Dianthus virgineus, we found a low genetic isolation paralleled by a high phenotypic differentiation among populations. In this species, genetic variation is related to geographic distance, whereas phenotypic variation does not result from genetic drift or gene flow limitations. Accordingly, we hypothesize that the phenotypic variation in this species is driven by selective forces, which act despite the presence of gene flow.Natural selection, genetic drift, and migration mainly drive evolution within species. By studying intraspecific variation at different elevations within the wild carnation Dianthus virgineus as study system. We genotyped genome-wide SNPs through ddRAD sequencing and quantified phenotypic variation through multivariate morphological variation. We assessed patterns of variation by testing the statistical association between genetic, phenotypic, geographic, and elevational distances and explored the role of genetic drift and selection by comparing the Fst and Pst of morphometric traits. We revealed a weak genetic structure related to geographic distance among populations, but we excluded the predominant role of genetic drift acting on phenotypic traits. A high degree of phenotypic differentiation with respect to genetic divergence at neutral loci allowed us to hypothesize the effect of selection, putatively fuelled by changing conditions at different sites, on morphological traits. Thus, natural selection acting despite low genetic divergence at neutral loci can be hypothesized as a putative driver explaining the observed patterns of variation.By relating genetic divergence at neutral loci, phenotypic variation, and geographic and environmental distances, it is possible to dissect micro-evolutionary scenarios involving natural selection and neutral evolution. In this work, we tested the patterns of intraspecific genetic and phenotypic variation along an elevational gradient, using  The micro-evolutionary forces acting within a species are mainly neutral processes, migration, and natural selection . As a geRelating neutral genetic variation to geographic and environmental differences sheds light on the mechanisms involved in the differentiation of natural populations . In partThe detection of phenotypic patterns of variation, and their association with genetic variation at neutral loci, can also help to understand micro-evolution within species . When phIn this context, plant species distributed along broad elevation ranges are natural study systems that provide insights into the patterns and mechanisms of intraspecific variation ,20. Typi2) and among populations (c) cannot be measured but only assumed [2 values and considering Pst-Fst comparisons as a tool to generate hypotheses on selection but not to test them. Thus, if Pst ≈ Fst, genetic drift can be imputed as the main differentiation mechanism for the trait for which Pst is calculated; if Pst is significantly different from Fst, either a divergent (if Pst > Fst) or stabilizing (if Pst < Fst) selection can be hypothesized [A feasible way to infer the presence of local adaptation in natural populations is to compare indices of neutral genetic differentiation (Fst) and indices of between-population differentiation of quantitative traits measured in wild populations (Pst) ,25,26. Tthesized , the forthesized ,33,34,35Dianthus virgineus L. [Dianthus virgineus is a Mediterranean species of wild carnations, mainly distributed in southern France and the Italian Peninsula, occurring along a wide elevational range [D. virgineus belong to a genetically uniform, recently originated (114–132 kya) evolutionary lineage [In this work, we explored the patterns of intraspecific genetic and phenotypic variation along an elevational gradient using ineus L. , an outcal range ,38,39. P lineage . In cent lineage . NeverthTo infer genetic relationships and structure, we genotyped genome-wide, neutral SNPs through ddRAD sequencing. To quantify phenotypic variation, we characterized multivariate morphological variation. Then, we assessed patterns of variation by testing statistical association between genetic, phenotypic, geographic, and elevation distances and explored the role of selection and genetic drift in phenotypic variation by comparing the Fst and Pst of the morphometric traits. In particular, if spatially restricted gene flow among populations is the main factor shaping intraspecific variation, we expect an association between phenotypic, neutral genetic, and geographic distances (IBD), but not with elevation, assuming that elevation and geographic distances do not covary. In this case, we also expect that the Pst values are not significantly different from the Fst of neutral loci. Alternatively, if environmental differences related to increasing altitude actually impose selection, we expect to retrieve a positive correlation between phenotypic variation and elevation. Moreover, this scenario would be supported by significant differences between Pst and Fst and a covariation between genetic fixation and phenotypic variation if ecological differentiation has happened. On the other hand, a statistical independence between these factors, combined with Pst values higher than Fst, would suggest that genetic drift is not the main force shaping phenotypic variation and that selection could be strong enough to overcome gene flow.Dianthus virgineus belongs to a species complex of 21 morphologically similar taxa occurring in open habitats of the central Mediterranean area along a wide elevational range [al range . All speal range ,43. In tal range ,45,46.D. virgineus grows in populations composed of a high number of adult individuals , occupying a broad area of occupancy; nevertheless, we found a lower number of narrowly distributed flowering individuals (<50) on Capraia Island. With the exception of the latter locality, we collected material from 20 randomly selected adult individuals per sampling site. All these populations were assessed as a diploid with 2n = 2x = 30 chromosomes [We selected as the study area a Mediterranean region from central Italy a, in whiomosomes . PopulatThe genetic analyses relied on genome-wide single nucleotide polymorphisms (SNPs) obtained using double digest restriction associated DNA sequencing (ddRAD-seq) . GenomicThe library preparation followed the protocol used by Westergaard et al. . BrieflyD. virgineus complex. The dDocent pipeline concatenates forward and reverse reads to generate sets of unique sequences that are then clustered into reference contigs by the software RAINBOW 2.0.4 [https://github.com/jpuritz/dDocent/blob/master/tutorials/Filtering%20Tutorial.md (accessed on 15 November 2020). The variants called in 50% of the individuals and with a minimum quality score of 30, a minor allele count of 3, and a minimum depth for a genotype call of 3 were initially kept. Four different individuals belonging to 4 different populations were discarded from the dataset due to a percentage of missing data higher than 45%. Variants with a 0.7 call rate and a minor allele frequency of 0.05 were kept. Additional filters to remove variants resulting from sequencing errors, paralogues, multicopy loci, or artefacts of library preparation were applied, as recommended in the pipeline. After keeping only single nucleotide polymorphism (SNP) variants, loci with significantly different allelic frequencies from the null model of Hardy–Weinberg equilibrium  within n et al. , eliminath TIGER .To explore the genetic structure of the studied populations, we inferred the number of genetically homogeneous clusters (K) by performing a Bayesian cluster analysis with STRUCTURE 2.3.4 ,63,64,65ST matrix  with hi package , and 99%D. virgineus complex [Dianthus, the variation in traits such as plant height, leaf dimensions, and calyx length is known to be associated with climatic variation and oscillations [https://www.r-project.org/index.html (accessed on 1 September 2023) and PAST (https://www.nhm.uio.no/english/research/resources/past/ (accessed on 1 September 2023)).Morphometric analyses were carried out to reveal quantitative patterns of variation and to detect phenotypic differentiation in adult plants among populations. We measured 24 quantitative variables regarding vegetative organs (leaves and stems) and reproductive structures  . These c complex ,42,72. Mllations ,72,73 anllations . Seven vllations ; https:/After data collection, we prepared the dataset for further multivariate analyses. Firstly, two individuals were removed from the dataset due to missing data (one from AR and one from CAP). Secondly, we removed variables that did not show significant differences among populations. We tested for the homoscedasticity of each variable with the Levene test (significance threshold α = 0.05), and then we performed univariate analyses on each morphometric character using an ANOVA or Kruskal–Wallis test and multiple comparison tests, Tukey–Kramer’s post hoc test, and Mann–Whitney’s U test with Bonferroni correction (α = 0.01), respectively. Thirdly, we built a dataset with mean values per population of each variable, and we filtered out highly correlated variables showing Pearson’s r >0.8. Finally, to avoid distortions in the multivariate analyses due to different absolute values, each variable was then standardized to z-score values. The overall variability in the refined dataset was explored with a Principal Component Analysis (PCA).p < 0.01), to understand if elevation could be a good predictor of the variation in the trait, regression  analyses were carried out.Spearman correlation tests between morphometric traits and elevations of populations were performed. In the case of a highly significant correlation (ST), geographic and elevational distributions, morphometry, and the seed germination of the studied populations, Mantel tests  were pe package . The elePstat R package [To estimate the differentiation of morphological traits among populations and to test the role of selection, we estimated Pst values using the  package . For eac package :(1)Pst=2 denotes the proportion of additive genetic effects on the variation within populations, whereas c is the proportion of additive genetic effects on the variation between populations. Contrary to Qst, when calculating Pst, no direct measurement of h2 and c can be obtained, and in a hypothetical scenario, c = h2 is assumed. Then, to understand if Pst can be used as a substitute for Qst, Pst is estimated using a range of c and h2 values [2 close to zero, meaning that c is much smaller than h2, which is likely due to the presence of environmental heterogeneity among population sites [2 when Pst is significantly higher than Fst, the stronger the evidence of a putative role of selection in shaping the studied trait.In (1) 2 values . Naturalon sites . Thus, t2 = 1), also assuming that the former was higher than the latter (c/h2 = 0.1). We assessed the robustness of Pst-Fst comparison by calculating Pst for an interval of c/h2 values, spanning from zero to two. For each Pst estimation, 99% confidence intervals were calculated by bootstrap resampling 1000 times. Genetic drift is not considered to have a relevant contribution to phenotypic differentiation if the confidence intervals of the Fst and Pst estimations do not overlap for c/h2 ≤ 0.5 [We calculated the Pst values for three morphological traits, selected according to the explorative multivariate analyses and the correlation test results with elevation. Firstly, we assumed that the proportion of additive genetic effects on the variation within and across populations did not differ , with 3.81% of the missing data and 2.22% of the mean genotyping error rate. The retained SNPs were unlinked and at Hardy–Weinberg equilibrium within each population.According to Evanno’s statistics, an explorative STRUCTURE analysis  suggests K = 9 as the number of the most homogeneous genetic clusters . However, basal leaf width and lower stem leaf width are highly correlated . Testing the association between calyx length and increasing elevation after removing the outlying AA populations results in a higher, negative, and significant correlation . Moreover, a linear model does not fit when considering the calyx length of AA , whereas it shows a good fit when this population is excluded . Plant height is not linearly correlated with increasing elevation , as EL shows values overlapping with those of high elevation populations .The results of the Spearman’s correlation tests are listed in ulations c. HoweveThe genetic, morphometric, elevational, and geographic distance matrices are shown in the p < 0.01, p = 0.138, p = 0.138, p = 0.013, p = 0.100, p = 0.210, The genetic distance at neutral loci is significantly correlated with geographic distance among populations . Thus, D. virgineus is widely distributed across the continental part of the study area [Dianthus are Lepidopterans [Our results suggest that the studied populations are connected through gene flow. Indeed, udy area . The maiopterans , highly opterans . In addiopterans ,45,46, pD. virgineus. Although the overall morphometric variation is not statistically correlated with elevational distances among populations, we measured a negative association along the elevational gradient of some vegetative and reproductive traits, in line with a general trend of decreasing plant size with increasing elevation [Dianthus. For instance, high-elevation