Passer domesticus), living in six insular and six mainland populations, and we aimed to determine whether their diversity or differentiation correlates with the diversity and the prevalence of infection of hemosporidian parasites. We found that island bird populations tended to have lower neutral genetic variability, whereas MHC variability gene was similar between island and mainland populations. Similarly, island populations tended to show greater genetic differentiation than mainland populations, especially at microsatellite markers. The maintenance of MHC genetic diversity and its less marked structure in the island populations could be attributed to balancing-selection. The greater MHC differentiation among populations was negatively correlated with similarity in blood parasites  between populations. Even at low prevalence and small geographical scale, haemosporidian parasites might contribute to structure the variability of immune genes among populations of hosts.Small and isolated populations usually exhibit low levels of genetic variability, and thus, they are expected to have a lower capacity to adapt to changes in environmental conditions, such as exposure to pathogens and parasites. Comparing the genetic variability of selectively neutral versus functional loci allows one to assess the evolutionary history of populations and their future evolutionary potential. The genes of the major histocompatibility complex (MHC) control immune recognition of parasites, and their unusually high diversity is genes which is likely driven by parasite-mediated balancing selection. Here, we examined diversity and differentiation of neutral microsatellite loci and functional MHC class I genes in house sparrows ( Habitat fragmentation is one of the greatest threats to the survival and persistence of wild populations  in the erosion/maintenance of genetic diversity. This issue is a central question in evolutionary and conservation biology. Threatened species are usually comprised of small and isolated and genetically depauperate populations that have undergone a reduction in numbers. However, it is difficult to infer the mechanisms leading to low genetic diversity without a comparison with populations with different demographic trends (e.gPasser domesticus). We also quantified the selective regime of each population by assessing their haemosporidian parasite communities and the prevalence of haemosporidian infection . Avian malaria is a widespread vectorborne disease of wild birds, extensively studied during the last decades  populations of house sparrow (The specific aims of this study were to determine (1) whether island populations have low microsatellite and/or MHC genetic variability than mainland populations, (2) whether genetic differentiation among populations was different depending on the type of marker used (microsatellites or MHC), and (3) whether haemosporidian parasites could explain differences or similarities in microsatellite or MHC genetic differentiation.μL) by brachial vein puncture. Blood was stored in 500 μL of Queen's Lysis Buffer . The theremocycling was performed using the following program: 94 °C 3 min, 30 cycles of 94 °C 20s, 20s for annealing (48 °C to 56 °C according to the locus), and 72 °C 40s, followed by a final extension of 72 °C 5 min. PCR amplifications were performed using fluorescent-labeled primers (6′). We used four different fluorochromes: PET for PdomD09, PdomB01, and Mjg1; FAM for PdomC11, PdomE09, and Ase18; VIC for PdomA08, PdomA04, and Pdo3; and NED for PdomF09, PdomH05, and Pdo5. Each locus was amplified separately, but eletrophoresed through an ABI3730 automated sequencer by combining in each run four differently colored loci. Allele sizes were determined using GeneMapper 3.0 software .A total of 449 individuals were genotyped using twelve microsatellite loci: PdomD09, PdomC11, PdomA08, PdomF09, PdomB01, PdomE09, PdomA04, PdomH05  containing DNA polymerase, buffer, and dNTPs. The PCR program began with 5-min initial denaturing at 95 °C followed by 35 cycles of 30-sec denaturation at 94 °C, 90-sec annealing at 56 °C, and 90-sec extension at 72 °C. A final elongation step was run for 10 min at 72 °C. To control for PCR contaminations, we used two negative controls (for PCR and for electrophoresis).A total of 389 house sparrows were genotyped by amplifying the major part of the exon 3 (208 bp compared to 268 bp for the entire exon) of the class I locus, which corresponds to the peptide-binding region (PBR)   with a nested PCR. This reaction amplified a 524-bp-long fragment of the cytochrome b gene of the two parasite genera . We also created alignments of our sequences and listed them in the public database MalAvi . As we found no genetic differentiation at microsatellite loci between the 2 years for any population, we decided to pool the data for all years.Among our 12 populations, five were sampled in two different years (Ouessant: 2007 [HE) using the software Genetix  was computed using a rarefaction index for the smallest sample size for one locus in one population  using FSTAT 2.9.3  with the software ESTIMATES 7.5 . We therefore report only FST in the rest of the manuscript, but supply DEST values in Table S2.Genetic differentiation between populations for microsatellite markers was measured using  Rousset . Genetic Rousset . We iden3 Goudet . We asse Hedrick , so we aFST/(1-FST) against the natural logarithm of the geographical distance between populations (Rousset FST). We also used a Mantel correlation to test whether the sea represented a barrier to gene flow (migration), as reduced gene flow would be expected to promote island–mainland population differentiation. We constructed a triangular matrix describing the presence (1)/absence (0) of water (the sea) between all population pairs and regressed it against the matrices of pairwise microsatellites and MHC FST values.For each marker type, we tested for isolation by distance using a Mantel regression of  Rousset , with stFST matrix, while controlling for microsatellite differentiation, using a partial Mantel test.For each sparrow population, we assessed the number of different avian malaria strains that were present and the prevalence of infection (proportion of infected birds). The number of strains was compared between insular and mainland populations with Mann–Whitney tests. Differences in prevalence  between insular and mainland populations were investigated using a general linear mixed model (GLMM) with population type (island and mainland), year, season, and sex as fixed factors, and population within type as a random factor. Parasite similarity was also correlated with the MHC S = 2W/(A + B), where W is the sum of the minimal number of infected hosts for each parasite strain between two populations, and A and B are the sum of infected hosts in populations A and B, respectively. In four populations, none of the sampled birds were infected. In these cases, the similarity index of pairs of populations with zero prevalence was set to 1 because we considered that the absence of the parasites homogenizes the selection pressures exerted by these specific parasites. The matrix of parasite similarity was then correlated with the matrices of FST based on both microsatellites and MHC, again using Mantel tests.To test the idea that genetic differentiation at the MHC depends on the dissimilarity between the parasite communities, we computed the Steinhaus similarity coefficient, Unless otherwise attested, statistical tests were performed in SAS 9.2 SAS,  and JMP HE) between island (median = 0.74) and mainland (median = 0.75) populations ; however, allelic richness was lower in insular (median = 6.24) than that in mainland ones (median = 6.65) .The number of microsatellite alleles varied from 5 to 26 and 42/769 exact tests  showed a significant linkage disequilibrium at the 0.05 level, but not after sequential Bonferroni correction. Eight of 144 population–locus tests showed a significant deviation from Hardy–Weinberg equilibrium, but again, none remained so after sequential Bonferroni correction. None of the 12 Hardy–Weinberg equilibrium global tests were significant. We found no other evidence that our 12 microsatellite markers were under selection (R) and mean number of MHC alleles per individual (MHC/ind) for each population are given in TableR  nor in MHC/ind  between island  and mainland populations .We observed 45 MHC alleles among our 12 populations. The maximal number of MHC alleles found in an individual was eight, suggesting that we amplified at least 4 MHC class I loci. Allelic richness  and 0.0445 (between Quimper and Molène)  were still significant after sequential Bonferroni correction. All 15 FST values between island pairs were significant. A total of 75% of the 36 pairwise FST values between island and mainland populations and only 6.7% of the 15 FST values between mainland pairs were significant. We did not observe evidence for isolation by distance ; however, there was a strong correlation between FST values and the presence/absence of sea between population pairs .Pairwise microsatellite FST values for MHC varied between −0.0109 (Ploemeur and Kerinou) and 0.0399 (Molène and Kerinou) , but they were strongly correlated with the presence/absence of sea between populations .Pairwise ou) Table. Twenty-ion Table. A totalFST and MHC FST was highly significant . We found 7 pairwise comparisons where MHC-based FST were above the upper limit of the 95% CI of microsatellite-based FST, 38 pairwise comparisons