlantic basin Acanthaster from distance geographical areas of the Indian Ocean64Due to the differences in nature and coalescence time between mitochondrial and nuclear markersf values into absolute population sizes, we note that Nf only counts mature females, and we have no information on the age of maturity of the species. Finally, results for the estimated number of individuals were within a range, and different markers in MCMC analysis already reported variation within a speciesHowever, our results based on the LAMARC Bayesian approach suggest a stable migration for a long periodE. sepositus did not follow a pattern of IBD when the disruptive effect of marine fronts between basins was eliminatedE. sepositus showed that both gametes and larvae immediately sink to the bottom of the experimental tanksE. sepositus42E. sepositus, the release of both floating and non-floating eggs has been observed in other Echinaster species during the same reproductive eventE. sepositus might explain the low differentiation between populations due to high connectivity promoted by floating and long-dispersal gametes and larvae. Additionally, the absence of IBD could be related to the stochastic nature of larval connectivity in nearshore species due to intermittent and heterogeneous processes of marine circulation, which vary over time, habitat availability and other biological features, such as competition and predatory pressureE. sepositus are needed to understand their relevance to the connectivity of populations and the potential evolutionary significance of divergent strategies of dispersal.The genetic structure of the populations in Echinaster, those effects may be related in some way with the low diversity and divergent structure found at these two particular sites. The genetic structure of populations in polluted areas may be due to the existence of selective sweepsInterestingly, two Mediterranean populations, Livorno and Cartagena, displayed sharp differences in genetic structure based on microsatellites, and appeared as separate clusters. Those differences were less noticeable in mtDNA sequences, which may indicate that the divergence of these two populations is a relatively recent process. These two particular populations are located close to large marine harbours, and coastal areas affected by industry. In the case of Livorno, just 11 km away from our sampling site there is a famous white beach produced by the industrial carbonate discharges of Rosignano Solvay. The load of heavy metals around the Livorno harbour has been demonstrated to impact genetic diversity of other marine species79IS values, which resulted in Hardy-Weinberg disequilibrium, might be related with inbreeding in some populations. The general patterns of genetic structure in E. sepositus, together with the low values of genetic diversity detected and potential inbreeding, make this species highly vulnerable to overfishing and environmental perturbations at a small to medium geographical scale71E. sepositus due to the stochasticity of the recruitment process that does not ensure gene flow or recruitment of larvae from nearby sites, thereby limiting the recovery of populations.Population decline was not detected at any of the sites analysed, but the significant FE. sepositus set the base for further studies to design management policies for sustainable exploitation of this iconic starfish. Any conservation strategy for this species should consider how genetic diversity is geographically distributed and the genetic divergence among the Atlantic, western Mediterranean and eastern Mediterranean basins.The genetic data presented here for E. sepositus were collected at 15 different locations: 9 in the western Mediterranean , four in the eastern Mediterranean  and two in the Atlantic Ocean  (Tube feet samples from 332 specimens of igantes) . The speigantes) . We usedwww.sigmaaldrich.com), following the protocol proposed by the manufacturer.DNA was extracted from all tube feet samples using a REDExtract-N-Amp Tissue PCR Kit  with a first step of 95 °C for 60 sec, followed by 35 cycles of 95 °C for 20 sec, 50 °C for 20 sec and 72 °C for 2 minutes, and a final extension of 5 minutes. Amplification products were purified and analysed on an Applied Biosystems 3730xl DNA Analyzer  at the Scientific-Technical Services of the University of Barcelona. Allele length was estimated relative to the internal GENESCAN 400HD ROX size standard (Applied Biosystems) using Peak-Scanner software. Alleles were scored using the MsatAllel 1.0 packageWe used eight specific microsatellite loci  already optimised for this speciesc oxidase subunit I gene (COI) was amplified with the primers F210-CO1 (5′-GTAATGCCAATTATGATTGG-3′) and COA (5′-AGTATAAGCGTCTGGGTAGTC-3′)www.macrogen.com) with the same primers used for amplification of the fragment. Sequences were edited and trimmed with MEGA v. 5.2 softwarewww.genbank.com).A fragment of the mitochondrial cytochrome www.fluxus-engineering.com/sharenet.htm) using an infinite site model. Haplotype frequencies in each population were plotted in geographic coordinates using the packages Maps and Mapplots in R91Haplotype number (Nh), nucleotide diversity (π) and haplotype diversity of COI, were calculated using the software DNASP v. 5.0o) and expected heterozygosity (He) were calculated using the hierfstat package in RIS was also calculated for each population and confidence intervals at the 0.05 level were tested after 100 bootstrapping resampling operations over loci with the same packageAllele number per microsatellite locus, allele richness, observed heterozygosity In order to infer major homogeneous genetic units (K) across the distribution range of the species, the software STRUCTURE v. 2.3a priori grouped within basins and sub-basins, which are considered different ecoregionsHierarchical analysis of the molecular variance (AMOVA) was performed with Arlequin v. 3.5ST for COI and FST for microsatellites, and the Jost’s D coefficients of dissimilarityST and FST values were computed in Arlequin v. 3.5ST and FST values. If mitochondrial and nuclear markers show similar trends in genetic divergence between populations, then the ΦST (from COI) and FST (from microsatellites) should be highly correlated. Additionally, the correlations between the ΦST and FST with the Jost’s D for COI and microsatellites, respectively, were also calculated to explore whether the two types of analysis yielded similar results. Dissimilarity genetic matrices based on Jost’s D coefficients were graphically represented using non-metric MDS for both kinds of markers with the Vegan R packagePairwise differences in genetic structure between locations were separately explored for COI and microsatellites by two different approaches; the ΦST and FST dissimilarity matrixes for COI and microsatellites, respectively, and a matrix of geographical distances. Geographical distances were considered as the shortest linear distance in kilometres by sea. The IBD analysis was performed with all populations with a stratification method to consider the divergence associated with the “basin” factor: Mediterranean and Atlantic basinsGenetic differentiation due to geographical distance (IBD) was calculated using the Mantel test procedure in the Vegan packagetheta), and demographic events, such as population growth or decline, were estimated with a Bayesian approach as implemented in LAMARC v. 2.1.9coda R packageMt = m/μ, where m is the migration rate per generation, μ is the substitution rate, and θ is the theta value. The theta value is defined as θ = 2Nf μ, where Nf is the effective population size for mitochondrial markers; we calculated Nf using a substitution rate of between 2.3% and 3.6% per million generations applied in other asteroids17323354theta values (θ) and growth rates (G) are defined from the evolution of different theta values: θt = θ1−Gt. The high computational requirements of LAMARC in both time and memory, which cannot be parallelised in a super-computer cluster, did not allow us to analyse microsatellites with this software; thus, other methods had to be applied for microsatellite loci.Migration between geographical areas, effective population size (expressed as 2utt) of the demographic changes from the coalescent methods. We used the same lineage substitution rate as before, between 2.3% and 3.6% per million generations; due to a lack of information on the generation time of E. sepositus, we assumed 1 year per generation, as applied to different echinoderms17323354Demographic changes based on COI sequences were also inferred with a mismatch distributionc), was calculated after 10,000 simulations, and population decline is inferred when Mc is greater than the mean M valueFor microsatellite loci, recent population size changes were explored using two different approaches: the software Bottleneck v. 1.2115How to cite this article: Garcia-Cisneros, A. et al. Low genetic diversity and recent demographic expansion in the red starfish Echinaster sepositus (Retzius 1816). Sci. Rep.6, 33269; doi: 10.1038/srep33269 (2016).