ecotypes, characterized by reduced plant size, have also been observed in D. carthusianorum L. in the Swiss Alps [D. virgineus complex proved that increasing elevation and decreasing temperature are negatively associated with the traits determining plant biomass  [The morphometric results point to the role of local environmental conditions in determining, possibly through selection, the phenotypic variation within levation ,22. Thisiss Alps . On broaf width) .D. virgineus. For instance, the non-linear pattern of plant height (showing a very high Pst) with elevation suggests that there may be other biotic and abiotic factors contributing to the variation in plant size. Concerning reproductive traits, calyx length decreases with elevation and shows conspicuous Pst values, but it is significantly shorter in individuals from Apuan Alps (AA), collected at just around 600 m a.s.l. The size of calyx, and of flowers in general, is functionally connected with pollination ecology in Dianthus [D. inodorus (L.) Gaertn., smaller flowers (often pistillate) are less likely to be affected by seed predators compared to larger, hermaphroditic flowers ([D. sylvestris Wulfen). Accordingly, the reduced flower size observed in AA may be due to strong selection, as it could be advantageous to avoid a fitness decline due to seed predators. These hypotheses on putative selective pressures acting on the phenotypic variation in D. virgineus will require future in-depth testing.The Mantel test between overall morphometric and elevation distances was not significant, suggesting that other environmental factors, not directly related to the elevational gradient, are acting on the morphological variation in Dianthus . For insflowers . These results do not allow a major role of gene flow limitations and genetic drift in shaping phenotypic variation among populations to be hypothesized. On the other hand, our results could support an alternative hypothesis in which the phenotypic variation in D. virgineus is the result of divergent selective pressures that are strong enough to counteract gene flow [Dianthus [2 values, the Pst values for the three considered traits overlap with the Fst values at c/h2 values approaching zero, suggesting that phenotypic differentiation can be used as a robust proxy for Qst to infer putative natural selection [Phenotypic differences among populations of ene flow ,32,35. Aene flow ,18,19. Tene flow , and it Dianthus . Nonetheelection . In addielection , and preD. virgineus. Although the studied populations show a weak genetic structure, differentiation at neutral loci parallels geographic distance but not elevation. Moreover, the significantly higher Pst values of the selected morphometric traits with respect to the Fst values do not support the role of gene flow limitations and genetic drift in shaping the observed phenotypic variation. Alternatively, selection under gene flow can be hypothesized to be a possible scenario to explain the intraspecific variation in D. virgineus.For the first time, we provided evidence supporting the putative role of phenotypic selection acting despite low genetic divergence at neutral loci in the widespread Mediterranean species


Prochilodus costatus a Neotropical migratory fish that has been threatened due to loss and fragmentation of its natural habitat since 1960s promoted by the expansion of hydroelectric power plant construction programs. This study evaluated the eco‐evolutionary parameters that cause oscillations in the demography and structure of P. costatus populations. An integrated approach was used, including temporal and spatial sampling, next‐generation sequencing of eight microsatellite loci, multivariate genetic analysis, and demographic life‐history reconstruction. The results provided evidence of the complex interplay of ecological–evolutionary and human‐interference events on the life history of this species in the upper basin. In particular, spawning wave behavior might have ecological triggers resulting in an overlapping of distinct genetic generations, and arising distinct migratory and nonmigratory genetic patterns living in the same area. An abrupt decrease in the effective population size of the P. costatus populations in the recent past (1960–80) was likely driven by environment fragmentation promoted by the construction of the Três Marias hydropower dam. The low allelic diversity that resulted from this event is still detected today; thus, active stocking programs are not effective at expanding the genetic diversity of this species in the river basin. Finally, this study highlights the importance of using mixed methods to understand spatial and temporal variation in genetic structure for effective mitigation and conservation programs for threatened species that are directly affected by human actions.The human activity impact on wild animal populations is indicated by eco‐evolutionary and demographic processes, along with their survival and capacity to evolve; consequently, such data can contribute toward enhancing genetic‐based conservation programs. In this context, knowledge on the life‐history and the eco‐evolutionary processes is required to understand extant patterns of population structure in  To hit these questions, this study used an integrative approach, including the genotyping of the populations using microsatellite loci and the comparison of contemporary and past Ne oscillations through BSP coalescence reconstruction. Thus, these mixed approaches are expected to allow us to identify the main processes that have shaped the contemporary demography and genetic patterns of P. costatus populations that include biotic stress, fragmentation of the habitat, and human interference.This study conducted a wide‐ranging spatio‐temporal investigation to evaluate the demography of , et al.  suggeste22.1Prochilodus costatus is a migratory fish that is endemic of the São Francisco River basin in Brazil that belongs to the Prochilodontidae family (Castro & Vari 2004). Wild females of Prochilodus costatus spawn, in total type, an average of 250,000 oocytes in the water to be fertilized externally, and development will occur as eggs float downstream  in L50 and L100 of 1.6 years and 3.8 years, respectively, and this species presented an estimated 6 cohorts . Several hydropower dams are distributed in the upper São Francisco Riverbed, with the Três Marias being the largest   Figure  over a p) Figure .ProC10, ProC18, ProC22, ProC36, ProC37, ProC44, ProC48, and ProC49 , which protocol was developed and followed using Pimentel et al.  and 5 µl 0.2 M NaOH, followed by 5‐min incubation for DNA strand denaturation. Next, the genotyping libraries were diluted using Illumina buffer to obtain a final concentration of 10 pMol DNA in a final volume of 1 ml. Sequencing was performed with 600 µl of the final solution, using the MiSeq platform (Illumina®).Genotyping was carried out using eight polymorphic microsatellite loci: l et al.  instruct2.3P. costatus ) using the high sensitivity prior in Bowtie 2  and the expected (He) heterozygosity, and to estimate the allelic richness (A), we performed a bootstrap through 999 replicates using the diveRsity package  by hydrological years ; (2) by areas ; and (3) by migratory/reproductive and nonmigratory/nonreproductive periods. Such combinations were independently analyzed by three distinct methods and softwares: AMOVA, pairwise ϴst  and then run the k‐means algorithm to detect the range of possible k‐clusters (without any a priori information of population); thus, the individuals are assigned into those k‐clusters using Bayesian information criterion (BIC). In a second step, a discriminant analysis (DA) is performed through dapc function using the number of retained PCs in order to maximize the variation of the obtained k‐clusters. Then, a membership probability of each individual is performed to be grouped into their respective k‐clusters through compoplot function that results in an assignment a probability (ranging from 0% to 100%) of an individual to belong into their k‐cluster .To evaluate the genetic structure of the Jombart, . To perfr ≤ 0.25 as unrelated, r between 0.26–0.5 as half‐siblings, and r ≥ 0.5 as full siblings as suggested by Jones, Small, Paczolt, and Ratterman  of the P. costatus populations was estimated using the linkage disequilibrium (LD) method . This phenomenon represents the loss of rare alleles and could be assessed by looking at the overall distribution of allele frequency classes  for 1,000 simulations.To assess whether the population showed the signature of a recent demographic event , the software BOTTLENECK v1.2.02  approach. This analysis simulated past demography using microsatellite data with a coalescent approach, to estimate changes in recent and ancestral Ne. This approach relies on motif distance frequencies between alleles to estimate variation in Ne over time   and 10% of the mutations greater than a single step. Then, we set four independent MCMC runs simulating for 10 million generations sampled at every 1,000 trees and 25% burn‐in. The sexual generation time for P. costatus was set to four years, as indicated by Santos and Barbieri  through NBAR function that attributes three global θ as means of effective size being: θ0 of 2.3384811908619 as Ne minimum, θ1 of 10.4918537554347 as Ne intermediate, and θ2 of 12.5949077154245 as Ne maximum. In the last 500 generations, changes to Ne were assessed. Inferences were made about recent past expansions and declines based on posterior distributions and their attributes , as suggested by Nikolic and Chevalet  (Table A was observed for PA2‐ITA (2014/2015), and highest A was observed for PA1‐MP (2014/2015) , 1,027 specimens of ow Table .k = 3) comparing the hydrological years  comparing the areas  comparing the periods , among individuals of sampling areas , between the migratory and nonmigratory periods , and among individuals between the migratory and nonmigratory periods   than between areas  and between populations , being 51% of the individuals are full siblings, 36% are half‐siblings, and 13% are unrelated.The relatedness analysis was significantly higher between pair of individuals sampled between years (3.2Ne = 215.4) and PA1 presented the lowest (mean Ne = 13.8) for LDNe, and for TpNe the PAO also presented the highest (mean Ne = 265.1) and PA1 the lowest (mean Ne = 10.1) that were obtained through NeESTIMATOR , which represent the site in the original riverbed of the São Francisco River basin, in the hydrological year 2016/2017. When the mean Ne for LDNe of all the populations was compared among the hydrological years, it increased from 42.06 (n = 378) in 2013/2014 to 61.67 (n = 394) in 2014/2015, followed by an abrupt decrease to 25.03 (n = 179) in 2015/2016 and 9.97 (n = 56) in 2016/2017  reached more range of variation  and PA2‐LAM‐2014/2015 (p ≤ .02) when the hydrological years were grouped, and in SFU (p ≤ .01) and PA2 (p ≤ .02) when analyses were performed with respect to area . The data demonstrated intra‐annual stability between 2013/2014 and 2016/2017 based on DAPC and AMOVA. However, despite similarity among the different methods used to assess temporal and spatial stability at sampling sites, intra‐year comparisons produced fewer significant differences compared to across years. Comparing the average He (He = 0.54) detected herein for the P. costatus populations with those founded by Carvalho‐Costa et al.  that was obtained through DAPC that may indicate that even with the construction of the Retiro Baixo dam in 2011 . Several studies of repopulation with native salmonids in various parts of the world have shown that stocking has little impact on the genetic makeups of populations . Thus, genetic pattern 2 might be the original pattern of the population. In comparison, genetic pattern 1 (observed throughout 2014/2015 and 2015/2016) was a consequence of the long drought period in the basin. However, only one site was sampled in 2016/2017, with additional sampling being required in the future to establish whether this hypothesis holds.Spawning waves are directly stimulated by ecological triggers for migratory fish to reproduce and enhance offspring survival during the optimal season  since it allows access to the decreasing of the Ne, the genetic variation levels, and its impact on wild population fitness under human interference , likely due to the environment fragmentation promoted by the construction of hydropower dams. Although stocking programs have been implemented to help address the impacts of dam construction in the area, these programs remain ineffective at expanding the level of genetic diversity, as low allelic diversity intrapopulation levels continue to be detected. Our results show that P. costatus populations are still subject to inbreeding depression, due to environmental changes caused by anthropogenic actions, raising the question of how long these populations will persist. This integrative study also emphasized the importance of using long‐term molecular methodologies to evaluate the eco‐evolutionary consequences of anthropic interference shaping the survival of wild organisms. This approach could be used to enhance mitigation programs and guide conservation decision‐making.This study highlighted the complex interplay of ecological and evolutionary events in shaping the life history of None declared.Supporting InformationClick here for additional data file.