where MHC-based FST were within the 95% CI of microsatellite-based FST, and 21 pairwise comparisons where MHC-based FST were below the lower limit of the 95% CI of microsatellite-based FST. We found a significant excess of MHC-based FST inferior to microsatellite-based FST (P < 0.0001).The correlation between microsatellite Plasmodium and Haemoproteus, 40 individuals harbored the infection with five different parasite strains. Prevalence in each population is given in TableF2,244 = 3.01, P = 0.096), or sex . Prevalence was higher in winter (12.94%) than that in autumn (3.54%) and spring (3.85%) , and was higher in mainland (15.46%) than that in insular populations (3.92%) .Among the 446 birds screened for haemosporidian r = −0.141, P = 0.25), nor with the presence/absence of sea between two populations . There was no correlation between microsatellite FST and parasite similarity between populations .We computed a Steinhaus similarity coefficient between pairs of populations Table to putatWe found that (1) genetic variability of neutral genetic markers was lower in island compared to mainland populations, as expected by genetic drift, whereas variability at the class I MHC gene was similar between island and mainland populations. Similarly, island populations appear to be more differentiated than mainland populations. We also found (2) stronger genetic differentiation between bird populations at microsatellite markers than for MHC genes, which could indicate that balancing selection overrides divergent selection in this class of MHC gene. Finally, we found that (3) similarity in the composition  of haemosporidian parasite community was correlated with the population differentiation based on MHC, even when controlling for conditional microsatellite-based differentiation. Below, we discuss each of these three main findings in more detail.(1) Microsatellite allelic richness was lower in islands than that in mainland populations. This is a rather common pattern that has been reported in several island/mainland comparisons, likely due to smaller population sizes and reduced gene flow in insular populations. Frankham  reviewedIsland populations were more differentiated than mainland populations at both genetic markers, suggesting a reduced gene flow between insular populations. The house sparrow is a sedentary species, with limited dispersal ability. Using recoveries, Paradis et al.  estimateFST computed on different genetic markers are somewhat problematic if these markers differ in their rate of molecular evolution and their diversity, which is the case for microsatellites and MHC genes. The use of a standardized genetic differentiation estimator (Hedrick D (DEST)  We found a stronger genetic differentiation between bird populations at microsatellite markers than for MHC genes. Disentangling the forces that drive the maintenance of variability in functionally important genes is challenging. Previous work conducted on nonmodel species has mostly aimed at comparing patterns of population differentiation based on neutral markers (supposedly reflecting only demographic forces) and selected genes . Such comparisons have provided useful insights on the selection acting on MHC. However, it should be kept in mind that direct comparisons of  Hedrick  that conST) Jost , which iOverall, differentiation based on MHC was weaker than for microsatellites. Weaker differentiation at MHC genes is in agreement with high overall MHC diversity relative to neutral diversity and with the idea that similar parasite pressures  tend to homogenize allele frequencies and reduce population differentiation  Finally, we found that similarity in the composition of haemosporidian parasite community was correlated with the population differentiation based on MHC. Identifying the selective agents responsible for the observed pattern is obviously not easy. Previous work has suggested that haemosporidian parasites might play a role in the maintenance of among population diversity of MHC genes (Jarvi et al. FST did not produce the same results. Interestingly, these results suggest that even within a small geographical scale, with relatively homogeneous environmental characteristics, fine-tuned differences in parasite-exerted selection pressures can affect population differentiation of immune genes.We investigated the avian malarial community infecting each of the studied populations, and we found, as expected, that the prevalence of infection was lower in insular populations compared to mainland (even though, it should be noted that prevalence was generally low, certainly reflecting local environmental features, Loiseau et al. In conclusion, our findings suggest that despite the erosion of neutral genetic diversity of birds living on islands, balancing selection on the MHC genes seems to be strong enough to counteract genomewide genetic drift. Balancing selection also appears to explain the weaker MHC genetic differentiation than for microsatellites, in the house sparrow populations. Even at low prevalence and across a limited geographical scale, haemosporidian parasites may help drive the structure of MHC diversity between populations.


These genetic markers failed to confirm the occurrence of two anchovy species in the Adriatic Sea, as previously postulated. However, we found fine-scale population structure in the Adriatic, especially in northern areas, that was associated with four of the 13 environmental variables tested. Geographic gradients in sea temperature, salinity and dissolved oxygen appear to drive adaptive differences in spawning time and early larval development among populations. Resolving adaptive seascapes in Adriatic anchovies provides a means to understand mechanisms underpinning local adaptation and a basis for optimizing exploitation strategies for sustainable harvests.The sustained exploitation of marine populations requires an understanding of a species' adaptive seascape so that populations can track environmental changes from short- and long-term climate cycles and from human development. The analysis of the distributions of genetic markers among populations, together with correlates of life-history and environmental variability, can provide insights into the extent of adaptive variation. Here, we examined genetic variability among populations of mature European anchovies ( A growing body of evidence indicates that marine organisms with potentially dispersive planktonic larvae can show genetically patchy distributions that may be uncorrelated with spatial complexity , 2. ThisEngraulis encrasicolus L.) populations in the Adriatic Sea. This species support large fisheries, as well as artisanal harvests along the coasts of Italy and the Balkan countries. The Adriatic is a semi-enclosed basin open to the Central-Eastern Mediterranean Sea and is divided into several areas with contrasting levels of productivity ; Cluster 2 (southwest Adriatic) [BAA+BAB]; Cluster 3 (southeast Adriatic) [KOT+MNA+MNB]; Cluster 4 (northeast Adriatic) [RIJ+JAB+DUG] and Cluster 5 (Tyrrhenian Sea) [SPE+CDG]. The Evanno method [K value from all simulations and graphical representation of multiple runs per K values were produced by the CLUMPAK Server (http://clumpak.tau.ac.il) [Runs in STRUCTURE were made assuming o method  in STRUCo method  was usedu.ac.il) .DAPC  is a mulThe geographic boundaries among sampled populations were identified with BARRIER 2.2 , which uet al. [Contemporary gene flow was evaluated with an individual-assignment test in GENECLASS 2.0 . For eacet al.  algorithhttp://popgen.sc.fsu.edu/Migrate/Migrate-n.html). A Bayesian method was applied [FST estimates among localities were used as baseline to calculate the mutation parameter Θ  and the historical migration rate, M . A Brownian motion model was used and mutation rates were assumed to be constant over a set of 1000 neutral loci. The MCMC procedure consisted of one long chain of 500 000 genealogies for each locus, with the firs 10 000 genealogies discarded as burn-in. The sum of M values from each location was used to determine directionality of gene flow.The second method used to detect gene flow and connectivity was based on the coalescent calculation of historical migration rates between sampling locations using MIGRATE-n 3.5.1 ( applied  and FST ST) between samples were compared to linear shortest sea-distances between samples with a permutation test of 10 000 iterations.The relationship between genetic and geographic distances between samples (isolation by distance) was tested using Mantel's difference matrix test implemented in Genepop (ISOLDE) . GeneticFST and expected heterozygosity (HE) under the island model of migration [HE between pair of samples under the following formula:Non-neutral loci were identified with four methods that are based on different statistical assumptions. First, the coalescent approach in LOSITAN  detects igration . Runs weigration , uses a igration  in Arleqigration  was usedigration , which iWe used a threshold of ±2.58 and assumed that LnRH values under neutrality were normally distributed after data standardization . The values of 99% of neutral loci are expected to fall within this range.http://gher-diva.phys.ulg.ac.be/web-vis/). In this database, chlorophyll and oxygen values were reported as averages over 1890 and 2008, whereas salinities and temperatures were reported as averages between 1900 and 2009. These values were used as independent variables in a redundancy discriminant analysis (RDA) implemented in CANOCO 4.5 [10 or