Acer saccharum Marshall) reaches its northern continuous distributional limit in northeastern North America at the transition between boreal mixed‐wood and temperate deciduous forests. We hypothesized that marginal fragmented northern populations from the boreal mixed wood would have a distinct pattern of genetic structure and diversity. We analyzed variation at 18 microsatellite loci from 23 populations distributed along three latitudinal transects  that encompass the continuous–discontinuous species range. Each transect was divided into two zones, continuous (temperate deciduous) and discontinuous , based on sugar maple stand abundance. Respective positive and negative relationships were found between the distance of each population to the northern limit (D_north), and allelic richness (AR) and population differentiation (FST). These relations were tested for each transect separately; the pattern (discontinuous–continuous) remained significant only for the western transect. structure analysis revealed the presence of four clusters. The most northern populations of each transect were assigned to a distinct group. Asymmetrical gene flow occurred from the southern into the four northernmost populations. Southern populations in Québec may have originated from two different postglacial migration routes. No evidence was found to validate the hypothesis that northern populations were remnants of a larger population that had migrated further north of the species range after the retreat of the ice sheet. The northernmost sugar maple populations possibly originated from long‐distance dispersal.Due to climate change, the ranges of many North American tree species are expected to shift northward. Sugar maple ( On the one hand, lower chilling requirements can result in early bud flush in spring under milder environments and greater susceptibility to spring frost damage  is a widespread, abundant and long‐lived (300–400‐year) deciduous northeastern American tree  bioclimatic domain where sugar maple is abundant. The discontinuous zone was in the balsam fir–yellow birch  Miller–B. alleghaniensis) bioclimatic domain, and some northern sites were located in the transitional area of the balsam fir–white birch  bioclimatic domain , with eight sites per transect  and saplings  were sampled in each site and analyzed. Samples were frozen (−80°C) or dried (using silica gel) until needed for genetic analyses.Tissue samples  were collected from 953 individual sugar maples in 2008 and 2009. As temporal variations in genetic differentiation may be present between different cohorts of regeneration . All samples were genotyped for 18 variable microsatellite loci using PCR and genotyping protocols as previously described by Graignic et al. . Five di2.3AT), mean number of alleles (A), mean observed (HO) and expected (HE) heterozygosity, and inbreeding coefficient (FIS) were estimated using fstat 2.9.3.2  and genetic distances , mean allelic richness (AR), HO, HE, pairwise FST, mean pairwise FST, and FIS were estimated using fstat. The AR was calculated using rarefaction method, based upon the size of the smallest number of samples. The indices were estimated for each population and on mature trees and saplings for each population separately. To assess whether population diversity parameters differed between zones and transects, AR, HO, HE, FST, and FIS were calculated by population  within each zone (discontinuous and continuous), transects , and zone in each transect separately using fstat .The mean number of alleles per locus  were compared to different characteristics listed below, using a linear mixed‐model analysis  stand characteristics, such as basal area of mature sugar maple or/and sugar maple sapling (m2/ha), density of mature sugar maple or/and sugar maple saplings (stems/ha), population size of mature sugar maple or/and sugar maple saplings (stems), (2) distance of each population to the northern limit , and (3) global models with a combination of one model of stand characteristics and the northern limit model. Stand characteristics are included in the analysis because in the central–marginal model, demographic processes are predicted to influence the patterns of genetic diversity. Basal area and density were calculated in a 0.16 ha quadrat on each site  and balsam fir–yellow birch  bioclimatic domains, and the distance of each sampled population to this limit was estimated with arcmap™ 10.0 ; negative values were assigned to sites located to the north of the northern limit, that is, in the balsam fir–white birch  bioclimatic domain. Correlations between variables were examined, and assumptions of normality and homoscedasticity were graphically tested on global models. Where needed, the data were square‐root‐transformed to meet assumptions. Fits for the 19 models were compared using the Akaike information criterion . We computed AICC and ΔAICC weights (ω) to determine the strength of evidence for each model , where required (ΔAICC ≤ 4), to calculate the parameter estimates and unconditional 95% confidence intervals (CI).Data were analyzed using version 2.13.1 of R  to the range in allele size (r), using m_p_val software  faster than the size range of those alleles (r), thereby resulting in a reduced M‐ratio (M = k/r). To determine the significance of a ratio, we compared our observed value to critical values (Mc) that were established using the critical_m program  below Mc, we assumed that a population had experienced a significant reduction in size  to account for a wide range of effective population sizes; and the parameters were set as recommended by Garza and Williamson  between the genetic distance [FST/(1 − FST)] or [DS/(1 − DS)] and geographic distance (km). Pairwise FST and Nei's genetic distance, DS  trees were constructed based on ew Page, .structure 2.3.4 , assign individuals to each K cluster, detect the population structure, and show whether geographic populations are assigned in genetic populations. Our analyses were based upon an admixture ancestral model with correlated allele frequencies, and a priori sampling locations were used as prior information (LOCPRIOR setting). LOCPRIOR was used to detect any further structure unidentified by standard settings  with a burn‐in of 100,000 followed by 200,000 MCMC iterations. The most likely value of K was determined using the ∆K criterion , 2 , 3 , and 4 ; and the level of differentiation between groups, between populations within the group, and within populations were tested with AMOVA.Genetic variation was examined using the hierarchical analysis of molecular variance (AMOVA) based on Φ2.9migrate‐n version 3.6  and the mutation‐scaled past immigration rates  using a Metropolis–Hastings algorithm to explore all possible genealogies. The mutation‐scaled population size (θ) for each population and the mutation‐scaled immigration rates (M) between each pair of populations were estimated using the Brownian motion mutation model for microsatellites. A coalescent simulation explored the likelihood space for θ and M. To limit the number of parameters, the amount of gene flow was estimated between four groups of populations from the transect 1 , 2 , 3 , and the most northern populations . The starting values of θ and M were generated from the FST estimates, as well as an exponential window prior set for both parameters . We replicated two long chains of 10,000,000 genealogies, which were recorded every 10 steps, after a burn‐in period of 10,000. The static heating scheme was set to four chains with temperatures of 100,000.0, 3.0, 1.5, and 1.0, and the swapping interval set at one. The program was run several times adding more sampling schemes and replications. The starting parameters and the resulting estimates were compared until the results were congruent.We used the Bayesian approach implemented in  Beerli,  to asses33.1FIS ≥ 0.227; Table FST estimates obtained before and after corrections for null alleles were similar . Consequently, all loci (18) were retained in the analysis.The total and mean number of alleles per locus ranged between 4 (SM60) and 31 (SM21A) and from 2.8 (SM60) to 14.8 (SM21A), respectively Table . Signifi3.2A), mean allelic richness (AR), mean expected heterozygosity (HE), and inbreeding coefficient (FIS) for all individuals and both cohorts  for saplings, mature trees, and all individuals pooled  from the discontinuous zone of transect 1 had the lowest mean number of alleles per locus  and in transect 1 (0.030 vs. 0.004) but not transects 2 and 3 (Table FST) was different between discontinuous and continuous zones both for mature trees (0.034 vs. 0.007) and saplings (0.029 vs. 0.003) in transect 1  for θ = 1  and in zones  using FST/(1 − FST). Tests were not significant when each transect was tested separately . Similar results were obtained using DS/(1 − DS) (Table Isolation by distance was observed among populations in Québec (S) Table .3.6The neighbor‐joining tree separated the populations of the transect 1  from the other two transects Figure . Howeverstructure output produced the highest value of ∆K at K = 4  and transect . Remarkable levels of gene flow were observed among populations from the three transects, with a slightly more scaled immigration rate (M) from the east (transect 3) than from the other two transects  was in the eastern area of Québec , and the smallest was in the center  and higher genetic differentiation (FST) between populations were detected in the discontinuous zone where populations are at the periphery of the sugar maple range in Québec. Furthermore, gene flow between zones was asymmetric, mostly directed outward from the populations of the temperate deciduous forest. A high level of admixture and a weak population structure were also detected. Most genetic variation was found within sugar maple populations, a frequent pattern for long‐lived perennials, outcrossing, and wind‐dispersed seeds plants , by Graignic et al. (HE = 0.689), and other species such as Acer mono Maxim. . Similar results were reported with microsatellites for Acer takesimense  and higher genetic differentiation (FST) between populations in the discontinuous zone that represents the periphery of sugar maple range in Québec  A.A. Weber ex Wigg  remained significant only for the western transect (transect 1). The reduction in Ar and Fst did not change from core to edge populations, indicating no direct effect of fragmentation on genetic diversity of sugar maple populations. Paleoecological records support the constant presence of sugar maple in mixed‐wood or boreal forests since its establishment  and had probably undergone a bottleneck in a distant past , and the highest FST (Table M‐ratio at θ = 1). Both neighbor‐joining trees and structure analysis clearly disjointed this population from all the others  had the lowest level of diversity  and mature trees (≥30 cm dbh). Graignic et al. , possibly due to higher fitness of heterozygous individuals  had a lower population size Table  and were5The inclusion of among‐edge population comparisons provided in this study provides new insights into the question of whether the “ecology marginality at the periphery” (central–marginal hypothesis; Eckert et al., None declared.N.G., F.T., and Y.B. conceived the ideas. N.G. collected samples, performed the laboratory work, analyzed the data, and led the writing of this manuscript. F.T. performed some statistical analysis and revised the manuscript.10.5061/dryad.36634.GenBank access numbers of microsatellites markers are listed in Table  Click here for additional data file.