Permeable phylogeographic barriers characterize the vast open ocean, boosting gene flow and counteracting population differentiation and speciation of widely distributed and migratory species. However, many widely distributed species consists of distinct populations throughout their distribution, evidencing that our understanding of how the marine environment triggers population and species divergence are insufficient. The sailfish is a circumtropical and highly migratory billfish that inhabits warm and productive areas. Despite its ecological and socioeconomic importance as a predator and fishery resource, the species is threatened by overfishing, requiring innovative approaches to improve their management and conservation status. Thus, we presented a novel high-quality reference genome for the species and applied a seascape genomics approach to understand how marine environmental features may promote local adaptation and how it affects gene flow between populations. We delimit two populations between the Atlantic and Indo-Western Pacific oceans and detect outlier loci correlated with sea surface temperature, salinity, oxygen, and chlorophyll concentrations. However, the most significant explanatory factor that explains the differences between populations was isolation by distance. Despite recent population drops, the sailfish populations are not inbred. For billfishes in general, genome-wide heterozygosity was found to be relatively low compared to other marine fishes, evidencing the need to counteract overfishing effects. In addition, in a climate change scenario, management agencies must implement state-of-the-art sequencing methods, consider our findings in their management plans, and monitor genome-wide heterozygosity over time to improve sustainable fisheries and the long-term viability of its populations. Highly migratory and widespread marine species usually present high gene flow levels between populations. However, the sailfish shows two highly divergent populations that lack gene flow between the Atlantic and Indo-Western Pacific oceans. These populations presumably evolved during the Pleistocene glacial cycles and were isolated for prolonged periods, but not sufficiently long for speciation to occur. The sailfish has lower genetic diversity than other marine fishes, although no recent inbreeding was detected. These results will improve the fisheries management for the sailfish, which requires establishing new and collaborative management plans. Besides, the results will enable comparative genomic studies and aid the assembly of high-quality reference genomes of closely related species.The open ocean lacks clear phylogeographic barriers that would prevent migration between populations. As a result, widely distributed marine species can sustain a circumtropical distribution counteracting local adaptation, population differentiation, and speciation. Yet, in practice, many marine species present deep evolutionary lineages that did not speciate, but have either confined or extensive distribution ranges with pronounced population differentiation between ocean basins. However, the factors underlying these speciation or population dynamics are not entirely understood, although habitat partitioning rather than allopatric isolation may have conducted these evolutionary forces .Istiophorus platypterus) is a highly migratory, circumtropical species characterized by its discernible sail-like dorsal fin, and like other billfishes (Istiophoridae and Xiphiidae), it inhabits a narrow range of environmental features preferring moderately warm and productive regions  and cost reduction have given rise to the genomics era, whereby genome-wide approaches rapidly replace traditional genetic marker approaches . High-quThe lack of genome-wide data for the sailfish compromises our understanding of its genome-wide heterozygosity, fishery stocks delimitation, and resilience to overfishing and climate change . Therefoa). Furthermore, Merqury estimated an assembly completeness of 95.86% with an assembly consensus quality value of 29.48, standing for an error rate of 0.11%. A high mapping rate of >99% for short reads and 95.66% long reads without contamination confirmed the assembly's completeness (fig. 2b). Short reads’ mapping statistics per sample are shown in The final chromosome-level de novo assembled genome has a length of 619.037 mega bases (Mb) divided into 404 scaffolds with contig N50 of 26.28 Mb, L50 of 11, and guanine-cytosine (GC) content of 40.87%. The largest 24 contigs (>1 Mb) make up over 99% of the total assembly length  online. fig. 2leteness  online. a and b). It also showed a possible substructuring between populations in the Southwest Indian (SWI) and Western Central Pacific (WCP) oceans. However, the ancestral admixture coefficients from K = 2 to K = 4, showed no evidence for substructuring between these populations. The ΔK method yielded the best value of K = 2 (c–e). K = 2 to K = 6 between and within each ocean basin. As a subproduct, we provided a subset of 200 highly informative SNPs for population analysis to nurture future fishery stock assessments for the sailfish  calling filtering, and linkage disequilibrium (LD) pruning comprised 428,675 unlinked sites. The discriminant analysis of principal components (DAPC) exhibited two highly divergent clusters between the ATL and Indo-Western Pacific (IDWP) oceans with linear discriminants of 99.88% in the first axis a and b. of K = 2 c–e. Suppsailfish  online.m = 0.0196 ± 0.0184) and Southwest Atlantic (SWA) (m = 0.0195 ± 0.0185) and from WCP toward SEI (m = 0.0159 ± 0.0152) and Southwest Indian (SWI) (m = 0.0159 ± 0.0152), respectively (g).In addition, we found that most short-term migration occurred within each ocean basin, not between them, reinforcing the existence of two populations. As much as in the ATL as IDWP, migration occurs westward from East Central Atlantic (ECA) toward Western Central Atlantic (WCA)  (a). Nevertheless, the raw genetic distance between these lineages (xyD = 0.264%) and the FST  are too low for them to be considered distinct species (The genomic maximum likelihood (ML) phylogeny shows that the genus ctively) a. Nevert species  online. pcadapt 700. However, only 82 outlier loci were detected simultaneously by the three methods (P < 0.001), and axis 1 was the most significant in explaining the data correlation , even though the four environmental variables were also significant (P < 0.001) (The three genome scans reported different numbers of outlier loci. Genome-wide differentiation scan (GWDS) reported 1,147 outliers, OutFLANK 567, and  methods  online.  methods  online. < 0.001)  online.a and b). Figure 5c–f exhibits the variation of the most significant environmental features.According to the RDAs, geographical distance plays a key role in explaining the differences between both populations, given that MEM1 strongly drives the variation between populations. Nevertheless, the RDAs showed that ATL outliers positively correlate to the mean chlorophyll concentration, mean sea surface temperature, and mean sea surface salinity but negatively relate to the mean dissolved oxygen. However, IDWP is positively correlated to dissolved oxygen and negatively correlated to the other variables a and b. eN) for most billfishes during the Middle Pleistocene (774–129 thousand years ago [Kya]), except for the swordfish that delivered a smooth increase in eN during the Late Pleistocene (129–11.7 Kya) . The genetic optimization for eN estimation (GONE) showed the most recent demographic history trajectories for each population; both populations displayed an abrupt eN plummet around 80 years ago, in the 1950s when the open ocean large-scale industrial fisheries began and had the highest fishery catch statistics reports .Pairwise sequentially Markovian coalescent (PSMC) showed an increase in the effective population size (1.7 Kya)  online, n cycles a and b.  reports . ATL sufeduction c.a). Heterozygosity ranged from 0.202% in WCP to 0.229% in WCA (b). The average of runs of homozygosity (RoH)-based inbreeding coefficients (FRoH) for the sailfish was 2.18%, with a higher inbreeding in ATL individuals (2.48%) than in IDWP (1.44%) (c). FRoH ranged from 2.76% on WCA to 0.92% in SEI (d). Sailfish individuals had an average of 691.09 RoHs, most of which were short fragments ranging from 1 to 50 Kbp (e), evidencing no recent inbreeding in both populations. The average genome-wide heterozygosity was 0.216%, and it was higher in ATL (0.228%) than in IDWP (0.203%) a. Hetero% in WCA b. The av (1.44%) c. FRoH r% in SEI d. Sailfio 50 Kbp e, evidena and b). The hierarchical analysis of molecular variance (AMOVA) scenario I rejected the null hypothesis of the absence of genetic population structure (ST = 0.558 (P < 0.001). The pairwise ΦST evidenced that structuring between the populations is higher from a mitogenomic perspective because the most significant values are between the ATL and IDWP regions , ranging from 16,519 to 16,539 bp with nucleotide frequencies of adenine (A: 27.4%), cytosine (C: 30.5%), guanine (G: 16.4%), and thymine (T: 25.8%). Mitogenomic outcomes showed differences from genome-wide nuclear data; DAPC displayed overlapping clusters with minor differences between ATL and IDWP a and b. tructure  online.  regions  online.b; fig. 4b; eN after 300 Kya (c and d). The migration rates show a stepping-stone dispersal from the IDWP toward the ATL Ocean, in which SWI connects with ECA and SWA (h).The topology of the fossil-calibrated tree showed that all extant billfishes diverged during the Middle Miocene, although the modern lineages diverged at the beginning of the Pliocene around 5.33 Ma. In addition, the sailfish lineages diverged at 1.81 Ma during the Early Pleistocene (2.58–1.80 Ma) fig. 5b;  online.  