Ln transformed for seascape analyses. The forward selection of environmental variables at each locus was performed with a Monte Carlo permutation test of 999 iterations (P < 0.01). When a locus was associated with more than one variable, a permutation test as above was repeated to establish the conjunct significance of these variables on the distribution of allele frequencies. The associations between allele frequencies in samples and environmental variables were further investigated with a Canonical Correspondence Analysis (CCA) in PAST 2.17 [A set of 13 environmental variables  is bathymetry. Hence, we collected samples from shallow coastal sites (<50m) and from open-water areas (>50m). We first performed a STRUCTURE simulation to test the effect of bathymetry. Two major groups of samples were defined based on the threshold of 50 m of the bottom depth and were used as a priori information. Cluster 1 included SLO+NAD+ANC+PEA+KOT +BAA+CDG, and Cluster 2 included the remaining samples. Second, we verified the consistency of clustering samples based on their sampling bathymetry (as performed in STRUCTURE) using an AMOVA test. Finally, estimates of genetic differentiation between samples were used to test for large genetic differences, as expected between divergent species.Based on the assumptions of Borsa et al. , the mosa et al. ) from opThe PCR success rate was high for the 14 microsatellite loci with only 1.17% missing genotypes. Although allele dropout and stuttering were minimal, null alleles were detected in 50 of 210 global tests. Null-allele corrections with the Brookfield algorithm improved the results, but 26 of 210 tests were still significant. Ten of the significant tests were for Enja148, which we excluded from the following analyses.NA) per sample over all loci varied from 11.4 alleles in JAB to 15.8 alleles in KOT. Allelic richness (RS), standardized with 19 individuals per sample, varied from 8.1 (DUG) to 11.2 (CDG)  . The sma and KOT . The smaectively .FIS was significant in 4 of 210 tests, and three values of FIS indicated homozygote excess (Significant departures from the Hardy-Weinberg (HW) proportions over all loci were found in 4 samples, MNA, MNB, BAB and ANC. e excess . Three le excess . Only 4 ST and FST over loci showed similar patterns. ƟST ranged from -0.004 (BAA vs. PEB) to 0.054 (NAD vs. JAB), and FST ranged from -0.004 to 0.058 between samples. Pairwise ƟST were significant (P < 0.0005) in 51 of 105 comparisons (GST ranged from -0.002 (BAA vs. PEB) to 0.057 (NAD vs. JAB) between samples, G'ST ranged from -0.002 to 0.030, and DEST values ranged from -0.004 (BAA vs. PEB) to 0.194 (NAD vs. JAB). Pairwise comparisons were significant (P < 0.0005) in 65 of 105 tests for GST and G'ST and in 66 of 105 tests for DEST  DUG+q values .In the DAPC analysis, the first 2 of the 150 Discriminant Axes (DA) were significant. These DA's defined three major genetic Clusters: i) DUG+JAB+RIJ (Northeast Adriatic samples), ii) NAD (Northern Adriatic sample) and iii) all remaining samples . The firM) ranged between 18.50 (MJAB→NAD) and 944.01 (MDUG→PEA). The directionality of historical gene flow suggested a pattern that was similar to sea-surface circulation  (data not shown).Even though differences appeared between populations, no isolation-by-distance over the length of the Adriatic was detected with Mantel's test between pairwise ϴP > 0.05)  correction. The Hierarchical Island Method (HIM) for clustering in STRUCTURE K = 2 postulated six outliers, with two loci (Ee10 and Ee2-507) as candidates for directional selection and four loci  for balancing selection  remained significant, showing the same pattern of selection as tests with K = 2  , but q velection ; Table 4th K = 2 ; Table 4th K = 2 .Tsamp), salinity at sampling depth (Ssamp) and at 10 m (S10) and dissolved oxygen concentration at 10 m (Oxyg10). At Ee2-507, alleles 279 and 291 were positively correlated and alleles 259 and 283 were inversely correlated with S10 (both P < 0.019) (Oxyg10 and Ssamp (P < 0.002); allele 169 varied positively and allele 147 inversely with Ssamp, and alleles 163 and 171 were positively and allele 159 was inversely correlated with Oxyg10 (Tsamp (P < 0.003) (Tsamp (P < 0.033)  identified significant correlations between allelic frequencies at four outlier loci  and four environmental variables, including temperature at sampling depth (< 0.019) . Signifih Oxyg10 . At locu< 0.003) . Finally< 0.033) .P < 0.05 and P < 0.001, respectively). The results indicated a positive association for allele frequencies in EMB, EMC and EMD and a negative association for allele-frequencies in sample EMA with salinity Ssamp and S10 (Tsamp). Finally, SPE and CDG were directly associated with dissolved oxygen content in the first 10 m (Oxyg10) and inversely associated with Tsamp.Both the first and second axes of the CCA ordination of samples with four environmental variables were significant  was significant (P < 0.05), it explained only a small amount of the total variation (1.79%). Divergence among groups (FCT = 0.0012) was not significantly different from zero (P > 0.05).The STRUCTURE analysis using the LocPrior function to cluster sampling sites by bathymetry showed three genetic clusters, as in the other STRUCTURE analyses . In geneet al. . FurtherThe results of our survey of microsatellite DNA variability among populations show that while anchovies in the Adriatic are genetically subdivided among regions to some extent, none of these populations represent separate species, as previously proposed. The mechanisms producing divergence between populations include both demographic processes influenced by ocean currents and fronts and natural selection that leads to locally adapted populations. Before exploring these factors in more detail, we offer the following evaluation of the study. While the sample sizes used in this study were not large, they provided sufficient statistical power to detect population differences. Statistical power was enhanced by the use of a large number of microsatellite loci encoded in nuclear genes. These markers provided complementary insights into population structure and local adaptation. We additionally used several approaches to the analyses of the microsatellite data that illuminated various dimensions of population structure.et al. [et al. [E. albidus Borsa, Collet et Durand, 2004, resembled shallow-water anchovies in low-saline areas of the northern Adriatic [E. encrasicolus [Borsa  and Borset al.  postulat [et al.  and on m [et al. , 63. TheAdriatic . The larasicolus . HoweverE. albidus in the low-saline plume of the Po River (NAD). Although these anchovies share some ecological and morphological traits with putative E. albidus in the Gulf of Lyon, Po River anchovies are genetically most similar to oceanic anchovies in the central Adriatic. The level of genetic differentiation between NAD and the other samples suggests a substantial genetic separation, but this level of divergence is not large enough to indicate the presence of two divergent species. The color and morphological variability observed among anchovy populations in the Adriatic Sea (and in the Gulf of Lyon) likely reflects plastic phenotypic responses to environmental variability in anchovies that are typical of anchovies in the Mediterranean Sea [Nevertheless, it might be argued that the genetic differences in the northern Adriatic Sea support the occurrence of nean Sea .FST and ϴST between Adriatic populations are typical of divergence between other population groups of anchovies or between populations of other pelagic fishes. The overall levels of genetic differentiation between populations in the Adriatic Sea were similar to those between anchovy populations in the eastern Atlantic [The small values of Atlantic , 66. As Atlantic . NeverthThe analysis of neutral makers with STRUCTURE resolved two major population groups: populations in northeastern coastal areas  and the remaining populations in the Adriatic and Tyrrhenian seas. The genetic uniqueness of northeastern anchovies may be due to adaptation to habitats in the numerous coves and embayments and around offshore isles , 18, 68.et al. [et al. [A north-south genetic discontinuity in the northern Adriatic Sea was previously reported by Bembo et al. , who fou [et al.  focused et al. [Instead, the oceanic front between the Istrian Peninsula and Mid Adriatic Pit may explain, in part, the north-south genetic discontinuity by limiting gene flow. The anticlockwise gyre off the Po River delta may also act as a barrier to gene flow , 18. Altet al.  to explaIndividual assignment showed that about 10% of the fish in our samples represented first-generation migrants from other populations. However, patterns of dispersals revealed by these tests have to be interpreted with caution, because small samples sizes and finite geographical sampling introduce some randomness into the results. The results also represent only a snapshot of a dynamic process that may change over time. Nevertheless, the results indicate that dispersals between populations are asymmetrical: some populations act as 'sources' and others act as 'sinks'. The extent that populations receiving migrants depend on immigration to persist has to be evaluated with long-term abundance data. With these caveats, most migrants originated from only a few source locations (mostly MNA and ANC samples) and moved to several nearby sink locations following a pattern of dispersion that is similar to major sea-surface currents in the