L. tigrinus. Here we report phylogeographic analyses encompassing most of its distribution , using a novel dataset comprising 2,143 bp of the mitogenome, along with previously reported mtDNA sequences. Our data revealed strong population strutucture and supported a west-to-east colonization process in this species’ history. We detected two population expansion events, one older (ca. 200 thousand years ago [kya]) in western South America and another more recent (ca. 60-50 kya) in eastern areas, coinciding with the expansion of savanna environments in Brazil. Analyses including L. tigrinus individuals bearing introgressed mtDNA from L. colocola showed a complete lack of shared haplotypes between species, indicating that their hybridization was ancient. Finally, we observed a close relationship between Brazilian/Uruguayan L. colocola haplotypes and those sampled in L. tigrinus, indicating that their hybridization was likely related to the demographic expansion of L. colocola into eastern South America.The pampas cat is a small felid that occurs in open habitats throughout much of South America. Previous studies have revealed intriguing patterns of morphological differentiation and genetic structure among its populations, as well as molecular evidence for hybridization with the closely related  Leopardus , composed by at least eight species of small and medium-sized cats that occur in a variety of habitats across the Neotropics, and whose diversification began 3 to 5 million years ago (MYA)  is considered one of the least known species of this genus , each containing subspecies-level units. A subsequent molecular study based on three mitochondrial genes  indicated strong genetic structuring among different pampas cat populations  and microsatellie loci for a data set focused on the central Andes. These authors found strong genetic differentiation among several regional populations, supporting the recognition of at least four Management Units (MU) for conservation purposes.The existence of highly structured populations in this cat species has been supported by recent molecular studies  in these earlier investigations, and thus their evolutionary relationships with those from western South America remain obscure.L. colocola present an additional layer of evolutionary complexity, as we have documented that they underwent a massive historical process of hibridization and unidirectional introgression affecting a congeneric species, Leopardus tigrinus , since it so far holds the only available record of this ancient episode of hybridization.In addition, according to L. colocola based on the analysis of mtDNA segments, aiming to characterize its phylogeographic patterns and demographic history, as well as to gain additional insights into its hybridization/introgression event with L. tigrinus. In particular, we pursued the following specific objectives: 1) to assess the genetic relationships between western L. colocola populations and those from eastern South America (Brazil and Uruguay); 2) to estimate the geographic origin and age of the mitochondrial DNA haplotypes introgressed into L. tigrinus, and 3) to analyze the correlation between the genetically identified groups and the morphology-based taxa proposed by In this context, the main goal of this present study was to investigate the evolutionary history of L. colocola individuals from Brazil, Argentina, Uruguay, Chile and Bolivia, as well as 28 L. tigrinus from central and northeastern Brazil (see Table S1). Blood samples were collected from wild animals captured for ecological studies, as well as from captive individuals , and were preserved in a salt saturated solution . Tissue samples were obtained from road-killed specimens and maintained in 96% ethanol. Samples of Leopardus pardalis (ocelot) and Leopardus wiedii (margay) were also included as outgroups in some of the analyses.We generated mtDNA sequence data from 40 TM Genomic DNA Mini Kit  following the manufacturers’ instructions. All DNA samples were quantified in a 1% agarose gel stained with GelRed®  using the LowMass DNA Ladder (Invitrogen).DNA extraction was performed using a standard phenol/chloroform protocol , 0.2 U Platinum® Taq Polymerase (Invitrogen) and 5-20 ng of genomic DNA. The addition of 0.2% Triton X-100 to the PCR reaction was used to remove PCR inhibitors in the case of the ND5, Cyt-b and control region segments. For these three segments, the PCR conditions were identical and began with one step of 94°C for 3 min, 5 cycles (Touchdown) of 94°C for 45 s, 55-51°C for 45 s, 72°C for 1 min 30 s followed by 40 cycles of 94°C for 45 s, 50°C for 30 s, 72°C for 1 min 30 s and final extension of 72°C for 30 min. Thermocycling conditions for the ATP8 gene consisted of an initial denaturing step at 94°C for 3 min followed by 35 cycles of 94°C for 45 s, 50°C for 45 s, 72°C for 1 min 30 s and final extension of 72°C for 10 min.PCR amplifications were performed in a final volume of 20 μL, containing 1X PCR buffer (Invitrogen), 0.2 μM of each primer, 0.2 mM dNTPs, 1.5-2.0 mM MgClPCR products were visualized on a 1% agarose gel stained with GelRed® (Biotium Inc.) and then purified using a protocol based on precipitation with ammonium acetate and isopropanol, or the enzymatic method with the enzymes Exonuclease I (EXO I) and Shrimp Alkaline Phosphatase (SAP). Both strands of each PCR product were sequenced using the DYEnamic ET Dye Terminator Sequencing Kit  and analyzed in a MegaBACE 1000 automated sequencer . For the mtDNA control region, due to the occurrence of a repetitive region which generates high levels of heteroplasmy . Shorter sequences for ATP8, ND5 and control region  reported by L. colocola distribution were also included   . Novel sDNA sequences were aligned using the MUSCLE algorithm  implemenTwo data sets were established for use in the analyses described below: (i) Data set A (DSA) with all sequences generated in this study as well as data reported by Phylogenetic analyses were performed with only one representative of each haplotype, and initially employed two different optimality criteria: Bayesian Inference (BI) and Maximum Likelihood (ML). The software jModelTest 0.1.1  was usedND5, Cytb, ATP8 and CR), using only a few divergent haplotypes and three node-based calibrations. These were based on the credibility intervals estimated previously (Leopardus: 1) the basal diversification of the genus (2.02 – 4.25 MYA); 2) the divergence between L. pardalis and L. wiedii (1.01 – 2.41 Mya); and 3) the basal divergence in the clade (L. jacobita + L. colocola + L. tigrinus + L. geoffroyi + L. guigna) (1.68 – 3.56 Mya). For these runs we used a uniform prior, an uncorrelated lognormal relaxed molecular clock and a Yule prior for the tree, and performed the same analysis separately for the two data sets (DSA and DSB). Trees were linked and the substitution model was set as unlinked, allowing the incorporation of the different substitution models estimated for each segment with jModelTest. We ran BEAST for 50,000,000 steps, sampling every 5,000 iterations, after a discarded burn-in of 50,000 steps. Convergence to the stationary distribution and sufficient sampling were assessed with TRACER. The mean substitution rate (per site per year) obtained in these analyses were as follows: DSA: ND5 = 2.52 x 10-8, Cytb = 2.48 x 10-8, ATP8 = 2.93 x 10-8, CR = 3.53 x 10-8; DSB: ND5 = 2.53 x 10-8, Cytb = 2.42 x 10-8, ATP8 = 3.87 x 10-8, CR = 4.89 x 10-8.Final phylogenetic analyses employed the relaxed Bayesian approached implemented in BEAST 1.6.2 , based oThe second set of BEAST analyses incorporated the mean substitution rates estimated in the first round for each mitochondrial partition, aiming to simultaneously estimate the phylogenetic tree and the TMRCA (the time to the most recent common ancestor) of each pampas cat phylogroup. In this round, we included one copy of all identified haplotypes and assumed a strict clock and a coalescent (constant size) tree prior. The analysis was performed separately for each data set (DSA and DSB) and was run for 100,000,000 steps, sampling every 10,000 iterations, after a discarded burn-in of 100,000 steps, taking into account the stabilization of the traces and the sampled parameters.http://www.fluxus-engineering.com/sharenet.htm) to depict phylogenetic, geographic and potential ancestor-descendent relationships among sequences. In parallel, population structure analyses were performed initially with the software BAPS   approach  scaled by the mutation rate (μ), along with pairwise migration rates (M) for all the defined populations. The number of migrants per generation was obtained by multiplying M by theta of the recipient population. We performed three independent runs of the Bayesian search strategy, including one long chain of 3,000,000 steps with a sampling increment of 100 , following a burn-in period of 3,000 sampled genealogies. The same analysis was conducted independently for both data sets (DSA and DSB), and the results were visualized in TRACER.To obtain estimates of matrilineal gene flow among different geographic populations, we used the coalescent-based Bayesian method implemented in LAMARC . This soL. colocola, we performed Fu’s Fs neutrality test , and designated this alignment as Data set A (DSA). Additonally, we added to these data the shorter sequences reported by L. colocola + L. tigrinus) in DSB, 58 of them being sampled only once, and 49 also being represented in DSA (Table S1). The concatenated data sets had high haplotypic and low nucleotide diversity, indicating a pattern of rapid and recent diversification. Interestingly, all 19 haplotypes identified in L. tigrinus samples were exclusive of that population, with no sharing with L. colocola individuals.We sequenced a total of 2,143 bp of mitochondrial DNA for 40 L. colocola and L. tigrinus were grouped into a monophyletic cluster with maximum probability based on both data sets. The TMRCAs estimated