300 Kya c and d.  and SWA h.Our new chromosome-level genome assembly supplies a novel and improved high-quality reference genome for the sailfish. In comparison to the recently published assembly from FST and ΦST values obtained by mtDNA are explained by the reduced eN of haploid genomes, which results in faster genetic drift and earlier lineage sorting  showed a much higher differentiation than genome-wide markers; the higher  sorting . Even th sorting .Our results corroborate these previous results, implying a single population within the ATL Ocean and another in the IDWP Ocean that accounts for the Indian and Western and Central Pacific oceans. The reconnaissance of these populations as distinct fisheries management units contradicts the current strategies adopted for the sailfish by the International Commission for the Conservation of Atlantic Tunas (ICCAT), the Indian Ocean Tuna Commission (IOTC), and the Western and Central Pacific Fisheries Commission (WCPFC), which traditionally manage the sailfish as eastern and western stocks in the ATL and Pacific oceans and as a single stock in the Indian Ocean. So, to counteract the overfishing effects on its populations , a new mSailfish exhibited different migration patterns for genome-wide and mitogenomic markers. Genome-wide short-term migration rates showed that migration occurs within the ATL and IDWP basins, with almost no migration between the ATL and Indo-Pacific oceans. However, the mitogenomic long-term migration rates connected these regions through a westward stepping-stone migration pattern from the WCP toward the ATL Ocean. Despite being a highly migratory species, no transoceanic migration between the ATL and Indo-Pacific is known for the sailfish; its displacements are more restrictive than other billfishes, and it may present site fidelity, cyclic annual movements, or both .Understanding the migration patterns between populations and their correlation with oceanic physicochemical is crucial for guiding management practices in commercially exploited species . Yet, maDetecting local adaptation can be challenging for highly migratory marine species formed by large and interconnected populations . Even soIstiophorus and the fossil-calibrated tree showed that the sailfish's mitogenomic lineages diverged around 1.81 Ma at the end of the Early Pleistocene (2.58–1.80 Ma). A few individuals from ATL clustered within the IDWP clade, as also evidenced by FST estimates  although the extant species diverged during the Pliocene (5.33–2.58 Ma), as proposed by stimates  online, stimates . TherefoThe Plio–Pleistocene transition (PPT) (∼3.3–2.8 Ma) paved the way for the interglacial cycles of the Pleistocene, resulting in cooler temperatures between the ATL and Indo-Pacific oceans . In addiAlthough based on mtDNA-based analyses, some ATL individuals clustered with Indo-Pacific individuals, the nuclear genome analyses indicate that individuals from the two ocean basins cluster as distinct units, with no evidence for past or ongoing gene flow. This mito-nuclear discordance appears consistent with a scenario of incomplete lineage sorting (ILS), whereby the random loss and fixation of a single locus (like mtDNA) can result in gene trees that are discordant with the species tree (true population history). This scenario would entail that the ancestral mtDNA haplotypes, which now occur exclusively in the ATL population, occurred originally widespread but were lost from the Indo-Pacific Ocean, possibly due to population bottlenecks during the Pleistocene glaciations. This scenario would explain the paraphyletic clustering of mtDNA haplotypes and the deep split times of the major mtDNA haplotype cluster. If the loss of mitochondrial haplotype diversity in the Pacific results from a population bottleneck, it would also be consistent with the observed differences in genome-wide heterozygosity between the populations (ATL: 0.228% vs. Indo-Pacific: 0.203%)  online.Istiophorus, which is currently represented only by I. platypterus, as suggested by Istiophorus to understand the taxonomic status of these highly divergent lineages.Although many widely distributed taxa are in monotypic genera or families, a circumtropical distribution may reduce the chances of allopatric speciation, which relies on a sufficient period of geographical isolation . HoweverBoth populations have similar demographic histories during the Late Pleistocene. ATL underwent a bottleneck event, due to the climatic changes over the Last Glacial Period (120–11.5 Kya), which led to a drop in global sea levels . In the eN expansion trend, regardless of whether a land bridge separated Western Pacific and Eastern Indian oceans during the Late Pleistocene , even among billfishes; it was smaller than the swordfish (0.365%) and the blue marlin (0.262%), although higher than the white marlin (0.102%) and the shortbill spearfish (0.177%)  online. eN , may sustain healthy levels of genetic diversity if management strategies were adopted to strengthen the efforts to cease overfishing and reduce fishing pressure within the maximum sustainable yield , it, along with the reduction in sequencing costs, will provide valuable and unprecedented information to detect oscillations in the genetic health of fishery stocks.To conclude, the new chromosome-level reference genome assembly will supply a powerful tool to improve sailfish management and conservation actions; coupled with our whole-genome resequencing data set and the subset of 200 highly informative SNPs, they will serve as a starting point for implementing a genomic monitoring plan. In this sense, sailfish fisheries management should incorporate time series estimations of genome-wide heterozygosity, inbreeding, Makaira nigricans), white marlin , and shortbill spearfish (Tetrapturus angustirostris) were collected from the ATL Ocean ; size distribution was analyzed in Agilent 2100 Bioanalyzer  and quantified by real-time PCR to ensure a minimum concentration of 3 nM, and then sequenced in a NovaSeq 6000 . Short-read data of an previously published Indo-Pacific sailfish (SRA: SRS7572558) and swordfish (Xiphias gladius) (SRA: SRR12883662)   were add©, Menlo Park, CA, USA) in continuous long-read (CLR) mode. The quality of the final library was assessed on the TapeStation System (Agilent Technologies©) using the Genomic DNA ScreenTape Analysis kit and the Invitrogen™ Qubit™ Fluorometer using the Qubit™ dsDNA high sensitivity (HS) assay kit . The high-quality high-throughput chromosome conformation capture (Hi-C) reads (SRA: SRR12883658) from To assemble the chromosome-level genome, we prepared a PacBio SMRTbell™ long-read library following the instructions of the SMRTbell™ Express Prep kit v2.0 sequencing on the Sequel System II  to annotate and hard mask known Actinopterygii repeats from the RepBase library, which comprises a database of representative eukaryotic repetitive sequences , applied trimming in a sliding window of 4 bp, discarded reads with ≤Q15, and removed polyG tails (http://broadinstitute.github.io/picard) using the recommended parameters for patterned flow cells. Qualimap v2.2.1 was used to evaluate mapping quality. Next, BAM files were processed to find intervals that needed realignment with RealignerTargetCreator and then realigned by IndelRealigner with Genome Analysis Toolkit (GATK) v3.8-1 (https://gatk.broadinstitute.org/). SAMtools was used to remove reads that did not pass the described filters above keeping only unique reads that mapped properly in the realigned BAM files. Reads mapped to scaffolds shorter than 1 Mb  and repetitive regions were also removed from the filtered BAM files.Fastp v0.23.2 evaluated the short reads for adaptors contamination, applied a low complexity filter and base error correction, restricted the presence of “yG tails . The filyG tails . PCR andd ± 5 × MAD, where d is the global site depth distribution median and MAD is the median absolute deviation. Sites with a P value < 1 × 10−6 for strand bias, heterozygous bias, or Hardy–Weinberg equilibrium were excluded. Only biallelic SNPs called with a P value < 1 × 10−6, minimum minor allele frequency (MAF) of 0.025, and shared for at least 90% of individuals were kept for further analyses.Genotype likelihoods from variant sites were estimated by SAMtools-modified Maq model in ANGSD v0.935 . MappingngsLD v1.1.1  (The Kinship-based INference (KING)  evaluatet v2.1.7 . The numt v2.1.7 , assuminters (K) . In ordehttps://github.com/stevemussmann/BA3-SNPS-autotune) was used with default settings for 1 million generations with 10% burn-in to tune-mixing parameters by implementing a binary search algorithm and conducting short exploratory runs to choose optimized parameters of migration rates, allele frequencies, and inbreeding coefficients. BayesAss3-SNPs ran for three independent runs performed through 100 million generations with 10% of burn-in with mixing parameters of 0.55 for migration rates, 0.55 for allele frequencies, and 0.075 for inbreeding coefficients.The contemporary migration patterns were estimated by BayesAss3-SNPs . BA3-SNPIQ-TREE v2.1.3  estimatepcadapt v4.3.3 , followed by a forward selection on adjusted R2 and P values of variables to guarantee non-correlated variables and to choose the most significant explanatory variables  were computed from a geographic distance matrix by adespatial v0.3–18  over time were estimated with the PSMC model  to infer the scaled mutation rate, the recombination rate, and the free population size parameters, respectively. We performed 100 bootstrap replicates by randomly sampling with replacement 1-Mb blocks from the consensus sequence for all individuals.Long-term changes in effective population sizes (MC model  using coMC model  for the eN changes over time. The site frequency spectrum (SFS) was estimated per population by ANGSD v0.935 and scaled by a substitution rate of 2.2 × 10−9 substitutions per site per generation estimated for the sailfish and 1.2 × 10−9 for the swordfish  was used as a proxy for inbreeding , estimating the allele frequency by recalculating INFO/AC and INFO/AN using the genotype likelihoods FORMAT/PL (“PL”). The proportion of the genome covered by RoH  among populations was inferred by MIGRATE-N v4.4.4 , defined as M = m/µ, where m is the immigration rate and µ is the mutation rate per site per generation. The runs were performed with an exponential distribution range of Θ 0–0.1  and M 0–1,000 . The MCMC settings were set as one long chain recorded by 300,000 steps incremented every 1,000 steps with a single replicate making up 300 million iterations and a static heating scheme of eight parallel chains with temperatures 1.0; 1.17; 1.4; 1.75; 2.33; 3.5; 7.0; and 1,000,000 with a burn-in of 30% per chain.The number of historical migrants per generation (N v4.4.4 , under aevad042_Supplementary_DataClick here for additional data file.