area. The evaluation of historical patterns of gene flow using Migrate-n suggests a similar scenario for both the magnitude and pattern of connectivity. This pattern of migration may also be related to the lack of divergence between Adriatic and Tyrrhenian anchovies because of reciprocal gene flow between the two seas. These results are in line with a recent analysis using SNPs markers showing genetic similarities between Adriatic and Western Mediterranean population of anchovies .While the small genetic distances between samples indicate high levels of gene flow, as expected for a pelagic species, the pattern of geographic differentiation and the associations of microsatellite loci with environmental variables indicate that gene flow may be countered by adaptation to local conditions. For example, the timing of spawning, relative to seasonal bursts of productivity can have a strong effect on year-class survival , 74. TheOur inferences about natural selection are based on non-coding microsatellite loci, which are unlikely to be the direct focus of selection, except when microsatellite alleles modify the functions of genes in which they are embedded , 76. AltNe) can produce transient genetic sweeps that lead to locally adaptive genotypes [Several life-history traits can interact to facilitate local adaptation. For instance, it is well known that this species has a high skew in reproductive success, which determines the level of genetic diversity in each cohort. Sweepstakes reproduction can promote selection at local scales, especially when coupled with large demographic variation in census size. During population bottlenecks, the reduction in effective population size  were positively associated with temperature variability, whereas samples from the Tyrrhenian Sea (SPE and CDG) were inversely associated with temperature variability. This is consistent with high seasonal fluctuations in temperature in northern and western Adriatic coastal areas, in contrast to greater thermal stability in the Tyrrhenian basin , 84. TemAdriatic anchovies are also influenced by variability in dissolved oxygen, which is inversely correlated with temperature, because oxygen saturation in water decreases at higher temperatures. Populations in the Tyrrhenian Sea (SPE and CDG) were positively associated with dissolved oxygen content, but coastal populations in shallow waters  were inversely associated with oxygen content. The dissolved oxygen content may interact with temperature to define local selective constraints. Higher water temperatures in summer generally lead to a drop in dissolved oxygen, producing hypoxic and anoxic conditions , especiaGenetic discontinuities among populations of anchovies may be related to the effects of environmental variables on the growth and survival of early developmental stages, as well as on adult reproductive success. The complex genetic patterns among populations of marine organisms have only recently been correlated with the influence of environmental variables on the genomes of several marine species . NumerouE. encrasicolus. Nevertheless, the individuals collected in the study area, which covered the entire Adriatic basin and part of the Tyrrhenian Sea, were partitioned into two major populations which were sometimes further sub-structured. Adriatic anchovy subpopulations show a patchy distribution of genetic variability that appears to reflect selective processes mediated by several environmental variables, including salinity, temperature and dissolved oxygen content. The relationship between environmental variables and the distribution of genetic differences among population groups is consistent with adaptation to local environmental conditions. Salinity, in particular, appears to be important in structuring northeastern Adriatic anchovy populations. Our geographical survey of microsatellite DNA variability reveals a more complex genetic structure among Adriatic anchovy populations than was previously detected, but does not confirm the presence of a second species of Engraulis. Further resolution of this biocomplexity will provide a basis for understanding adaptive mechanisms and a basis for optimizing management strategies for sustainable harvests.An important result of this study indicates that anchovies throughout the Adriatic belong to a single species, S1 FigK < 10; figure on the left) and the delta K probability obtained after the use of Evanno method [K < 9; figure on the right). Additionally, a simulation was carried out dividing sampling sites based on bathymetry using a threshold of 50 m to detect population structure associate with potential occurrence of different anchovy species in the covered area .The simulations were run using i) all 13 loci, ii) only neutral loci and iii) only candidate outliers with and without the assistance of prior information . Each simulation shows the mean logarithmic probability with standard deviation (SD) obtained for each K tested (1 < o method  (2 < K <(DOCX)Click here for additional data file.S2 FigFST and HE in each marker (microsatellite locus) analyzed. Blue dots falling within light grey area represent neutral markers whereas those within the yellow and the red areas represent outlier markers under balancing and directional selection, respectively.Blue dots represent interpolation between mean values of (DOCX)Click here for additional data file.S3 FigFST and HE in each marker (microsatellite locus) analyzed. Red, green and purple lines represent 1%, 5% and 95% percentiles of FST null distribution, respectively.Dots represent the interpolation between mean values of (DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableG'ST and GST while in S4B Table the values from DEST.In S4A Table are showed values from (DOCX)Click here for additional data file.S5 TableThe numbers within the table refer to the individuals that were allocated outside the sampling localities examined in this study (receiving sampling locations). The original sampling sites detected for each migrant are reported along the first row (native sampling locations). The S5B Table provide values for the demographic estimator Theta (Θ), while S5C Table shows migration rates (M) of historical gene flow. The sampling location in the first line represent the source locations while the sampling location in the first column the receiving locations.(DOCX)Click here for additional data file.S6 TableValues in red are those exceeding the threshold of significance set at ±2.58.(DOCX)Click here for additional data file.S7 TableSamples were divided into groups according to the bottom depth.(DOCX)Click here for additional data file.


Troglohyphantes vignai  and Pimoa rupicola , the latter showing minor adaptation to hypogean life. We sampled populations of the model species in caves and other subterranean habitats across their known geographical range in the Western Alps. By combining phylogeographic inferences and Ecological Niche Modeling techniques, we inferred the biogeographic scenario that led to the present day population structure of the two species. According to our divergent time estimates and relative uncertainties, the isolation of T. vignai and P. rupicola from their northern sister groups was tracked back to Middle–Late Miocene. Furthermore, the fingerprint left by Pleistocene glaciations on the population structure revealed by the genetic data, led to the hypothesis that a progressive adaptation to subterranean habitats occurred in T. vignai, followed by strong population isolation. On the other hand, P. rupicola underwent a remarkable genetic bottleneck during the Pleistocene glaciations, that shaped its present population structure. It seems likely that such shallow population structure is both the result of the minor degree of specialization to hypogean life and the higher dispersal ability characterizing this species. The simultaneous study of overlapping spider species showing different levels of adaptation to hypogean life, disclosed a new way to clarify patterns of biological diversification and to understand the effects of past climatic shift on the subterranean biodiversity.We designed a comparative study to unravel the phylogeography of two Alpine endemic spiders characterized by a different degree of adaptation to subterranean life:  In thisHere we designed a comparative study aimed at providing insights on the origin and the evolution of the hypogean biodiversity. Specifically, we focused on two Alpine endemic species co-occurring in caves and other subterranean habitats across most of their known distribution range and exhibiting different levels of adaptation to subterranean life.Troglohyphantes vignaiT. vignai shows adaptations to the hypogean life, namely loss of pigmentation, reduction of the eye apparatus, thinning of integuments and heavy spination. T. vignai was described from the cave Buco di Valenza  by T. rupicapra, which was distinguished from T. vignai by small morphological details of the epigynum. T. rupicapra was described on material from Grotta superiore delle Camoscere . According to the species description, and as later observed by T. rupicapra shows a higher degree of troglomorphism compared to T. vignai, namely higher depigmentation, reduction of eye diameter and loss of functional eyes, and lowering of the profile of the cephalothorax. The species validity of T. rupicapra was questioned by T. rupicapra = T. vignai, currently accepted in the The first model species,Pimoa rupicola  , is an Alpine-Apenninic endemic element, recorded almost