for this clade with DSA and DSB were very similar, with a mean ranging from 759 to 874 thousand years ago (kya), and 95% credibility intervals between 516 kya and 1.163 Mya. Three samples from central Brazil were placed in a basal cluster relative to all L. colocola and L. tigrinus haplotypes, corresponding to two individuals (bLti72 and bLti96) previously recognized as L. guttulus by L. guttulus, supports the inference of a possible area of sympatry between this species and L. tigrinus in central Brazil.All phylogenetic trees constructed with both data sets and both criteria (BI and ML) were congruent with respect to their main topological features. Therefore, only the results of the final analyses, using the relaxed Baysian approach implemented in BEAST, are shown . HaplotyL. colocola and all L. tigrinus  were restricted to areas in the southern portion of western South America (south of latitude 19º S), including Argentina, Chile and southern Bolivia. Cluster 6 was comprised uniquely by haplotypes sampled in the northern portion of western South America (north of latitude 19º S). Cluster 5 grouped the haplotypes most related to the eastern samples, and presented a mixed composition with individuals sampled in the northern and southern portions of western South America. In particular, this cluster contained samples from latitudes 17 – 25° S, in a partially intermediate geographic position between the northern and southern groups.L. colocola segregated from southern Brazil and Uruguay, and also from L. tigrinus  had a star-shaped pattern, suggesting a possible demographic expansion, which could be connected to the inferred haplotype diversification.The haplotype networks indicated a similar geographic structure to that observed in the phylogenetic analyses for eastern South America (SA) populations, with central and northeastern tigrinus ,B. InterL. colocola. For DSA, one haplotype originating from northern Chile (H49) was the most basal, while for DSB, the basal position was occupied mainly by haplotypes from southern portions of the Andes .As also evidenced in the phylogenetic analyses, haplotypes from western SA appeared as the most basal lineages in the phylogeographic structure of L. colocola geographic distribution. The results from BAPS analyses performed with DSB were initially unstable and identified 4-5 genetic groups. In all of the five runs performed, two groups were stable and in agreement with the phylogenetic and network analyses, including mostly haplotypes sampled in eastern SA. Haplotypes from western SA were subdivided into 2 or 3 different groups, with an unstable allocation of individuals among different runs.Genetic structure and gene flow analyses were only performed for DSB, due to its broader coverage of the Considering that these samples comprised mainly the shorter sequences reported by ST indices. All resulting values were statistically significant and considered high, indicating a strong genetic differentiation between these clusters  and two of the three western populations (NW and CW), with SW being the western group most closely related to the eastern groups. The lowest ΦST values were found, in general, in comparisons between different western groups, except for NW vs. CW. This was quite intriguing, given the geographic proximity between these groups, but the results obtained for the Mantel test analysis also reflected this pattern. In spite of significant correlations among genetic and geographic distance , the correlation was relatively low mainly due to the occurrence of haplotypes with higher genetic distance but very close geographic origin .Levels of genetic differentiation among these five groups were evaluated through Φclusters . The higHistorical patterns of migration among populations were estimated for a scheme with only four populations. This included the groups identified with the BAPS analysis, with the exclusion of the CW group due to its small sample size. The haplotypes assigned to this group were allocated to the two other subpopulations of western SA according to their geographic origin in relation to latitude 19° S. Effective migration was estimated to be less than one migrant per generation, indicating limited dispersal and gene flow . Two preL. colocola. For this, we used only DSB. The mismatch distribution for the entire data set resulted in a bimodal graph, which would be consistent with a heterogeneous genetic composition of the data set. The analyses conducted with western and eastern regions independently revealed a unimodal pattern  only for the former, with a mode around 30 differences suggesting a rapid and relatively old population expansion . On the other hand, Fu’s FS neutrality test resulted in negative and significant values for both populations analyzed independently and also for the entire data set , indicating possible events of demographic expansion in all the assessed data sets.Mismatch distribution analyses and neutrality tests were used to test the hypothesis of a recent population expansion in The Bayesian Skyline plot showed a constant population size until about 300 kya, with a strong signal of population expansion in recent times. For western SA, we observed a strong signal of population expansion starting around 200 kya . On the L. colocola distribution, in agreement with previous studies of this species , Cerrado , and Chaco  . SeveralIn spite of the migration results, recent gene flow between the five lineages identified here seems to be relatively low, taking into account their high genetic differentiation, especially between eastern and western regions of South America. In fact, although haplotype co-occurrence was identified at some localities, the five lineages identified here were almost completely segregated geographically.Among the three major groups identified in western South America, the northwestern one was mainly associated to the Montane Grasslands of Peru and northern Bolivia, while the southwestern one was documented in a variety of biomes, including the temperate grasslands (Patagonian Steppe), Mediterranean Forests and Montane grasslands of Argentina, southern Bolivia and Chile. The boundary between these two populations apparently lies around parallel 18-19° S as was also documented by Vicugna vicugna) .In eastern regions of pampas cat occurrence, the two identified populations were also subdivided according to different biomes. Northern populations are restricted to two Brazilian biomes, Pantanal , and one more recent in eastern SA (60-50 kya). These episodes were supported by the skyline plots and neutrality tests, even though they were not as visible in the mismatch distribution analyses. This latter result could be attributed to an insufficient sampling, or a more complex demography history in addition to a simple event of population expansion. In fact, the haplotype networks depicted few cases consistent with an expansion pattern, indicating a more complex history of pampas cat populations. The estimated ages for these demographic events, in addition to the results gleaned from the networks, phylogenetic and migration rate analyses, support the inference that ancestral haplotypes from pampas cat occurred in western South America, especially in the southwestern portion of the continent. Nevertheless, additional evidence from the haplotype networks illustrate the complexity of pampas cat mtDNA history, with lineages/groups found in the north and central-west areas apparently derived from those found in central Brazil. This phylogeographic pattern suggests a demographic colonization from southwestern to central Brazil, possibly via the dry corridor, with a posterior colonization of the western regions in the northern part of the continent.The estimated age for both events of population expansion were coincident with two independent Pleistocene colder periods . This QuL. colocola is coincident with a colder and drier period in the country, which was associated with a retraction of forested environments and expansion of subtropical grasslands . Therefore, we postulate that hybridization/introgression events occurred in central Brazil soon after the colonization of eastern South America, thus being connected with the first demographic expansion detected in this study. The L. colocola haplotypes present in the individuals that arrived in eastern SA seem to remain only  in the L. tigrinus population, and may have become extinct in the current eastern populations of L. colocola.The demographic history of L. tigrinus and L. colocola, leading to complete mitochondrial introgression into the former species, occurred before, during, or after the divergence of L. tigrinus and L. guttulus is still uncertain. Available estimates for divergence of these species are 103 kya – 1.038 Mya , hybridization and introgession from L. colocola could have occurred at an early stage of the divergence between the two tigrina species, and thus could have played a role in this divergence process.Whether hybridization between Leopardus colocolo, Leopardus pajeros and L. braccatus. This proposed classification was followed by Three distinct species have been proposed for the pampas cat by L. colocolo recognized by L. c. wolffsohni from the highland steppes of northern Chile and L. c. colocolo from forests of central Chile. According to our results, samples collected in northern Chile harbored a haplotype that is clearly connected to populations found in the southwestern region on the eastern slope of the Andes, in a group that is recognized as L. pajeros by . In addition, L. c. colocolo, present more complex relationships. While one sample was clearly associated to the southwestern group, in a pattern similar to that obtained from northern Chile across all analyses, two other samples were more closely assigned to southern or northwestern populations, depending on the analysis performed. These results indicate a more unstable position of this