Introduction: Several polymorphisms altering the NAT2 activity have already been identified. The geographical distribution of NAT2 variants has been extensively studied and has been demonstrated to vary significantly among different ethnic population. Here, we describe the genetic variability of human N-acetyltransferase 2 (NAT2) gene and the predominant genotype-deduced acetylation profiles of Brazilians.Methods: A total of 964 individuals, from five geographical different regions, were genotyped for NAT2 by sequencing the entire coding exon.Results: Twenty-three previously described NAT2 single nucleotide polymorphisms (SNPs) were identified, including the seven most common ones globally . The main allelic groups were NAT2*5 (36%) and NAT2*6 (18.2%), followed to the reference allele NAT2*4 (20.4%). Combined into genotypes, the most prevalent allelic groups were NAT2*5/*5 (14.6%), NAT2*5/*6 (11.9%) and NAT2*6/*6 (6.2%). The genotype deduced NAT2 slow acetylation phenotype was predominant but showed significant variability between geographical regions. The prevalence of slow acetylation phenotype was higher in the Northeast, North and Midwest  of the country. In the Southeast, the intermediate acetylation phenotype was the most prevalent (40.3%) and, in the South, the prevalence of rapid acetylation phenotype was significantly higher (36.7%), when compared to other Brazilian states (p < 0.0001). Comparison of the predicted acetylation profile among regions showed homogeneity among the North and Northeast but was significantly different when compared to the Southeast (p = 0.0396). The Southern region was significantly different from all other regions (p < 0.0001).Discussion: This study contributes not only to current knowledge of the NAT2 population genetic diversity in different geographical regions of Brazil, but also to the reconstruction of a more accurate phenotypic picture of NAT2 acetylator profiles in those regions. NAT1 and NAT2), encoding for phase II xenobiotic-metabolizing NAT1 and NAT2 enzymes, represent one of the very first landmark examples of how genetic variation among individuals and across populations may affect drug safety , hydralazine, dapsone, procainamide, amifampridine, various sulphonamides and other clinically important drugs  are depeg others . Moreoveinsonism , while iinsonism .NAT2 is a highly polymorphic gene on chromosome 8p22, with the majority of single nucleotide polymorphisms (SNPs) located within its 873 bp intronless coding region (http://nat.mbg.duth.gr/). NAT2*4 reference allele, which is the most common functional (or “rapid”) allele across global populations , rs1799930 , rs1799931  and rs1801279 . The remaining three common SNPs have no effect on the phenotype. Those are rs1041983 , rs1799929  and rs1208   , and areNAT2 variants has been extensively studied and has been demonstrated to vary significantly among different ethnic backgrounds  with diverse histories of colonization and settlement. The difficulty of exploring the country at the beginning of colonization made it possible for large urban centers to exist close to the coast and rural populations and native communities in the interior of the country, that although demographically less representative, still exist and maintain a strong Native American origin.The genetics landscape can be observed in studies carried out in Brazil by NAT2 allelic distribution in populations characterized by a high degree of ethnic admixture, requires further investigation , Pernambuco (PE) and Rio Grande do Norte (RN), in the Northeast (n = 79), Espírito Santo (ES) and Rio de Janeiro (RJ), specifically the municipalities of Rio de Janeiro and Campos, in the Southeast (n = 339), and Paraná (PR) in the South (n = 10). The selection of patients in different regions was random, and the determination of genotype/haplotype was blinded.After written informed consent, 964 unrelated individuals from five different geographical regions spanning ten states of Brazil were enrolled in this study. They comprised 240 active tuberculosis (TB) patients from the Federal University of Rio Grande do Sul (RS) in the South, 106 asymptomatic Healthcare Workers (HCWs) from Anuar Auad Infectious Disease Reference Hospital of the State Health Secretariat of Goiás State (GO) in the Midwest, and 618 leprosy patients from different units of the State Health Secretariat of Northeast, North and Southeast of Brazil, including the states of Acre (AC), Rondônia (RO) and Tocantins (TO), in the North  and a written informed consent prior to enrolment was obtained from each subject. The procedures followed were in accordance with the ethical standards of the responsible committee and with the Helsinki Declaration of 1975.® DNA Blood Kit , according to the manufacturer’s specifications. After extraction, DNA samples were stored at −20°C.A volume of 1 mL of venous blood was collected from each volunteer and stored at −20°C. Genomic DNA was isolated from 200 µL of frozen whole blood using the QIAampNAT2 genotyping was performed by sequencing of a 1,093 bp PCR-amplified DNA fragment spanning the entire coding exon of the gene, as previously described , according to the manufacturer’s specifications and subsequently used for direct Sanger sequencing. Because of the size of the amplified product , and in order to read the entire coding sequence of NAT2, two internal oligonucleotides , using SeqScape v.2.6 software (Applied BioSystems).Sequence data of each sample were analyzed for SNP identification after alignment with the sequence of the NAT2, including Sanger sequencing, cannot always discriminate between the two haplotypes of each genotype.SNP frequencies were determined by chromosome counting, and deviations from Hardy-Weinberg equilibrium were tested by Arlequin v.3.0 . Haplotyp < 0.05.Statistical analysis was performed using the SPSS  software version 17.0 for Windows. The chi-square test was used to determine whether there was a significant difference between the observed and expected frequencies, regarding the distribution of NAT2 variants in the analyzed cohort versus other populations. Statistical significance was assumed at NAT2 was performed for 964 samples (1928 chromosomes) from ten different states, across five geographical regions of Brazil: GO (Midwest), AC, RO and TO (North), RN and PE (Northeast), RJ and ES (Southeast), and PR and RS (South). Following amplification and sequencing of the entire intronless coding exon of NAT2, optimal quality sequences of 500–800 bp in length were produced. Their analysis allowed detection of NAT2 SNPs, followed by the determination of alleles and genotypes, as well as the prediction of acetylator phenotypes.Genotyping of The distribution and regional frequencies of detected SNPs are provided in NAT2. SNP c.33C>A, previously reported in Brazil . NAT Gene Nomenclature Committee and five of those contained the c.33C>A SNP previously described in Brazil  and six slow alleles  were observed , *12A (7.4%), *13A (7%), *12B (2.3%), *12C (0.7%) and *11A (0.2%). The most common slow alleles were NAT2*5B (29.3%) and NAT2* 6A (17.1%), in all states except RO. Those two slow alleles, together accounted for 46.4% of haplotypes in the population studied. The six most frequent defective alleles accounted for 56.8% of the NAT2 haplotypes found in the Brazilian population.Based on their SNP composition, it was possible to deduce the acetylation phenotype for the majority of the In total, 116 different allelic diplotypes (genotypes) were identified in the populations studied from the five geographical regions of Brazil . RegardiThe 21 genotypes, which demonstrate a population frequency at least 1% in Brazilian samples, are described in NAT2*4/*4 showed significant difference (p < 0.001) between the RJ and ES states in the Southeast, as well as between RJ and all other regions, i.e., the South (p = 0.014), the Northeast (p = 0.045), the Midwest (p = 0.002) and the North (p = 0.014). Genotype NAT2*12A/*12A was found only in RS (9.2%) and GO (0.9%) states, and its frequency varied significantly in the South relative to other regions (p < 0.001). Genotypes NAT2*12A/*12B and NAT2*12A/*12C were found only in the RS state in frequencies of 5% and 1.7%, respectively, and varied relative to other regions (p < 0.001). Genotype NAT2*13A/*13A was also found in RS (3.3%) and RJ (3%) states, and its frequency varied significantly relative to other regions (p < 0.001). After the genotyping was completed (p < 0.001). Similarly, a difference was also seen between the Southeast and Northeast region (p = 0.014).There were significant differences in the frequencies of common genotypes between states and regions. Genotype ompleted , it was ompleted . The SouNAT2 genetic variability in other geographical areas. An overview of representative studies reporting on the frequencies of common NAT2 haplotypic groups across global populations is presented in To gain further insight into how the Brazilian population compares with others, we also surveyed studies recording NAT2 coding exon in 1928 chromosomes sampled from Brazilians in five different geographical regions of the country. The activity of the NAT2 enzyme encoded by this highly polymorphic gene has important implications for health risk assessment of environmental agents, given the critical importance of this enzyme in activating some types of aromatic amines and hydrazines, while detoxifying others. Consequently, it has been established that variation in the acetylator phenotype may lead to an increased risk for drug adverse reactions or lack of therapeutic efficacy  is mainly found in African populations and is absent in European descendants  is predominant in East Asian populations  but is much rarer (1%–5%) in European descendants , c.481C>T and c.803A>G has also been correlated with ethnicity. Those SNPs are usually prevalent in European descendants and Africans. In contrast, SNP c.590G>A  shows a rather homogenous frequency distribution across global populations  and RN (Northeast) states, only six of the seven most frequent SNPs were detected. SNP c.191G>A was not found in both states. Considering all identified SNPs in relation to their predicted effect on NAT2 enzymatic activity, it can be concluded that the frequency of SNPs associated with low enzymatic activity was higher in RN (77%) than in RS (44.6%).Distribution of ulations . In our The c.282C>T is widely distributed across worldwide populations  and the NAT2 in the Brazilian population corroborates previous observations of our group. NAT2 between residents of RJ (Southeast) and GO (Midwest) states, demonstrating the heterogeneity of the Brazilian population. The authors attributed their findings to the influence of different ethnic backgrounds introduced into the Brazilian population during colonization. Although that early study was performed in only two states (RJ and GO), it demonstrated the importance of involving additional regions of Brazil. In a subsequent study  state. More importantly, that study confirmed the association between the NAT2 slow acetylator phenotype and drug-induced adverse reactions in TB patients treated with therapeutic schemes including INH. The results supported those of studies previously performed with ethnically less diverse populations. It also characterized NAT2 variability in different regions of Brazil, including the different ethnicities represented in the gene pool of Brazilians, a population with admixed European, African and Native American roots.The variability distribution of nt study , the patNAT2, where alleles are characterized by the combination of one to six different SNPs, haplotype characterization is fundamental for the prediction of the acetylation phenotype. After haplotype reconstruction, we identified 44 alleles/haplotypes, distributed variably between states. The most frequent allele was NAT2*5B (29.3%), followed by NAT2*4 (20.4%) and NAT2*6A (17.1%), in line with the findings of According to the molecular genetics of NAT2*4 was the most frequent in all states, except for RS (South) where the rapid acetylation phenotype was attributed mainly to the NAT2*13A and *12B alleles. In our population, the reference NAT2*4 allele showed frequencies like those found in European populations and was significantly more prevalent compared with central African (p < 0.001) and South African (p = 0.025) populations. The distribution in the whole sample varied for functional alleles