continuously from the Graian Alps to the Tuscan Apennines  and the nuclear second internal transcribed spacer region (ITS-2). The popularity of this markers stem from a generally high level of variation, which permit to reconstruct relationships among and within spider species, making them particularly suitable for population and biogeographic studies , while for Pimoa we only sampled Alpine populations, thus excluding French and Apenninic populations. The complete list of localities is reported in P. rupicola and P. n. sp. and from 1 to 7 in T. vignai  gene and a 400 bp region of the nuclear second internal transcribed spacer region (ITS-2) gene were amplified using polymerase chain reaction (PCR). We utilized the primers C1-J-1490  and the ITS-28s (5′-TCCTCCGCTTATTGATATGC-3′) for the ITS-2 (One leg was removed from each specimen for DNA extraction. Whole genomic DNA was extracted from the samples using the SpeedTools Tissue Extraction Kit (Biotools) following the manufacturer’s protocol. A 676 bp region of the mitochondrial cytochrome oxidase subunit I (he ITS-2 .Taq polymerase (Promega), 5 µL buffer (Promega), 2.25 µL MgCl2 (Promega), 0.2 mm of each dNTP, 0.5 µL of each primer and 1.5 µL of DNA sample. PCR conditions for amplification were as follows: initial denaturing step at 95 °C for 5 min, 35 amplification cycles  fallowed by a final extension at 72 °C for 5 min. For certain populations of Pimoa rupicola , a slightly different annealing protocol for the cox1 was utilized . PCR products were visualized on agarose gels.PCR amplifications were carried out in 25 µL reaction volume in a final concentration of 0.1 µL http://www.macrogen.com). The DNA sequences obtained were preliminary assembled and edited using Geneious 7.1 (http://www.geneious.com).PCR product were cycle-sequenced at Macrogen, Inc. , using the Q-INS-I strategy with default options. We explored best partitioning schemes and substitution models simultaneously using PartitionFinder v.1.0.1 .Standard genetic diversity indices  were estimated with the PopGenome package  in R envUIN 3.01 . SignifiUIN 3.01  with a cCox1 and ITS-2 gene fragments in Geneious and excluded taxa with partial sequences. Gaps in the ITS-2 were recoded as absence/presence characters using the simple method proposed by Troglohyphantes nigraerosae and Pimoa edenticulata as outgroups to root the respective trees based on the results of ongoing analyses on the two genera .Maximum likelihood (ML) and Bayesian inference (BI) were used to infer the gene trees and the concatenated tree for each genus. For this analysis we only included unique haplotypes. We concatenated per 500 bootstrap replicates.ML analyses were performed in RAxML v.7.4.2  with theBI analyses were conducted in MrBayes v.3.2  with twocox1 alignment, we generated a ML tree  and we converted it to an ultrametric tree with the help of PATHd8  for any of the two lineages, we relied on informed priors of the substitution rates of the cox1, based on available information for spiders , including new unpublished records discovered during the present study. Given the low number of localities for T. vignai, we only inferred the ENM model for the Pimoa lineages. The dataset was corrected for potential spatial autocorrelation and haphazard sampling (www.worldclim.org). We obtained downscaled and calibrated Paleoclimatic data for the Last Glacial Maximum  from three different simulations available from Global Climate Models . The climatic preferences of the two species were investigated via Principal Component Analysis (PCA) in the Vegan R package  in each LGM forecast.We generated presence-only models with the Maximum Entropy Distribution Models available in MaxEnt , as implea sensu. To genecox1 gene of 676 bp was obtained for 37 specimens of Troglohyphantes vignai in 8 localities, corresponding to 14 unique haplotypes. The cox1 data set had 79 segregating sites and 9 parsimony informative sites. The overall mean distance (p-distance among haplotypes) was 0.0495 ± 0.0059. Sequences of the nuclear intron ITS-2 were obtained from the same individuals. The alignment was 400 positions long, 10 additional absence/presence gap characters were scored, corresponding to 10 ITS-2 sequence types. The ITS-2 had 16 segregating sites and 9 parsimony informative sites. The overall p-distance among caves was 0.0389 ± 0.0020. We obtained 676 bp cox1 sequence fragments of 119 Pimoa individuals from 25 localities. The 93 individuals from 19 localities of Pimoa n. sp. yielded 43 haplotypes (35 segregating sites and 7 parsimony informative sites) and the 27 individuals from 6 localities of P. rupicola yielded 7 haplotypes. The average p-distance within populations in Pimoa n. sp. and P. rupicola was 0.0076 ± 0.0017 and 0.0052 ± 0.0017, respectively, and the maximum p-distance between the two lineages was 0.1164 ± 0.0111. The nuclear ITS-2 sequences were obtained from 118 Pimoa specimens. The final alignment included 411 positions and 4 additional gap characters. Individuals of Pimoa n. sp.  and P. rupicola  yielded 34 and 10 sequence types, respectively. The average p-distance within populations in Pimoa n. sp. and P. rupicola was 0.0102 ± 0.0027 and 0.0035 ± 0.0017, respectively, and the maximum p-distance between the two lineages was 0.0701 ± 0.0121.The new sequences obtained in the present study are available in GenBank . A fragmcox1 and the ITS-2 for each locality are summarized in Pimoa n. sp. showed high levels of nucleotide diversity and low levels of haplotype diversity in most of the populations, while both P. rupicola and T. vignai showed low levels of haplotype and nucleotide diversity. This pattern was especially obvious in P. rupicola, since most individuals within populations were identical.The standard genetic diversity indices calculated for the FST values calculated for the localities of the three species are reported in FST values between localities revealed contrasting patterns of gene flow. In T. vignai, pairwise FST values between localities were always higher than 0.8, and significant (p < 0.05), except for localities #14 and #16. A relatively strong population structure was also found in P. rupicola. Pairwise FST values between localities were always higher than 0.6, although significant comparisons involved exclusively southernmost localities (#28 and #29). Pimoa n. sp. showed instead a more shallow population structure, with several FST values below 0.5, generally corresponding to nearby localities.Pairwise Pimoa haplotypes were resolved as two independent networks, corresponding to Pimoa n. sp. (1) and P. rupicola (2), respectively, separated by 42 steps. P. rupicola haplotypes were limited to single populations, except H26 and H27, which were shared across populations. Generally, populations had low haplotype diversity, and in one case (#26) there was one single haplotype. Two divergent haplotypes (11 steps), however, were found in locality #29.Pimoa n. sp. haplotypes from nearby localities clustered together or were separated by only few steps (1–4). Several haplotypes  were shared among closely located localities . In some instances, however, haplotypes from distant localities were found to be very similar (few steps), e.g., locality #1 and #2 with #12. Moreover, two haplotypes (H18 and H22) were found in individuals occurring in distant populations.In T. vignai, haplotypes were not shared between localities. Haplotypes from closely located populations were generally separated by few mutations. Haplotype diversity was low within populations, and localities #7, #14 and #16 showed single haplotypes. Two localities (#9 and #25), on the other hand, had more than two haplotypes. The haplotypes of T. vignai sensu rupicapra were separated by 17–20 steps from the nearest locality (#14), which is actually the locus typicus of T. vignai . A higher number of steps (38–56) separated this cave from the remaining localities.In T. vignai yielded in similar tree topologies, and most branches were highly supported  > 0.95, bootstrap support (BS) < 75%; T. vignai was split in two main clades: one including the southern populations , and the second one including the remaining northern populations . The Bayesian and the ML analyses also recovered similar tree topologies in Pimoa , one including all sequences/localities of P. rupicola and the other including all sequences/localities of Pimoa n. sp. Troglohyphantes vignai cox1 sequences were resolved as 7 coalescent clusters , and for the ITS-2 was 0.0024 (95% HPD = 0.0005–0.0031). The split between T. vignai and T. nigraerosae was traced back to approximately 7.2 million years ago . Diversification of the extant T. vignai lineages occurred 2.9 million years ago (95% HPD = 5.4–1.5 Ma), while the diversification of the extant northern populations  occurred approximately 0.5 Ma (95% HPD = 0.9–0.2 Ma). The substitution rate estimated for Pimoa cox1 was 0.0217 substitutions per lineage/million years (95% HPD = 0.011–0.033), and for the ITS-2 was 0.006 (95% HPD = 0.002–0.013). The basal split between Pimoa n. sp. and P. rupicola was estimated to have occurred around 5.7 Ma (HPD = 12–2 Ma). The origin of the extant diversity of each lineage was estimated approximately at 0.4 Ma for both lineages .The species trees and the embedded Pimoa linages with PCA, using bioclimatic variables (see Table 1 of Pimoa species cluster in two groups according to the second axis (PC2), which combines bioclimatic variables referable to continentality. According to the original definition  and in