population, requiring a larger sample to define its phylogenetic position more precisely.On the other hand, samples evaluated here from central Chile, corresponding to L. pajeros, seven subspecies were recognized by L. pajeros distribution was represented by three subspecies: L. p. thomasi from Ecuador, L. p. garleppi from Peru and L. p. steinbachi from Bolivia. Unfortunately, we did not have access to samples representing L. p. thomasi and L. p. steinbachi, but the geographic distribution of L. p. garleppi is in great concordance with the northwestern group identified here, with only a proposed extension of this distribution to regions of northwestern Bolivia. According to L. p. thomasi and L. p. garleppi have similar pelage patterns and cranial morphology and occurr in the same habitats, and could thus be assigned to the same geographic population. L. p. steinbachi presents only slight morphological differences from L. p. garlepii, and given its geographic proximity to the limit of the northwestern group found in this study, this taxon might also be a synonym of garleppi.For L. pajeros includes the subspecies L. p. budini and L. p. crespoi from montaneous areas of northwestern Argentina, L. p. pajeros from central Argentina with a possible boundary in the Pampa Province of Argentina, around latitude 38°S, and L. p. crucinus from southern Chile and Argentina. L. p. crespoi is only known from the type specimen and L. p. budini due to morphological and habitat similarities. The geographic distribution of these two subspecies approximates the central-western population identified in our study, in a transitional area between highland steppes and lowland dry forests. The geographic distribution of L. p. pajeros strongly corresponds to the southern group found in our analyses, with only an extension of its distribution over that which would be recognized as L. p. crucinus, approximately around latitude 41° S. However, additional sampling from areas south of this area would be important to evaluate the existence of an additional subspecies in southernmost Argentina and Chile.The central and southern distribution of L. braccatus: L. b. braccatus for Central Brazil and L. b. munoai for southern Brazil, Uruguay and northeastern Argentina. According to that author, morphological differences in skulls are not clear for these subspecies, but there are relevant distinctions in coat pattern and body size. In agreement with this view, our results indicate a strong genetic differentiation between these populations, indicating that they consist of two separate evolutionary units.The eastern populations of pampas cat were subdivided by L. colocola, which were largely consistent with currently recognized subspecies (Overall, our study allowed the identification of at least five genetic groups in


MCM6 gene and varies widely in frequency among different human populations. Although, evolution of LP-related genetic variants was investigated in many groups of Sub-Saharan African, Middle Eastern, and European ancestry, only few studies have focused on populations from North Africa and no data are especially available from the Tunisian one. For this reason, there is an urgent need to investigate the frequency patterns at these loci in Tunisia since this adaptive trait is implicated in health.The ability to digest lactose after weaning, namely, lactase persistence (LP), is encoded by polymorphisms in the LCT/MCM6 genes and including the two functional variants − 13,910 C > T and − 22,018 G > A were genotyped in 117 Tunisian individuals using the Sequenom Mass Array technology. The observed nucleotide and haplotype patterns of variation were then compared with those of several African, European, and Mediterranean human groups for which comparable data were publicly available. Admixture analysis on a 5 Mb genomic region surrounding the LCT/MCM6 loci was also performed by extracting genotypes from a previously generated genome-wide dataset in order to deepen the reconstruction of the evolutionary history of these loci.Forty SNPs covering the We found that lactase non-persistence (LNP)-related alleles and haplotypes were predominantly present in the examined population. A clear differentiation between Tunisian, African, and North European/North Italian samples was found, while the Tunisian population showed more genetic affinity to Central and South Italian groups.Our study provided a first report of LP-associated alleles and haplotypes in the Tunisian population. We highlighted a gradient followed by LP diffusion from Europe to North Africa. Based on the rich historic background of Tunisia, we suggest that this adaptive trait was introduced in that geographic region by a relatively recent gene flow.The online version of this article (doi:10.1186/s12263-017-0573-3) contains supplementary material, which is available to authorized users. LCT gene in some groups of North European descent, pastoralists from Africa, the Arabian Peninsula, and Central Asia .Patterns of LD at the genotyped loci were investigated using the software Haploview 4.2 . HaplotyGenotyping failed for four of the collected samples and individuals with more than 30% missing genotypes were excluded. One SNP (rs749017) for which experimental problems occurred during PCR reaction was also removed. The remaining 39 multiplexed SNPs showed call rates higher than 99% and minor allele frequencies (MAF) >5%. No loci showed a significant departure from Hardy-Weinberg equilibrium (HWE).Allele frequencies for the typed functional SNPs −13,910 C/T and −22,018 G/A were calculated for all populations , nucleotide diversity was calculated for the overall Tunisian population showing high values of diversity (0.400 ± 0.211)  and Western European populations , followed by those compared to the African groups . In contrast, the lowest differences were observed when we compared TN with Northern Italian samples .Finally, no significant differences were found when Italian groups with a more Southern European ancestry  were considered  and a non-African-like (green) component. K = 3 further distinguishes an Asian-like (green) ancestry fraction from a European-like one (blue), while K = 4 identifies an additional component (yellow) that is mainly shared among East, Sub-Saharan, and North African clusters. Remarkably, the Tunisian population, and at a lower extent, the Algerian samples appeared to be clearly differentiated from the other African populations. They showed more than 95% contribution from different ancestral populations along different K values. When using the cross validation error, the mean log probability for the successive increase of K levels continues to decrease substantially as K increases. Although, higher values of K reveal additional population-specific ancestries until K = 4. For this reason, we focused on K = 2 through K = 4.We applied the unsupervised ancestry inference algorithm implemented in the ADMIXTURE software on the obtained pruned datasets. As the number of ancestral clusters increased, we observed the emergence of specific population clusters Fig. . At K = The analysis of LD patterns that was carried out on the Tunisian dataset showed the existence of a long block of relatively high linkage disequilibrium 266 kb), which includes the two functional variants − 13,910 C/T and −22,810 G/A  showed the functional allele − 13,910 T  that carry the reference − 13,910 C and − 22,080 G alleles. The most frequent haplotype (H1) in the merged dataset  at Our study highlighted an important prevalence of alleles and genotypes related to LI in the Tunisian population, as well as in South European and African groups Tables  and 2. AThe present study further highlighted the close affinity of the Tunisian population to the South European and Mediterranean populations than to African ones if we consider the LP-related genetic variants. Our data thus support the hypothesis that this adaptive trait was introduced to Tunisia by a relatively recent gene flow and did not evolve locally and independently as occurred for the bulk of Sub-Saharan African groups.To draw a first picture of the potentially LP-related patterns in the Tunisian population, LD analysis  carrying the reference LI alleles was the most frequent in all the Tunisian and Italian subgroups, as well as in most African populations. However, the second most frequent haplotype (H19) that carries the adaptive alleles showed the highest frequencies in Northern European populations  Additional file 2: Figure S1.Figure S2. Discriminant analysis of principal component (DAPC) in Tunisian groups. DAPC analysis confirmed results of PCA. Figure S3. Linkage disequilibrium patterns (LD) of the investigated genomic region in Tunisian population. Each number in squares indicates the r2 index of LD between the corresponding SNPs. a Tunisian population. b Northern Tunisian group. c Central Tunisian group. d Southern Tunisian group. Table S1. Summary statistics for the studied populations. Table includes number of chromosome, nucleotide diversity, number of haplotypes, and haplotype diversity. Table S2. Results of allelic frequencies comparison between Tunisia and African and European populations. A chi-square test was used to test allelic differences; to reduce the false discovery rate of multiple testing, Bonferroni’s multiple comparison adjustment was performed. The statistical significance reached a p value less or equal to 5%. Table S3. Haplotype list in Tunisian population. A list of 23 different haplotypes generated by PHASE software, distributed between Northern, Central, and Southern Tunisian subgroups. Table S4. Haplotype list in the merged dataset. A list of 45 different haplotypes generated when comparing our data with other populations. (ZIP 608 kb)Principal component analysis (PCA) plot of genetic variation in Tunisian groups. Distribution of northern, central, and southern samples in the space of two first component of LP-related genotypes.