other than NAT2*4, i.e., NAT2*12A (7.4%), NAT2*13A (7%), NAT*12B (2.3%) and NAT2*12C (0.7%). In fact, the distribution of those alleles showed some noteworthy features across different sub-populations. For example, allele NAT2*13A had a low frequency in most of states, except for RS (9.2%) and RJ . Moreover, among the two RJ municipalities, the frequency of NAT2*13A was almost four times higher in Rio de Janeiro (16.5%) versus Campos (4.4%). The NAT2*12A allele, which had a low frequency in the majority of the states, was encountered in higher frequencies in Campos (7.9%) and RS (21.7%). The NAT2*12B allele was found only in Rio de Janeiro municipality (0.2%), TO (0.7%) and RS (8.7%), while the *12C allele was found only in RO (0.7%), TO (0.7%), ES (2.2%) and RS (1.9%). Interestingly, those four alleles were not found at all in RN or in PR.Among the functional alleles, NAT2*5 and NAT2*6 were the most frequent defective alleles in the Brazilian population. NAT2*5B showed relatively lower frequencies in RO (11.4%) and RS (22.4%); for all other states, the allele had higher frequencies (∼30% or higher). Moreover, the high frequency (61%) of NAT2*5B in PR was found to be significantly different when compared to the Midwest (p = 0.025) and the Northeast (p = 0.045) regions (∼30%). The allelic frequency of NAT2*5A was highest in RO (15.7%), differing significantly from AC and TO , and all other regions (p < 0.001). Finally, the second most frequent defective allele, NAT2*6A, varied significantly when the Midwest and Northeast (∼26.5%) were compared with the Northern AC state (12.5%), as well as with the South (9.9%) and Southeast (16%) regions. The Asian allele NAT2*7B showed its highest frequencies in the North (7.4%) and Northeast (5.1%) regions. Significant differences were observed between the South (1.2%) and North (7.4%), Northeast (5.1%) and Midwest (4.2%) regions. Finally, the NAT2*14B allele demonstrated significant difference in frequency between the South (undetermined frequency) and all other regions (∼1% or higher).Haplotypic groups NAT2*5 has been reported as the most frequent in all European and African populations, and this was the case for our population too. Interestingly, although the frequency of this allelic group was significantly lower in Brazilians, compared with populations of European or African descent (p < 0.005), it was nevertheless significantly higher than in the Japanese, Chinese, Korean, Thai and Native American populations (p < 0.001). The NAT2*6 was the second most frequent low-activity allelic group in our population, and it was well-distributed in our cohort. The frequency of this allelic group in the Brazilian population was significantly lower than in the European (p < 0.001), Thai (p < 0.001) and Sudanese (p < 0.001) population. The NAT2*7 allelic group, also associated with low NAT2 activity, is mainly found in Asia and Central America, and its frequency was significantly lower in our Brazilian population than in the Japanese, Thai, Chinese, Korean and Native American (p < 0.001), but was higher than in some Caucasian populations. Finally, the NAT2*14 allelic group, which is more prevalent in Sub-Saharan Africa with a frequency above 6%, was also identified in our population in a lower frequency of about 2%. These findings clearly demonstrate that the contribution of different ethnic groups in Brazilian genetic background has an impact in the distribution of NAT2 alleles throughout the country’s territory.The low-activity allelic group NAT2 allelic diplotypes (genotypes), it was possible to infer the acetylator phenotypes for individuals across the different states of Brazil. We observed that populations in the Northeast (RN and PE), North  and Midwest (GO) displayed a higher prevalence of slow acetylators . On the other hand, in the Southeast, the most frequent phenotype was the intermediate acetylator (40.3%). Interestingly, the Southern state of RS presented the highest frequency of rapid acetylators (38%), with a significant difference compared to other states of Brazil (p < 0.0001). On the other hand, there was no significant difference in the phenotypic profile between the North and Northeast, suggesting homogeneity between the two regions. However, both regions showed a significant difference in the observed phenotypic pattern, when compared with the Southeast region (p = 0.04). More remarkably, when compared with the South region, all other regions of the country demonstrated significant difference (p < 0.0001), as the frequency of rapid acetylators in the South was considerably higher. Our findings demonstrate a gradual shift from higher prevalence of the slow acetylator phenotype in the North and more central parts of the country, to a higher prevalence of intermediate acetylators in the Southeast and of rapid acetylators in the South.From the subsequent determination of NAT2 gene concerning SNPs, alleles and genotypes in the Brazilian population; second, the distribution of these diverse alleles varies among subpopulations across the country and third, these distribution patterns have an impact on the observed acetylation status, in line with previous literature (NAT2 genotypes and susceptibility to some unfavorable outcomes of certain drugs, this study importantly contributes to the reconstruction of a more accurate phenotypic picture of the NAT2 acetylator profiles in different geographic regions of Brazil. We understand that our findings can be useful in planning therapeutic regimens containing drugs metabolized by NAT2 in these regions of Brazil.In conclusion, our results can be summarized in three fundamental observations: first, they reveal a significant diversity of the terature . Given t


Chrysodeixis includens  is an economically important pest of soybeans (Glycine max (L.) Merr.) in the southeastern United States. It has characteristics that are of particular concern for pest mitigation that include a broad host range, the capacity for annual long-distance flight, and resistance in some populations to important pesticides such as pyrethroids and chitin synthesis inhibitor. The biology of SBL in the United States resembles that of the fellow noctuid fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), a major pest of corn and several other crops. FAW exhibits a population structure in that it can be divided into two groups (host strains) that differ in their host preferences but are broadly sympatric and exhibit incomplete reproductive isolation. In this paper, strategies used to characterize the FAW strains were applied to SBL to assess the likelihood of population structure in the United States. Evidence is presented for two SBL strains that were defined phylogenetically and display differences in the proportions of a small set of genetic markers. The populations exhibit evidence of reproductive barriers sufficient to allow persistent asymmetry in the distribution of mitochondrial haplotypes. The identified molecular markers will facilitate studies characterizing the behaviors of these two populations, with relevance to pest mitigation and efforts to prevent further dispersal of the resistance traits.The noctuid moth soybean looper (SBL),  Phytophagous insects can be classified into three categories based on feeding behavior: monophagous insects feed on a single plant species, oligophagous insects feed on several species within closely related families, and polyphagous insects have the ability to feed across higher phylogenetic groupings. Polyphagous agricultural pests capable of using a multiplicity of plant species present added challenges for mitigation strategies that include an increased number of habitats that can support pest populations. This makes extrapolating the source of migratory populations more difficult and complicates projections of future infestations. Fortunately, a more extreme phytophagy is relatively rare, perhaps because of unfavorable competition with more specialized, better-adapted species . This woChrysodeixis includens , and fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), are noctuid moth species that appear to exhibit this type of oligophagous feeding behavior. SBL has a broad host range that includes over 70 plant species but exhibits the most economic damage as a defoliator of soybean, while also causing significant yield losses in cotton, bean, and tomato crops [Heliconius, where divergence in some parts of the genome occurred contemporaneously with persistent admixture [The soybean looper (SBL), to crops . FAW hasto crops ,5. The Cto crops ,7. This dmixture .There is also evidence for at least two genetically distinct populations of SBL al-though the data are more suggestive than conclusive. Collections in Brazil were characterized via inter-simple sequence repeat (ISSR) analysis, in which geographical populations were compared in terms of microsatellite-based polymorphisms . PrincipTriosephosphate isomerase (Tpi) gene can differentiate closely related moth populations such as the FAW host strains [Helicoverpa species of H. armigera and H. zea [E and Z pheromone strains of Ostrinia nubilalis [Tpi product is a housekeeping function and so is unlikely to drive population divergence, leaving its physical linkage to other Z-chromosome functions as the most likely explanation for its effectiveness as a genetic marker.In this study, we tested for genetically differentiated groups in the North American SBL population. This was carried out by applying a methodology developed and successfully used to detect and characterize the FAW strains in wild populations. The strategy is based on two premises. The first is that while moderate gene flow resulting from incomplete reproductive isolation may not allow population-specific mitochondrial haplotypes, differences in the relative proportions of haplotypes might form and be detectable. The second is that the Z-chromosome may play a disproportionate role in speciation ,12 with  strains , the sibd H. zea , and theubilalis . The TpiCytochrome oxidase subunit I (COI) gene previously used to differentiate geographical FAW populations [Tpi gene, the analog of which in FAW produced a phylogenetic description of the two host strains [Based on these observations, SBL specimens were collected from multiple locations in the U.S. over multiple years and characterized for a segment of the mitochondrial ulations , and a h strains ,20. EvidSpecimens were collected from soybean in field plots either through pheromone trapping  or as larvae picked directly from soybean A. In addA mix of genomic and mitochondrial DNA from individual specimens was purified from individuals as described in , and stoCOI gene sequence were identified by aligning the well-characterized FAW COI sequences with the SBL mitochondria genome sequence (GenBank LR797832). The primers used to amplify the sCOIB segment were sC891f (5′-TACACGAGCTTATTTTACTTC-3′), and sC1457r (5′-ATATCATTCAATAGAAGAGG-3′). PCR amplification was performed in a 30 µL reaction mix containing 3 µL of 10× manufacturer’s reaction buffer, 1 µL of 10 mM dNTP, 0.5 µL of a 20 µM primer mix, 1 µL of the DNA template (between 0.05–0.5 µg), and 0.5 units of Taq DNA polymerase . The thermocycling program was 94 °C (1 min), followed by 33 cycles of 92 °C (30 s), 56 °C (30 s), 72 °C (30 s), and a final segment of 72 °C for 3 min. Briefly, 96 PCR amplifications were performed at the same time using either 0.2 mL tube strips or 96 well-microtiter plates. Segments of the SBL The sTpiI140 segment was amplified by the primer pair sTpi891f/sTpi1140r using the same protocol as that used for sCOIB. All primers used for PCR and DNA sequencing were synthesized by Integrated DNA Technologies . These included sC891f (5′-TACACGAGCTTATTTTACTTC-3′), sC1457r (5′-ATATCATTCAATAGAAGAGG-3′), sTpi412f (5′-ATGGCCTGAAAGTCATTGCCTG-3′), and sTpi1140r (5′-GCAGACACATTCTTAGCCAGCC-3′).For fragment isolations, 6 µL of 6× gel loading buffer was added to the amplification reaction and the entire sample run on 1.8% agarose horizontal gel containing GelGreen  in 0.5× Tris-borate buffer . Fragments were visualized on a Blue LED transilluminator  and cut out from the gel. Fragment isolation was performed using Zymo-Spin I columns  in accordance with the manufacturer’s instructions. COI haplotype that was maternally inherited. The sCOIB haplotype classes used in this study are defined by SNPs sC1035 and sC1272 that are each polymorphic for C/T and produce four observed combinations , with C1035T1272 and T1035C1272 being the predominant forms. To simplify the analysis, the sCOIB mitochondrial haplotype categories in this paper were defined solely by sC1035 polymorphisms and designated as either C1035COI or T1035COI if a C or T was present, respectively.Each specimen was anticipated to have a single Tpi hapotypes are more complicated as the location of the Tpi gene on the Z-chromosome means that two gene copies are present in males (Z/Z) and so heterozygous allele combinations are possible, while females (Z/W) carry only a single copy. Because DNA sequencing is performed on PCR fragments directly produced from the specimen, heterozygosity for the Tpi segment will result in the simultaneous sequencing of two different alleles. This will produce chromatographs with overlapping curves at the sequence mismatch. Particularly disruptive are heterozygous indels (insertions and deletions) because these can shift the DNA sequencing frame of the two DNA strands, resulting in continued mismatch downstream of the indel. The SBL https://www.geneious.com, accessed on 15 June 2023) [The isolated fragments were analyzed via DNA sequencing using the appropriate primers . DNA comparisons and alignments were performed using the Geneious Pro 10.1.2 ne 2023) . All sCO . All sCne 2023) . The anane 2023)  and diagCOI gene, a fragment designated as sCOIB, from which nine SNPs were identified. Only two of the nine SNPs were polymorphic in more than one sample, sC1035 and sC1272, and these produced four pairwise combinations, with the C1035T1272 and T1035C1272 haplotype categories predominating . The data indicate that the T1035COI mitochondrial haplotype and i65delTpi allele segregate in combination at frequencies too high for what would be predicted from random mating.Comparisons of the distribution of the sCOIB mitochondrial haplotypes and sTpiI140 in our collections uncovered evidence of a possible linkage between i65dellections . The simTpi allele frequencies by state and comparing its frequencies in the C1035COI and T1035COI populations. Because the colony and field collections differed with respect to artificial rearing and source locations, the two data sets were analyzed separately as well as in combination. In each analysis, states with fewer than ten specimens of either the C1035COI or T1035COI haplotypes were pooled. Both the colony and field-collected specimens showed a significantly higher association of the i65delTpi allele with the T1035COI mitochondrial haplotype  to as large as 32% (FL). As with the individual data set analyses, the observed difference between the means was statistically significant.Given the similarity in their results, the data from the colony and field collections were combined and the i65delg pooled C. DespitTpi also allowed the detection of heterozygotes between i65delTpi and i65+Tpi (designated as i65del/+). The presence of both alleles in the same sequencing reaction will produce a chromatograph with a predictable superimposition of curves resulting from the frameshift beginning at the i65del breakpoint  relative to what would be expected (eH) from a population at the Hardy–Weinberg equilibrium, based on the equation isF = (eH − oH)/eH. A isF equal to 0 indicates a state of equilibrium where eH = oH, with values becoming increasingly positive if heterozygosity is suppressed (high inbreeding) and more negative if the observed heterozygosity is greater than expected. An added complication is that because Tpi is on the Z-chromosome, heterozygotes of two Tpi alleles can only occur in SBL males as females being ZW will only carry one copy of the gene. Our initial analysis was therefore limited to the pheromone trap collections that exclusively attracted adult males. The pooled data produced a isF of 0.30 while the mean isF of the six collections was 0.41 (isF (−0.11).The frequency of heterozygotes was used to calculate Wright’s local inbreeding coefficient, was 0.41 A. Both hisF. The results are consistent with this reasoning with both pooled and mean isF values being higher than that observed with the pheromone trap collections  gave similar results even though the gender of the specimens was not determined. The likely presence of female specimens reduces the opportunities for i65del/+ hybrids and therefore should lead to overestimates of lections B. In theTpi allele  carrying i65delTpi in ClanBSBL compared to none out of 492 in ClanASBL  and nucleotide diversity was eight-fold greater (0.024 vs. 0.003).Neighbor-joining phylogenetic analysis of the SBL sTpiI140 sequence from 550 specimens collected from multiple U.S. locations and times produced a tree that could be divided into two groups based on the distribution of i65deli allele A. The trASBL NJ, . Based oTpi and T1035COI markers  and the other predominated by C1035COI i65+Tpi (ClanASBL).Additional evidence for two distinct SBL populations was derived from the fact that the nuclear Z-chromosome and the mitochondrial genome are separate genetic entities that segregate independently of the other. This means that under conditions of random mating, the i65del markers  indicateTpi and i65+Tpi populations. The isF calculations showed consistent evidence of high levels of inbreeding diagnostic of the Wahlund effect that describes a reduction in observed heterozygosity as populations diverge into multiple groups [Tpi and i65+Tpi populations as indicated by the isF data provides compelling explanation for the asymmetric distribution of the T1035COI and C1035COI mitochondrial haplotypes across these groups.This suggestion is supported by our ability to identify i65del/+ heterozygotes, which allowed the quantification of the hypothesized reproductive restrictions between the i65dele groups  (Table 3SBL and ClanBSBL that seem by analogy to FAW to be good candidates for diverging populations represents an important advance in understanding SBL population structure.Given the similarities between FAW and SBL with respect to their overlapping host range, geographical distribution, and climate suitability, it is plausible that SBL is facing similar selective pressure to diverge into two populations. This can be difficult to demonstrate, however, as populations at intermediate stages of speciation are, by definition, still capable of some cross-hybridization with potentially high levels of gene flow. In addition, the genes driving divergence are likely to be low in number early in the speciation process and many of the polymorphisms that become asymmetrically distributed by chance will likely vary by region. Given these issues, our phylogenetic identification of ClanACOI and Tpi polymorphisms for two sympatric SBL populations with only a limited capability for cross-hybridization. These findings complement results from a companion paper demonstrating geographical differences in haplotype distribution that may reflect how SBL migration is impacted by the timing of soybean agriculture. Together, they describe a more complicated population biology in the United States than previously thought with potential relevance to the dispersion of resistance traits and projections of SBL migration.In summary, evidence was presented based on the distribution of


Vaccinium vitis-idaea (Ericaceae) show genetic differentiation from nearby zonal subalpine populations, and high functional trait distinctiveness, in agreement with genetic patterns. We genotyped almost 30,000 single nucleotide polymorphisms using restriction site-associated DNA sequencing and measured eight functional traits indicative of individual performance and ecological strategies. Genetic results showed high differentiation among the six populations suggesting isolation. On siliceous bedrock, ice hole individuals exhibited higher levels of admixture than those from subalpine populations which could have experienced more bottlenecks during demographic fluctuations related to glacial cycles. Ice hole and subalpine calcareous populations clearly separated from siliceous populations, indicating a possible effect of bedrock in shaping genetic patterns. Trait analysis reflected the bedrock effect on populations’ differentiation. The significant correlation between trait and genetic distances suggests the genetic contribution in shaping intraspecific functional differentiation. In conclusion, extra-zonal populations reveal a prominent genetic and phenotypic differentiation determined by history and ecological contingency. Therefore, microrefugia populations can contribute to the overall variability of the species and lead to intraspecific-driven responses to upcoming environmental changes.In the mountain terrain, ice holes are little depressions between rock boulders that are characterized by the exit of cold air able to cool down the rock surface even in summer. This cold air creates cold microrefugia in warmer surroundings that preserve plant species probably over thousands of years under extra-zonal climatic conditions. We hypothesized that ice hole populations of the model species  During glacial periods, many boreo-alpine species experienced range retractions and southwards shifts in the face of ice expansion2. During interglacial periods, these species recolonized the higher latitudes or elevations as a consequence of rising temperature4. While plant species thereby mainly tracked suitable environments, another phenomenon of extinction avoidance during both glacial and interglacial periods has been reported: Local spots characterized by stable microclimatic conditions, the so-called microrefugia, enabled long-term persistence of small populations within largely unfavorable geographical regions5. In the fast-warming climate of the Anthropocene6 the existence of cold microhabitats within a warmer surrounding is even more important for the persistence of boreo-alpine plant species at the retracting margin of their area8. The persistence of these cold-adapted species over long periods of time within such local spots is expected to result in the genetic and phenotypic differentiation of their isolated populations, making them of particular interest for species conservation11.Quaternary climatic fluctuations were responsible for species’ extinctions and range shifts, which strongly influenced the present distribution of plant species12. Plant functional traits are individual phenotypic characteristics that give information about important plant functions, e.g., photosynthetic capacity, growth, and competitive ability13. Given the correlation between functional traits and individual fitness and success, they would easily differ among populations experiencing various environmental conditions.Genetic differentiation can be mirrored at the phenotypic level. However, phenotypic differences among populations can also be the result of phenotypic plasticity, which allows an immediate response of organisms to the environment16. They are characterized by depressions between rock boulders, which exhibit cold exiting air able to cool down the rock surface of the holes even during summer. This phenomenon, and the resulting peculiar extra-zonal climate is an exceptional consequence of landslides, which have presumably originated during repeated post-glacial events17. It depends on air flows descending from the upper edge of the landslide to the base becoming cooler and denser while passing through the cold humid rock blocks. The air that exits the ice holes accumulates over concave landforms about five meters deep, creating as permanent cold air pool that allows the growth of subalpine and alpine species which are absent in the surrounding area19. Populations of subalpine and alpine species growing today in ice holes might represent relict populations that have been left behind with the shifts of the species main range toward higher altitudes during the Holocene20. The isolation of these small populations for thousands of years may have left signatures in their genome9. Indeed, isolation maintained over long periods can lead to genetic differentiation of marginal populations as shown for Vaccinium vitis-idaea L. (Ericaceae) in Japan21. In line with this, further studies showed that genetic diversity is more strongly preserved in areas where the species persisted for long periods such as glacial refugia22. On the contrary, genetic diversity is expected to decrease during range expansion such as in case of colonization of formerly glaciated areas, due to repeated bottlenecks24. Up to our