P. rupicola (0.908 ± 0.089). Overall, the suitable areas predicted by the model were congruent with the known distribution of the two species, at least in the Western Alps and Apennines. Current predictions identified suitable areas for P. n. sp. around the medium mountain belt (500–1500 m a.s.l.) of the Central and Western Alps, from the Camonica valley (province of Bergamo) down to the margin between Cottian and Maritime Alps . In respect to the known distribution, the predicted range of P. n. sp. extended northwards over the known limit of the species  to the Ligurian eastern coast and Tuscan Apennines . Additional suitable areas were also found in Tuscany, Lazio and Corsica. The projection of the distribution model to the environmental condition of the Last Glacial Maximum (LGM) revealed that most of the current suitable areas were unsuitable in the LGM (P. n. sp. (RF1) and two for P. rupicola (RF2 and RF3). All refugia corresponded to areas that were devoid from glaciers  to the Late Miocene (Messinian), namely from 13 to 3.5 mya. More precisely, the isolation of T. vignai and P. rupicola from their northern sister groups (T. nigraerosae and P. n. sp.) dates back 7.2 and 5.7 mya, respectively. This time period approximately corresponds to the closure of the Gibraltar Strait and the onset of the so-called Messinian Salinity Crisis  down to Ligurian  Alps. Conversely, the distribution of the northern sister group T. nigraerosae is adjacent southern Graian Alps  occurred approximately during the Pleistocene glaciations. In this respect, it is worth noting that subterranean localities inhabited by T. vignai lie at the periphery of the Pleistocene glaciers  (In this area the distribution of rechini) . The simT. vignai, for which we did not detect any evidence of current population expansion, the topology of the chronogram obtained for Pimoa lineages hints at a recent expansion following a bottleneck  associated to the southernmost offshoots of the Alpine glacial masses. Notably, similar areas have also been classified as peripheral refugia for several Alpine plants, such as Phyteuma globulariifolium  and the Tuscan coast (RF3), including the northern part of Corsica. Even though the presence of P. rupicola in Tuscany is confirmed by literature records  may not fully reflect patterns of gene flow . Similar complete niche partitioning between congeneric subterranean spiders has been reported elsewhere , provide further support for the synonymy between the two species proposed by T. vignai sensu rupicapra have to be regarded as a population of T. vignai isolated in an area characterized by different climatic conditions (major Mediterranean influence). Such isolation could tentatively be related to the higher development of troglomorphism in T. vignai sensu rupicapra, as already observed by T. vignai, corresponding to the northern and the southern clade  within Pimoa rupicola, which are further delimited both by genitalic morphology  and different ecological requirements , to shed further light on the processes that shaped the present diversity of Alpine subterranean fauna.The parallel study of populations of subterranean species, especially when showing different levels of adaptation and overlapping ranges of distribution, may disclose new ways to understand patterns of biological diversification. Future research may include new highly variable nuclear markers and analytical tools, and also consider other taxa showing similar distributions (e.g., 10.7717/peerj.1384/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.1384/supp-2Supplemental Information 2Click here for additional data file.


Phylogeographic studies of aquatic insects provide valuable insights into mechanisms that shape the genetic structure of communities, yet studies that include broad geographic areas are uncommon for this group. We conducted a broad scale phylogeographic analysis of the least salmonfly Pteronarcella badia (Plecoptera) across western North America. We tested hypotheses related to mode of dispersal and the influence of historic climate oscillations on population genetic structure. In order to generate a larger mitochondrial data set, we used 454 sequencing to reconstruct the complete mitochondrial genome in the early stages of the project.Our analysis revealed high levels of population structure with several deeply divergent clades present across the sample area. Evidence from five mitochondrial genes and one nuclear locus identified a potentially cryptic lineage in the Pacific Northwest. Gene flow estimates and geographic clade distributions suggest that overland flight during the winged adult stage is an important dispersal mechanism for this taxon. We found evidence of multiple glacial refugia across the species distribution and signs of secondary contact within and among major clades.P. badia. This work adds to the ever-growing list of studies that highlight the potential of next-generation DNA sequencing in a phylogenetic context to improve molecular data sets from understudied groups.This study provides a basis for future studies of aquatic insect phylogeography at the inter-basin scale in western North America. Our findings add to an understanding of the role of historical climate isolations in shaping assemblages of aquatic insects in this region. We identified several geographic areas that may have historical importance for other aquatic organisms with similar distributions and dispersal strategies as The online version of this article (doi:10.1186/s12862-015-0553-4) contains supplementary material, which is available to authorized users. Molecular studies in a phylogeographic context provide insights into the evolutionary history of the taxon of interest, and as studies across taxa accumulate, inference of broader deterministic processes is possible. Such studies are particularly valuable in understanding factors that impact complex, diverse communities as seen in freshwater aquatic systems. Insects account for much of the diversity present in freshwater communities . With a While aquatic insects have been reasonably well studied for many decades, relatively few molecular studies are conducted given the number of aquatic taxa. Studies that do exist are often limited in geographic scale . Yet stuA significant obstacle to conducting molecular studies in aquatic insects is the lack of genomic information for many taxa of interest. Until recently, researchers have been confined to using markers available through universal or degenerate primers such as the “barcode” region of the mitochondrial DNA (mtDNA) cytochrome oxidase I (CO1) gene , 12. ForPteronarcella badia. This herbivorous stonefly is moderately sized and occurs in mid-elevation mountain streams across western North America. It is one of two members of the genus Pteronarcella within the family Pteronarcyidae (Plecoptera). The species is readily identifiable in the field in its immature form and as an adult (except where its range overlaps with its sister species P. regularis (Hagen) in the Pacific Northwest, there the immature stages cannot be distinguished). When it is present it often occurs abundantly. The broad western North American distribution of P. badia makes it an effective organism to study phylogeographic patterns in this region, which has few studies of aquatic insects from a similar geographic scale to date (but see [b (CYTB), and NADH dehydrogenase 6 (ND6). We combined sequence data from these five mtDNA genes with nuclear rDNA 28S to form a dataset designed to address the following research questions: (1) What is the population structure of P. badia? (2) What are the dominant modes of dispersal in P. badia? We specifically test whether dispersal through hydrologic connectivity or overland movement (putatively through flight during the winged adult stage) appears to be more important in determining genetic structure. (3) How have historical climate oscillations influenced population structure? We test for evidence of multiple glacial refugia, timing of interclade divergence, and patterns of demographic history to estimate the influence of historical climate cycles on the population structure of P. badia.Here we present a phylogeographic study of the least salmonfly . DePteronarcys princeps (GenBank #NC_006133.1) [tRNAArg, the only gene unidentified by MOSAS. No mt genome rearrangements relative to the ancestral Pancrustacean genome [Drosophila yakuba [Our 454 pyrosequencing produced six mtDNA contigs that represented 96.3 %  of the total genome. PCR-based Sanger sequencing of the gaps yielded the remaining 569 bps with the majority (>500 bps) of the missing sequence coming from the A + T rich control region. Nucleotide composition showed overall A + T richness that is typical in insect mt genomes  with tot06133.1)  confirmea yakuba . The comFollowing alignment and trimming, our data set consisted of 2518 bp of mitochondrial and 880 bp of nuclear DNA sequence. PCR amplification for one or more genes was unsuccessful in 13 of the 265 in-group individuals included in the analysis (mostly museum specimens with putatively degraded DNA), so they were not included in the population genetic analyses. Translation of mtDNA sequence into amino acids did not reveal any unexpected stop codons. In scanning chromatograms for ambiguous bases, we observed at least one ambiguity in 42 of the approximately 750 mitochondrial sequences generated for this study . Although a total of 119 ambiguities were observed in these 42 sequences, all but six ambiguities were