Calydorea crocoides occurs in a restricted area at altitudes from 800 to 1500 m in southern Brazil and is considered endangered. A study assessing genetic diversity, cytogenetic features and ecological niche was performed aiming to characterize C. crocoides by multidisciplinary approaches. Molecular data highlighted that most of the total variation  was found within populations and the parameters of genetic diversity were high at the species level . Gene flow (Nm) was estimated in 0.97 individuals per generation. Cytogenetically, C. crocoides presents a bimodal karyotype and low asymmetry. DAPI banding pattern was uniform, but the CMA-signal evidenced a pericentric inversion in the population ESC688. The species presents high pollen viability and two different morphologies of pollen grains. Our data showed high levels of polymorphism maintained in this species that could ensure conservationist practices in which the main goal is to preserve the evolutionary potential of the species through the maintenance of genetic diversity.Evolutionary analyses have been widely used for evaluation of genetic diversity of natural populations and correlate these data to the fitness of the species, especially in the case of threatened species.  Natural populations can be affected by several relevant factors such as mating system, population density, microhabitat selection, besides historical events, as disturbance and colonization. Thus, investigating these factors is of interest for population geneticists  is a species found in grasslands, fields with herbaceous slopes of hills, at high altitudes in Campos de Cima da Serra, and has restricted distributed in this region (Table S1). The most recent publication listing the endangered flora of the state Rio Grande do Sul classified C. crocoides as “endangered” , considering as criteria its geographic range and continuous decline of the extension and quality of its habitat  evaluate the levels of genetic diversity in natural populations, (b) determine the degree of differentiation among populations, (c) characterize them cytogenetically, (d) analyze pollen viability of the populations, and (e) identify the potential distribution focused on suitable habitats for the species. These results are useful for proposing a threat status review for C. crocoides in the IUCN Red List, as well as to know if it is necessary to initiate certain in situ conservation strategies.Considering that Calydorea crocoides were collected at 10 sites located in the Subtropical Highland Grasslands of southern Brazil , 1 μL primer (10 pmol/μL), 0.2 μL Taq DNA polymerase (5 U/μL) . A negative control was added and run by replacing template DNA with H2O. Amplifications were performed using a Biocycler MJ96G thermocycler  with an initial denaturation step of 5 min at 94 °C, followed by 40 cycles of 1 min at 94 °C, 45 s at 48 °C, 2 min at 72 °C, and 5 min final extension step at 72 °C. PCR products were analyzed on 1.5% agarose gels and stained with GelRed . The size of the fragments was estimated using Ladder 100 bp . Similar to the approach employed by Leaves dried on silica gel from 25–30 specimen per population were used for total genomic DNA extraction following the modified protocol of PPB), observed number of alleles (Na), effective number of alleles (Ne), the Shannon’s information index (I) (h) (Nm) (hs), assuming a full model, here defined as average panmictic heterozygosity. To assess the genetic similarity of individuals across populations, we computed the Jaccard similarity coefficients for all pairs of individuals, and an UPGMA dendrogram was generated using the program NTSYSpc version 2.1N . Total variance was partitioned within populations or among population levels. Pairwise Nei’s genetic identity was also estimated in GenAlEx to assess the similarity of the sampled populations. The number of permutations for significance testing was set at 999. A Mantel test with 999 permutations was also computed using GenAlEx 6.The amplified fragments were scored for band presence (1) or absence (0), and a binary qualitative data matrix was produced. Genetic parameters, including the percentage of polymorphic bands (ndex (I) , Nei’s g (I) (h)  and geneh) (Nm)  were calion 2.1N . The denion 2.1N  to deterK) in a sample. The most likely number of populations (K) was estimated under the admixture model and correlated allele frequencies, with no prior information on population origin. The program was run for 50,000 iterations, after a burn-in length of 1,000,000 iterations, to test population subdivision from K = 1 to K =16, and thereby checking for any possible subdivision. Fifty runs were carried out for each K to quantify variation in likelihood, as a means of checking whether different runs could produce different likelihood values. Individual and average admixture proportions (Q) for each population in each genetic cluster found by the program were recorded for the model. As an aid in identifying the number of clusters of individuals (K), the results generated by STRUCTURE were subsequently analyzed by the program STRUCTURE HARVESTER version 0.6.7 . Root meristems were collected from bulbs of plants in pots and pre-treated with 2 mM 8-hydroxyquinoline solution for 24 h at 4 °C and subsequently fixed in fresh 3:1 (v/v) ethanol-acetic acid solution. Slides were prepared based on modified protocols of a classical squash technique  or of a 3 (CMA3) and 4’,6-diamidino-2-phenylindole (DAPI) with some modifications: chromomycin staining was done for 1.5 h, followed by DAPI for 45 min. Segments of the chromosomes displaying enhanced (+), neutral (°) or reduced (–) fluorescence are denoted in the text following the fluorochrome name or fluorochrome combination. Metaphase chromosomes were observed and photographed with a fluorescence microscope Olympus BX51  coupled with a DP72 digital camera and an imaging software DP2-BSW (Olympus). Metaphase plates with the best chromosome spreading and similar condensation levels were chosen. Measurements and karyotype analysis of 28 cells were made with the software KaryoType version 2.0 (Staining of chromosomes was performed according to the protocol of n) and basic (x) chromosome numbers, karyotype description included three karyological parameters, which properly adress karyotype assymetry according to CA  and equatorial diameter (E) of 20 pollen grains per slide were performed to determine the pollen morphology according to The material used for pollen analysis consisted of inflorescences of 5-10 individuals per population , which were collected in the field and fixed in an ethanol: acetic acid solution (3:1) for 12-24 h at room temperature. Slides were prepared following the method of Calydorea crocoides found between the years 2004 to 2016 were used to construct a distribution map for the species (Table S1). The map was constructed using the program DIVA-GIS version 7.5 , at a resolution of five minutes per pixel. These variables come from monthly values of temperature and precipitation, represented by means, seasonality, and extreme temperature and precipitation conditions throughout the year, all these being widely used in ecological niche modeling studies (Table S1) using DIVA-GIS version 7.5 4] with an average of 13.86 per primer , I = 0.2771 (range from 0.1970 to 0.3276) and PPB = 51.16 (range from 36.08% to 61.86%). Assuming Hardy-Weinberg equilibrium, the average Nei’s gene diversity was h = 0.1873 and varied between 0.1331 and 0.2233  among populations, and the rest of total genetic variance  was attributable to within-populations diversity (ST (0.24) indicated genetic differentiation among populations. Average gene flow was Nm = 0.87 individuals per generation. A pairwise matrix comparing Nm and ΦST between populations was estimated and emphasized that the populations ESC218 and ESC661 showed greater genetic differentiation; pairwise gene flow ranged from 1.01 to 2.82 .The results provided by the analysis of molecular variance (AMOVA) showed significant genetic variation  or populations potentially admixture (p = 0.97). In accordance with the STRUCTURAMA results, the pairwise genetic identity of sampled populations was close to 1, showing a high degree of similarity among populations (Table S2). Pairwise Nei’s genetic identity range from 0.769 (population ESC692 versus DR71) to 0.905 (ESC677 versus ESC688).According to Evanno’s method, the STRUCTURE analysis indicated  cluster . These g cluster . HoweverC. crocoides showed