knowledge, our study is the first to investigate this hypothesis for inter-glacial microrefugia in the Alps.In the mountain terrain, ice holes represent montane cold inter-glacial refugiaVaccinium vitis-idaea is one of the most frequent subalpine species, characteristic of the extra-zonal vegetation of ice holes of calcareous as well as siliceous bedrock. This species has been analyzed at the phenotypic and/or genotypic level in different countries by 13 extant studies, however, only two of them linked the genetic and phenotypic diversity focusing on agronomically important traits  shown by all populations indicates an excess of heterozygotes   clearly clustered the populations by bedrock, with CCc and VGc populations appearing rather intermixed and forming a group separated from siliceous populations. Subalpine siliceous populations MNs and SRs appeared to be quite homogeneous. Ice holes population APs appeared intermixed with LSs.Structure analysis revealed seven main genetic clusters (K = 7). Subalpine siliceous populations MNs and SRs appeared well separated from the others being characterized by one prevalent structure cluster Fig. . Three iWhen habitat was considered as the highest grouping factor, AMOVAs recorded most of the variance within populations, i.e., 88.14%, whereas the habitat classification did not contribute to explain any of the genetic variance , is not protected according to IUCN standards. The plant collection and use was in accordance with all the relevant guidelines. V. vitis-idaea leaf samples were collected at six localities, in three ice holes and in three subalpine sites located in the south-eastern Italian Alps  and Lases , lie on siliceous bedrock while the third, Cornacalda , lies on calcareous bedrock. To make the bedrock of each locality clear, a letter indicating its siliceous (s) or calcareous (c) nature was added to the site acronym. The three ice holes, although being all located in large block fields, differ rather strongly from each other in terms of topography. In particular, the calcareous site differs substantially from the two siliceous sites . The calcareous ice hole is more detrital, with less soil accumulating among the blocks . The siliceous ice holes are more similar to each other despite topographical differences. The APs ice hole is located in a small circular depression surrounded by broadleaf deciduous woodlands . Likewise, the LSs ice hole is surrounded by broadleaf deciduous woodlands but has a more elongated shape .The sampled species, i.e., lps Fig. . The icehttp://meteo.provincia.bz.it/), 11 °C at LSs  and 10 °C at CCc . The MAT for the subalpine sites is 3 °C at MNs , 1 °C at SRs  and 4 °C at VGc . The mean annual precipitation is 815 mm at APs, 1043 mm at LSs, 822 mm at CCc, 1150 mm at MNs, 800 mm at SRs and 1450 mm at VGc.The subalpine sites are located at about 2000 m a.s.l.. As the ice hole sites, the subalpine sites are on either siliceous or calcareous bedrock. The siliceous bedrock sites are located nearby Passo Manghen, on the Lagorai chain , and on Monti Sarentini . The site on calcareous bedrock is located in Val di Garés  . Sampled individuals were distant within each locality, as the species V. vitis-idaea can extend by clonal growth for more than 20 cm per year through horizontal stems47. For MNs and VGc, individuals were subsequently reduced from six to five, owing to technical problems in DNA extraction. For each individual, we harvested 20 fully expanded leaves: Ten of these were immediately used for functional trait measurements while the other ten were stored in silica gel for the genetic analyses.Sampling was carried out at the peak of the growing season 2016, namely in July in the ice holes and in August in the subalpine sites. We sampled six healthy, adult individuals because of the small area of ice holes  adapting a protocol that was described in previous studies50. Starting from a concentration of 200 ng of DNA, each individual sample was digested at 37 °C for 2 h and then heated at 80 °C for 20 min for enzyme inactivation. Eight different P1 adapters were ligated to the digested DNA, pooling together individuals with different adapters to form five sub-libraries. Samples in each sub-library were sheared by sonication using a Bioruptor Pico  to an average size of ca. 400 bp with three cycles of 30 s “on” and 60 s “off” at 4 °C and then purified with the MinElute Reaction Cleanup Kit (Qiagen). After double-size selection 0.55 × right and 0.7 × left with the SPRIselect Reagent Kit , five different P2 adapters with index barcodes were ligated, one per sub-library. At this point, all samples were pooled in a unique library, in a way that they could be distinguished by different P1 and P2 combinations. Before and after PCR amplification with the Phusion Master Mix (NEB), cleaning and size selection only on the left side (0.75 × before and 0.7 × after PCR) were performed again. The final library was sequenced as 125 bp paired-end reads on an Illumina HiSeq at Vienna BioCenter Cora Facilities .Genome size was estimated by flow cytometry. DNA was extracted from 50 mg of dry material from each individual plant using the DNeasy Plant Mini Kit  with addition of up to 3.1% polyvinylpyrrolidone (PVP) to the AP1 lysis buffer to enhance the final yield. The samples were incubated overnight at 65 °C with the mixed buffer and RNase Ahttps://github.com/wtsi-npg/illumina2bam). Quality filtering and demultiplexing based on inline barcodes was performed with process_radtags.pl available in the STACKS 2.0 Beta 9 package51 specifying PstI as the restriction enzyme, rescuing RADtags and barcodes and rejecting low quality reads. For detecting the latter, in a very simplified way, the program uses a sliding window approach to obtain the average quality of a read and its probability of being corrected. If the quality drops below a certain threshold , the read is discarded as low quality read. More details on the filtering process are explained in the supplement file ustacks, cstacks and sstacks using a minimum of five reads to call a stack (m = 5), allowing for two mismatches between four stacks to assemble a locus in each individual (M = 4) and one mismatch between loci to build the catalog (n = 1). Paired-end information was introduced during the tsv2bam step and processed with gstacks, calling variant sites in populations and genotypes in every individual. The catalog was filtered blacklisting loci carrying more than ten SNPs to avoid retention of any merged paralogs using a custom bash script. Using the populations program from STACKS 2.0, we selected loci shared by at least 75% of the individuals (r = 0.75) and present in all the populations (p = 6). As a first step, only one SNP per locus was called to minimize linkage and the options genepop, vcf and structure were selected to produce the respective files for the following data analyses. As a last step, population was run again keeping unchanged the above-mentioned parameters but calling all SNP per locus selecting the RADpainter option in preparation for fineRADstructure52. The STACKS code used for the analyses is reported in the supplement file The raw reads were demultiplexed based on index barcoded P2 adapters using the Picard BamIndexDecoder tool, which is part of the Picard Illumina2Bam package  connected to a digital camera  at 400 × magnification. SD (number of stomata × mm−2) and SL 55 were estimated for five fields of view per impression, i.e., ten per leaf, using the software DeltaPix, v.3.2.× . Nutrient traits, i.e., LNC and LPC, were measured from the remaining five leaves per individual by the salicylate method and molybdenum blue method through a continuous flow autoanalyzer . See Table Eight leaf functional traits, namely leaf area (LA), specific leaf area (SLA), leaf dry matter content (LDMC), leaf nitrogen content (LNC), leaf phosphorous content (LPC), N:P ratio (N:P), stomatal density (SD) and stomatal length (SL) were measured. LA, SLA, LDMC and the stomatal traits were measured for five of the ten harvested leaves per individual. The other five leaves were used for nutrient analyses. For each of these traits, individual values were obtained averaging the values of the five leaves measured per individual. To measure LA, SLA and LDMC we determined the fresh weight and surface area of each of these five leaves . The same leaves were then dried at 70 °C for 72 h, weighed again and used for stomatal analysespopulations program was used to perform a Principal Component Analysis (PCA) in R with the adegenet package v. 2.1.156. With the adegenet package the file was also adapted to obtain the number of migrants (Nm) based on private alleles through the pairwise comparison of populations in R with the genepop package57. For each pair of populations, pairwise FST was calculated with the software VCFtools58 using the vcf file obtained by populations program. To deeply investigate the structure and the relationships of the populations inferring their recent shared ancestry, both fineRADstructure and Structure analyses were run. The RADpainter file produced from populations was used as input file for the fineRADstructure package that was run with standard options and the structure file produced from populations was used as input file for the software Structure59 (v. 2.3.4) that was employed using the admixture model with correlated frequencies between populations. For each K (from 1 to 12) 10 replicates, 100,000 burn-ins and 200,000 iterations were computed. The K value that fitted the data the best was inferred through Structure Harvester v. 0.6.9460. In R, pophelper package 61 was used to define the admixture proportion resulting from the 10 simulations at the best K and to represent the results through barplots. After conversion of the structure file from populations with PGDSpider v. 2.1.1.362, we applied ARLEQUIN v. 3.5.2.263 to assess the genetic structure of the populations. It allowed the calculation of AMOVAs to estimate genetic diversity of populations and higher-level groups, i.e., habitat and bedrock. When habitat was considered as the higher-level group, the three ice hole populations, i.e., APs, LSs, and CCc were distinguished from the three subalpine populations, i.e., SRs, MNs and VGc. In contrast, when bedrock was the higher-level group the four populations occurring on the siliceous bedrock, i.e., APs, LSs, SRs and MNs were distinguished by the two occurring on calcareous bedrock, i.e., CCc and VGc.The population genetics were analyzed by several different methods described as follows. The genepop file produced with the adegenet and poppr64. Geographic distances were calculated as straight-lines including elevational differences. The geographic distance matrix was then tested for statistical correlation with the genetic distance matrix in R through a Mantel–Haenszel test with 100,000 permutations in the ade4 package v. 1.7-1065.Finally, the effect of isolation by distance (IBD) on the genetic structure was investigated through the comparison of geographical and genetic matrices. The genetic matrix was obtained by using the R packages V. vitis-idaea growing in the two different habitats, a PCA was performed on functional trait data of LA, SLA, LDMC, LNC, LPC, N:P, SD and SL in R . Furthermore, to make the intraspecific variability of each trait evident, univariate two-way factorial ANOVAs were performed with each functional trait as dependent variable and habitat, bedrock, and their interaction as independent variable. Significance of differences of means among sites were assessed by Fisher’s LSD post-hoc test. To correlate the functional with the genetic distance, a multidimensional scaling analysis was performed on the distance matrix of the abovementioned traits through the R package bios2mds66. The Mantel–Haenszel test with 100,000 permutations was then applied to assess the statistical correlation between the genetic and the phenotypic distance matrices. For a conceptual model of relationships among intraspecific functional trait variation, abiotic heterogeneity and genetic diversity we refer to67 and the conceptual figure therein.The functional traits’ analysis is done in two steps. First, the analysis of phenotypic traits’ differences, and second, the correlation between the functional traits’ differences and genetic difference. To detect phenotypic differences of individuals between populations of Supplementary Information 1.Supplementary Information 2.