due to low quality score bases positioned within 50 bases of the end amplified fragment, where calls were being made on a single  sequence. Thus, we only found six ambiguities across all sequences for which both forward and reverse sequences showed the same ambiguous signal. Given this result, we find it unlikely that our conclusions are being appreciably influenced by the presence of pseudogenes.ST of 0.91 (p < 0.001). The nuclear locus showed far less variability than the mtDNA sequences, with only 15 genotypes present, and all individuals from 31 of the 38 sample localities sharing the same genotype  and an overall nucleotide diversity of 0.025. Of the 252 individuals sampled, 151 unique haplotypes were present with an overall haplotypic diversity of 0.991 ± 0.002. TCS haplotype networks revealed very high levels of population structure with multiple sub-networks failing to connect with the connection limit set to 50 steps. High levels of population structure were also evidenced by an overall ΦTree reconstruction in RAxML produced a topology with good to excellent nodal support for all major clades see Fig. . Six deeP. badia phylogeny, the Old Rio Grande and Old Colorado Plateau clades, comprise haplotypes sampled from seven localities in the southeastern portion of the species range . Despite being highly divergent at the mitochondrial locus (up to 5.2 % pairwise distance), no changes separate these clades at the nuclear locus. The Pacific Northwest clade includes all individuals sampled from the western edge of the species’ range, from northern California to southern Washington. We excluded Thomas Creek, CA from the full data set as only COI and CYTB sequences were available for the single specimen from that locality. However, secondary analysis of COI and CYTB placed this locality in the Pacific Northwest clade. For this reason we show Thomas Creek as belonging to the Pacific Northwest clade on the distribution map shown in Fig. The two clades that originate from the initial divergence in the The Northern Rockies clade includes all individuals from all localities in Montana and southern Canada. Three additional localities in Canada obtained from museum specimens  were excluded from the analysis due to missing sequence data, however a separate analysis that included only sequences from COI and CYTB showed that they also fall in the Northern Rockies clade , all haplotypes from the Red River and Pecos River in the southern Rocky Mountains (BS = 94 %), all haplotypes from Deer Creek, Mammoth Creek, Parowan Creek, and 3 haplotypes from Salina Creek (BS = 98 %), and all the Alaskan localities (BS = 72 %). Within the Old Rio Grande Clade, the Salina Creek haplotypes form a well-supported clade (BS = 100 %), as do all haplotypes from the Chama River, Conejos River, and Sauguache Creek (BS = 95 %).Comparison of the total evidence tree (3398 bp) to the topology produced using only COI (745 bp after trimming) showed that while COI recovered the major clades present in the total evidence topology, relationships between clades were not identical and three of the six clades had less than 70 % bootstrap support in the COI topology.SC = 0.88, p = >0.001), differences between sample localities explained 64.87 % of the genetic variation , and differences within localities explained 8.98 % of the variation . Each major clade, and several subclades contained localities from multiple drainage basins . Near identical haplotypes were observed in localities without hydrologic connections in the Widespread and Northern Rockies clades. In the Widespread clade, near identical haplotypes  were present in the Diamond Fork River (Colorado River drainage), the Hoback River (Columbia River drainage), and Kisaralik River (Kuskokwim drainage). In the Northern Rockies clade, near identical haplotypes  were observed in Blodgett Creek (Columbia River drainage) and Lee Creek (Hudson River drainage).AMOVA results showed that 26.15 % of genetic variation was explained by differences between drainage basin (Φp = 0.33), Old Rio Grande (p = 0.16), Pacific Northwest (p = 0.46), and Northern Rockies (p = 0.25) clades. Isolation by distance was significant for the Widespread clade when the Alaskan samples were included (p = 0.0015), and also when they were excluded (p = <0.001).Estimates of gene flow suggested that migration rates between localities in adjacent drainage boundaries may be comparable to, or higher than migration rates between geographically proximate localities with a direct hydrologic connection. The gene flow analysis is summarized in Table P. badia diverged from its sister species P. regularis approximately 2.65 million ybp, near the end of the Pliocene Epoch  dated to 1.59 million ybp, or the middle Pleistocene, with the latest diverging Widespread clade beginning approximately 830,000 ybp. The Widespread subclade containing all localities from Alaska is well supported within the Widespread clade , also indicative of a rapid demographic expansion. A negative Tajima’s D value further indicated recent expansion; however it was not statistically significant .The Bayesian Skyline Plot detected a change in effective population size in the late Pleistocene, showing a slight decrease beginning ~100,000 ybp and continuing until ~ 60,000 ybp, followed by a rapid increase from ~60,000 ybp to present  of genetic variation was explained by differences in drainage basin. This result provides initial evidence that hydrologic connectivity is not a major determinant of genetic structure across broad geographic scales in P. badia.Despite having a winged adult stage, some aquatic insects  have beeP. badia; however, connectivity across broad distances within drainages is not necessarily expected. For example, although high order, low-elevation streams provide a hydrologic connection between patches of mid-elevation habitat suitable for P. badia, these low elevation connecting rivers may not meet the ecological requirements of the species , thereby preventing in-stream dispersal of aquatic forms. Low elevation regions may also be sufficiently expansive as to prevent dispersal of winged adults to opposite slopes of large drainages [We emphasize that the above stated conclusion, that hydrologic connectivity is not a primary driver in the genetic structure of this species, is scale dependent. Our sampling density allowed us to test for dispersal across tens to hundreds of kilometers. At local levels (tens of kilometers), contemporary dispersal among networks of stream corridors likely results in high levels of connectivity. Larger tracts of connectivity would be expected in regions of the landscape with a greater density of habitats meeting the ecological requirements of rainages , 20.a priori groups according to sub-drainage. The geographic distribution of our sampling localities, however, did not allow us to designate a priori groups according to sub-basin across the study area, while retaining sufficient replicates for statistical analysis of all the groups. While it is possible that grouping according to sub-drainage basin would explain a greater percentage of mtDNA diversity than our AMOVA as presently organized, it is clear from the geographic distribution of the various clades  either must have colonized by way of overland dispersal in the last ~20,000 years since the whole of the Hudson Bay drainage was glaciated during the Last Glacial Maximum (LGM), or closely tracked the recession of periglacial lakes formed by the melting Laurentide ice sheet. Beyond the Northern Rockies clade however, all other clades except the Western Great Basin  also show closely related haplotypes that span drainage boundaries. The results of our gene flow analysis provide empirical support to the observed patterns of inter-basin movement discussed above.The presently observed distribution of haplotypes in the Widespread clade suggests that this lineage achieved overland connectivity from the southern Rocky Mountains to Alaska in the relatively recent history of the species, as recently as 100,000 ybp according to our dating analysis. This pattern would only be observed if overland dispersal across drainage boundaries were a dispersal mechanism in the group. The Northern Rockies clade provides what appears to be a recent example of inter-basin transfer with very closely related haplotypes being present in both the upper Columbia (Kootenay River), and Hudson Bay drainages . While post-glacial colonization into the upper Columbia drainage (Kootenay River) could have proceeded from populations residing at lower elevations in the same drainage, P. badia is not structured exclusively along drainage basin boundaries, as predicted for organisms that are extreme headwater specialists, the headwater model of dispersal [P. badia. We expect headwater dispersal to be of particular importance in areas where drainage basin boundaries fall within (or near) the elevational distribution of the species, as observed for the boundary between the Green River and Hoback River localities. Our analysis also found isolation by distance to be a dispersal related mechanism shaping genetic structure. While isolation by distance was non-significant for the Northern Rockies, Old Colorado Plateau, Old Rio Grande, and Pacific Northwest clades, a significant correlation was found in the Widespread clade, which contains over half of the localities sampled in the present study, and has the largest geographical footprint. Finally, although overland dispersal of the species is clearly evident, the