that all accessions are diploids (2n = 14). Presence of cytotypes was not observed for this species. Mitotic metaphase chromosomes and haploid idiogram are shown in C. crocoides was included in category 2A. Analyses of intrachromosomal (MCA) and interchromosomal (CVCL) karyotype asymmetry, however, showed values of 22.81 and 26.62, respectively, corresponding to relatively low karyotype asymmetry (°/DAPI+). Clusters of GC-rich DNA (CMA+/DAPI°) were observed on chromosomes 13 and 14 (pair VII), comprising the secondary constriction. Nonetheless, most accessions presented CMA+ bands on the short arm of both chromosomes, except ESC 688 which presented such on the short arm of one chromosome and on the long of another  showed values above 90% . MeasureC. crocoides were identified  Baker, C. basaltica Ravenna, C. longipes Ravenna and C. approximata R.C. Foster  and retain residual gene flow among them (Nm > 1) (K = 1), since gene flow was detected even for the most differentiated populations (ESC218 and ESC661). However, considering that C. crocoides is a geophyte, part of the estimated gene flow may be a reflection of an extensive genetic connection between populations in the past. Currently, southern Brazilian grasslands have been highlighted as threatened by habitat loss and fragmentation, due to severe and long-term human disturbance, and studies have highlighted the need to implement conservation strategies  . The popNm > 1) , consistNm > 1) , the estC. crocoides analyzed are diploids with 2n = 14. The basic chromosome number x = 7 plus some morphological characters are considered synapomorphies for Tigridieae , this region is nowadays threatened by antropogenic activities, such as the introduction of alien species, or over-use by fire and cattle grazing  to the “vulnerable” (VU) status in the IUCN Red List, especially considering that the species is threatened by current or potential levels of exploitation of habitat and by the effects of introduced taxa. Our data provide an glimpse into the variability and evolution of C. crocoides that can serve as a starting point for further biological and evolutionary studies. Along this perspective, we intend to analyze the main factors involved in the dispersion power in C. crocoides and to compare it with C. campestris that presents a wider geographic distribution.The results reported in this study showed that the surveyed populations of


Symphodus melops), which are transported long distances to be used as cleaner fish in salmon farms, escape and hybridize with local populations. Recently, increasing numbers of corkwing wrasse have been reported in Flatanger in Norway, north of its described distribution range, an area heavily relying on the import of cleaner fish from Skagerrak. Using genetic markers identified with 2bRAD sequencing, we show that, although the Flatanger population largely is a result of a northward range expansion, there is also evidence of considerable gene flow from southern populations in Skagerrak and Kattegat. Of the 40 corkwing wrasses sampled in Flatanger, we discovered two individuals with clear southern genotypes, one first-generation hybrid, and 12 potential second-generation hybrids. In summary, we provide evidence that corkwing wrasse escape from fish farms and hybridize with local populations at the leading edge of an ongoing range expansion. Although the magnitude and significance of escapees warrant further investigation, these results should be taken into consideration in the use of translocated cleaner fish.The genetic impact of farmed fish escaping aquaculture is a highly debated issue. However, non-target species, such as cleaner fish used to remove sea lice from farmed fish, are rarely considered. Here, we report that wild corkwing wrasse ( TransfeSalmon farming may also promote inadvertent gene flow in populations of species of wrasses (Labridae) in Norway and the UK, where wild wrasses are caught and used as cleaner fish to mitigate the increasing problems of sea lice infestations in the farmed salmon ,7. TheseCtenolabrus rupestris) and the corkwing wrasse (Symphodus melops), are the most commonly used wild cleaner fish, with 39% and 52% of the total Norwegian official landings 2016, respectively . A recent study found relatively low genetic divergence between wild goldsinny populations in farming areas in mid-Norway and populations in southern Norway and Sweden, suggesting inadvertent gene flow [In Norway, two species, the goldsinny wrasse  sequencing method 2b-RAD  to simul2.2.1.et al. [With the help of commercial fishermen and local researchers, we collected corkwing wrasse from Flatanger in mid-Norway; from two locations in western Norway: Austevoll and Stavanger (western population); and from three locations at the Skagerrak–Kattegat coast: Kristiansand, Strömstad and Kungsbacka (southern population) . Fin cliet al. . Additio® Blood & Tissue Kit (Qiagen) with optional RNAse treatment (200 mg RNAse), and purified and concentrated with standard ethanol/isopropanol precipitation. DNA quantity and quality  were assessed using Qubit® ds DNA BR AssayKit (Invitrogen–ThermoFisher Scientific) and on a 1% agarose gel. 2b-RAD libraries were prepared following a protocol modified from Matz & Aglyamova [https://github.com/ellikafaust/S.melopsPopGen). All individual DNA samples were tagged with unique barcodes and then pooled in sets of 24 per sequencing lane, including technical replicates of four individuals to control for methodical artefacts. Pooling was done by sampling site, where each sample  was divided in two independent pools that were sequenced in separate lanes. This was done to minimize the risk of mixing up samples during library preparation, while having two independent pools to account for any lane bias. Single-read, 50 bp target length sequencing on Illumina HiSeq2500 platform was conducted at the SNP&SEQ Technology Platform in Uppsala, Uppsala University.Genomic DNA was extracted using DNeasyglyamova , availab2.2.https://github.com/z0on/2bRAD_denovo). First, low-quality reads and redundant sequences (i.e. restriction sites and duplicates) were removed. Remaining fragments were then clustered into rad tags, allowing up to three mismatches among reads (identity threshold 91%) and with a minimum depth of 20 reads. Individual genotypes were called, following the criteria of Mindp = 5 , hetero = 0.8 , aobs = 20  and strbias = 20 (strand bias cut-off). Four technical replicates per lane were used to control for methodical artefacts using the recalibrateSNPs.pl script. Variants that had been identically genotyped between the replicates were used as reference for non-parametric quality recalibration of all variants, estimating their probability of being ‘true’ SNPs. Loci with recalibrated quality below 20 and alleles with quality below 20 were removed. Only variants with less than 75% heterozygotes and less than 50% missing data were kept for thinning (removing) of the dataset. SNPs occurring on the same RAD-tag were removed, leaving only the SNP with the highest minor allele frequency (MAF) in each RAD-tag. Technical replicates and poorly sequenced individuals  were removed. Finally, we removed loci that were missing in more than 30% of the individuals or with a global MAF below 1%. Initially, different levels of minor allele frequency  were tested. As the different datasets did not change the outcome of the analyses (data not shown), we only present results from loci with maf > 1%, maintaining the most number of loci, while still removing genotyping errors and uninformative polymorphisms [The bioinformatic analysis of the DNA sequences followed a modified de novo pipeline from Pierre de Wit  using scorphisms .2.3.2.3.1.Ho) values deviated from expected heterozygosity (He) was assessed by calculating FIS according to Weir & Cockerham [p-values calculated according to the complete enumeration method [q < 0.05) from HW proportions in more than one of the samples were subsequently removed. Weir & Cockerham's FST was estimated for each population pair and over all samples using diveRsity. Statistical significance of FST values was assessed using Fisher's exact probability test with 5000 Monte Carlo replicates, followed by FDR correction.We used the R package diveRsity  in R v. ockerham . Deviatin method  and adjun method . Loci th2.3.2.Missing genotypes can induce patterns of similarity or differentiation that are easily confused with genetic structure. To detect such biases, we clustered individuals based on their identity-by-missingness in PLINK v. 1.9 ,38 whereK. Once K is defined, it estimates the posterior probability of each individual's genotype to originate in each cluster. STRUCTURE analyses were performed assuming uncorrelated allele frequencies, allowing admixture and with no locprior. The burn-in period was set to 10 000 and the number of Markov chain Monte Carlo (MCMC) repetitions to 50 000. Clusters K from 1 to 7 were run three times per K. The different runs were merged for visual analysis with CLUMPAK [K) were estimated using STRUCTURE HARVESTER [K (mean lnP(K)) and the modal value of Delta K. The second individual-based clustering method (PCA) uses a multivariate exploratory approach that makes no prior assumptions about how many populations exist or boundaries between populations. Allele frequencies were centred but not scaled and missing data were replaced by mean allele frequencies with the function scaleGen in ADEGENET [To estimate and visualize genetic differentiation among individuals, we applied two individual-based clustering methods, STRUCTURE v. 2.3.4  and prin CLUMPAK . CalculaARVESTER  by calcuADEGENET ,46. PCA 2.3.3.FST were tested for linkage disequilibrium (LD) in Genepop on the web [To remove potential bias in hybrid analysis, 200 SNPs with the highest overall  the web  using 10 the web . We used the web  which es the web .FST estimates and no LD. The data were analysed using the uniform prior option, default genotype proportions and the burn-in period was set to 50 000 and the number of MCMC sweeps after burn-in to 300 000.Finally, we investigated the occurrence of hybridization in the northern-most location Flatanger in mid-Norway with the software NEWHYBRIDS. Individuals from Skagerrak  and western Norway (Austevoll and Stavanger) were included in the runs as the pure parent genotypes using the ‘z’ and ‘s’ options. The analysis was performed using the same 200 loci as for the simulated data, displaying the highest overall 3.3.1.FIS estimates indicate heterozygote excess in all samples (mean FIS ranging from −0.344 to −0.052). Fifteen loci deviated significantly (q < 0.05) from HW proportions in more than one sample and were subsequently removed, leaving 4357 SNPs for final analysis. No more than eight loci deviate significantly from HW proportions in any of the western or southern samples. However, a much higher number of loci deviate from HW proportions in the Flatanger sample. Almost all of the loci display negative FIS values, indicating heterozygosity excess. Furthermore, wrasse from the western population display an overall higher genetic diversity  compared with wrasse from the southern population . The Flatanger population shows the highest genetic diversity . Global genetic differentiation, estimated as FST = 0.0789, is significantly (p < 0.05) different from zero. Pairwise FST estimates (S4) demonstrate higher genetic differentiation between the western and southern populations (FST = 0.101–0.1312) than among the southern samples (FST = 0.0023–0.0030) or between the western samples (FST = 0.0065). Overall, Flatanger is genetically more similar to the western population (FST = 0.0243–0.0277) than the southern population (FST = 0.1163–0.1258).From the total 48 technical replicates (four for each pool of 20), we called 237 090 SNPs (average 4939 ± 126 s.d. per replicate pair). Of these, 9% ± 0.05% s.d. were inconsistent between technical replicates. Data filtering resulted in a total of 4372 polymorphic SNPs, and none of the 240 individuals had to be removed due to missing data. Of the 57 600 missing data comparisons, only 479 pairwise comparisons have an identity of missingness higher than 20% (max 47%), and no obvious patterns of identity by missingness can be observed (S3). 3.2.FST estimates and previous studies [K = 2, and partially to a third cluster (purple) for K = 3. However, two individuals from Flatanger (FKH48 and FKH50) were assigned to the southern population (blue). Another individual from the Flatanger sample (FKH67) was assigned equally to both populations, suggesting admixture.STRUCTURE analyses suggest the existence of two, potentially three, genetically differentiated clusters ( studies . Most iny-axis) splits the Flatanger population from the western population, placing Flatanger closer to the southern population than the western. Succeeding components explain less than 1% of the total variance each, and are not shown. The same two individuals from Flatanger (FKH48 and FKH50) which were assigned to the southern cluster in the STRUCTURE analyses group with the southern cluster in the PCA. The Flatanger individual which was assigned equally to both clusters in the STRUCTURE analyses (FKH67) is closer to the southern cluster than any of the other individuals from the Flatanger or western population.To estimate and visualize genetic differentiation among individuals without prior assumptions about the population model, we conducted a PCA . The fir3.3.a), indicating gene flow across the break. Simulated data demonstrated high efficiency, accuracy and power to detect individuals from all of the six hybrid classes given the battery of 200 loci used  demonstrate that the Flatanger as a whole is genetically most similar to Austevoll, followed by Stavanger, while almost 10-fold more differentiated from the southern sampling locations. We observe similar patterns of genetic differentiation in the individual-based clustering methods for a majority of individuals from Flatanger  in addition to the hybrids discovered in Flatanger. Stavanger and Kristiansand are the two samples collected closest to the genetic break on the western and the southern side, respectively. Except for Flatanger, we did not detect any indication of hybrids in any of the other samples further from the genetic break, indicating the existence of isolated populations [Two individuals (FKH48 and FKH50) exhibit high similarity to the southern population while differentiating from all western and Flatanger individuals. They clearly cluster with southern individuals in STRUCTURE and PCA, suggesting a southern genotype. A third individual (FKH67) did not cluster with either southern or western populations, and was classified as a F1 hybrid (50 : 50 western:southern) by NEWHYBRIDS . Furtherfigure 4ulations .et al. [The relatively high number of southern--western hybrids in Flatanger is, therefore, convincing evidence of escapement and hybridization of cleaner fish sourced from Skagerrak and/or Kattegat. Recently, Jansson et al.  showed tet al. . CombineThe onset of gene flow between previously isolated populations may have genetic, physiological and ecological consequences. The corkwing wrasse in Flatanger most likely colonized the area within the last two decades . This an4.3.The effects of hybridization between genetically distinct populations are hard to predict and depend on many factors. Fitness can increase as a result of introducing favourable alleles and genotypes (overdominance), or because of deleterious alleles being sheltered (heterosis) . The thrPopulations on the boundary of a species range exist in conditions similar to the habitats just outside the distribution range, making them more likely to carry genotypes that are able to colonize new habitats . As the In the face of climate-induced changing environments, conservation of populations on the leading edge should be prioritized to maximize future adaptive potential ,61,62. W5.We provide the first evidence that translocated wild corkwing wrasse used as cleaner fish in salmon farms escape and hybridize with local populations at the northern limit of its distribution. These findings provide important information for aquaculture management and conservation of wild populations of non-target species, and have implications for the increasing use of cleaner fish as parasite control in fish farms, which is both poorly documented and regulated. Moving genetic material between isolated populations could drastically alter the genetic composition, erode population structure and potentially result in loss of local adaptation, hampering the species expansion. The geographical extent and magnitude of introgression and the ecological consequences remain unknown for this and other wrasse species. It is urgent to address these gaps of knowledge, as there is no immediate sign of reduction of the current practice in Norway, and wrasse are increasingly being deployed in other areas such as the UK.