very high overall ΦST of 0.91 (p < 0.001) indicates that dispersal events between geographically distinct localities are rare. This conclusion is also consistent with the presence of several well-supported subclades that are comprised of specimens from a single geographic locality, or multiple geographically proximate localities  between distant localities (100–500 km distant) on the same river. While the geographic distribution of the clades and the gene flow analysis show that mtDNA diversity for ispersal  appears ies Fig. .P. badia from its sister species P. regularis at the end of the Pliocene approximately 2.5 million ybp. This divergence date is coincident with the of xerification of the Columbia basin associated with the Cascadian orogeny [P. badia. Still, we note that the precision of the dates here discussed should be re-evaluated as more calibration data become available for pteronarcyid stoneflies.Our dating analysis places the divergence of  orogeny , a vicar orogeny , 23, inc orogeny . This fiPteronarcella badia as well, and may have given rise to many of the presently observed patterns of genetic structure. The presence of several, highly differentiated mitochondrial lineages confined to discrete geographic regions corroborates evidence for multiple glacial refugia seen in other aquatic groups distributed across glaciated regions [Several studies have shown Pleistocene glacial cycles to be one of the main drivers in shaping genetic structure of various aquatic insect species across Europe , 7, 25.  regions , 26. In  regions , 23.P. badia into northern (Alaskan) refugia [Additional evidence of past glacial cycles affecting present genetic patterns is our unexpected finding that all haplotypes from Alaskan localities occur as a subclade within the Widespread clade, despite their being geographically closer to the Northern Rockies and Pacific Northwest clades Fig. . Our div refugia , 28 and  refugia , 22 and P. badia in northern British Columbia and the Yukon through extensive personal contacts with aquatic biologists in southeastern Alaska, British Columbia, Yukon territories, or through a careful search of Canadian stonefly checklists [This raises the question: Is there presently connectivity between the Alaskan and southern groups? No evidence of present genetic connectivity exists. The Canadian sample localities that are closest geographically to Alaska contain haplotypes of the genetically distant Northern Rockies clade Figs.  and 4. Fecklists , 30 and P. badia to contract northward into the Bering refugia, or into refugia south of the glacial ice sheets, in general our analysis of past population demographics with Fu’s Fs and Bayesian skyline plot data  occur in the southern most limits of the present species distribution. According to our divergence dating, these lineages in the southern Great Basin and Rio Grande drainages began differentiating in the early-middle Pleistocene ~1.5 million ybp. However, haplotypes from the most recently diverged Widespread clade are also abundant in the southern limits of the species distribution; and in three instances occur at the same localities as Old Colorado Plateau and Old Rio Grande haplotypes . This observation is consistent with recent southward expansion of the Widespread clade resulting in secondary contact with the Old Colorado Plateau and Old Rio Grande lineages.Although the presence of the Cordilleran and Laurentide ice sheets would have caused the distribution of ata Fig.  show eviFinally, while cooling patterns would have allowed for southern expansion, the retreat of the continental ice sheet following the LGM has resulted in a subsequent expansion of the Northern Rockies clade into formerly glaciated latitudes in Canada. Northern, post-LGM expansion of the Old Colorado Plateau and Old Rio Grande lineages is also an alternative explanation for the pattern of secondary contact between those clades and the Widespread clade described in the previous paragraph. Thus, both warming and cooling cycles associated with recent glacial maxima are likely drivers in the recent range expansion of the group.P. badia is consistent with patterns seen in other western North American taxa [While our mitochondrial data reveal the presence of several highly divergent clades (3–5 % pairwise distance), the nuclear locus only shows distinctiveness in the Pacific Northwest and Western Great Basin clades Fig. . This iscan taxa , 31.P. badia reveals a complex history of isolation and multiple invasions among some areas and a cryptic lineage in the Pacific Northwest. The study provides evidence of multiple glacial refugia and suggests that historical climatic oscillations have been important mechanisms in determining genetic structure of insects in western North America. Our ability to generate a large mitochondrial data set through mitochondrial genome reconstruction greatly improved nodal support of our mitochondrial gene tree and allowed us to make stronger inference of relationships between lineages and timing of divergence events. We emphasize that due to the limited signal in the nuclear locus we examined, our findings are based almost entirely on mitochondrial data. As such, many of our findings will need to be re-evaluated when additional, informative, nuclear loci become available. This work adds to the ever-growing list of studies that highlight the potential of next-generation DNA sequencing in a phylogenetic context to improve molecular data sets in understudied groups.Our phylogeographic analysis of P. badia through species checklists [P. badia nymphs and adults from 30 localities throughout its known distribution in the western United States. Where densities were sufficiently high, we collected at least ten individuals per locality. Samples for seven additional localities in Canada and Alaska were sent to us by remote collectors or obtained from the stonefly collection at the Monte L. Bean Life Science Museum at Brigham Young University. Although the species is known to occur in both Alaska and southern regions of Canada, we were unable to find confirmed localities for the species in northern British Columbia, northern Alberta or the Yukon, indicating an apparent gap in the known species distribution.We determined the approximate range of ecklists , 30 onliecklists , personaP. regularis from both personal collecting efforts and the Monte L. Bean Life Science Museum. All samples collected through personal efforts were preserved in 100 % EtOH and stored at −80 °C until DNA could be extracted. Ten individuals per locality were included in the phylogenetic analysis. We determined that 10 individuals per locality would adequately sample haplotypes based on a pilot study data, which showed low haplotypic diversity within localities but high haplotypic diversity between localities. For localities where fewer than ten individuals were obtained, we used all available individuals for that locality. DNA was dissected from leg or thoracic muscle tissue and extracted using the Qiagen® DNeasy™ protocol. All DNA sequences were uploaded to GenBank as accession numbers KU180827 through KU182359. Collection information is summarized in Table For outgroup sampling, we obtained up to ten individuals from two localities of the sister species P. badia individual collected on the Diamond Fork River (Table Pteronarcys princeps (Hagen) mt genome [P. badia contigs, we designed PCR primers flanking gap regions and amplified the gaps using DNA extracted from the same individual that was sequenced via 454 pyrosequencing. To amplify part of the A + T rich control region not recovered in the Newbler assembly, we used Phusion High-fidelity DNA Polymerase  on an Eppendorf Mastercycler® pro  in a 12.5 μL reactions containing 3 μL of DNA template, 2.25 μL nuclease free water, 0.5 μL dNTP’s, 0.5 μL each primer, and 6.25 μL Phusion polymerase using the following thermal profile: 2 min. at 95 °C, followed by 35 cycles of 30 s @ 95 °C, 30 s annealing @ 50 °C, and 2 min. extension @ 72 °C, with a final elongation step of 4 min at 72 °C. We used MOSAS [We pooled whole genomic DNA extracted from a single er Table  with DNAt genome  as a refed MOSAS  to idented MOSAS  as impleP. badia and the closely related P. princeps in an attempt to select markers that would give maximum resolution to our species-level data set. We chose 28S as the second locus because it showed variation at six positions in a pilot study that considered a sub-sample of individuals from geographically distant localities. All primers used for data set generation are listed in Table We targeted portions of five, mitochondrial protein coding genes for PCR amplification: ATP6 (506 bp), COI (844 bp), COIII (711 bp), CYTB (795 bp), and ND6 493 bp); as well as a portion of nuclear ribosomal 28S  for 275 individuals  from 40 sample localities  across western North America. Genes from the mitochondrial locus were amplified via PCR using primer pairs designed from the annotated mt genome, which we generated through 454 pyrosequencing. We chose primer locations flanking regions that showed moderate to high variation in an alignment between the mt genomes of  bp; as wμ96 filter plates. . The purified DNA was sequenced in 10.5 μL reactions using the ABI Big Dye terminator standard protocol  and sequenced at the BYU DNA Sequencing Center using an ABI 3730XL automated sequencer (Applied Biosystems). We edited DNA sequences using Geneious v5.5.5 and aligned using MAFFT v6 [We performed PCR with a Peltier PTC-225 DNA Engine Tetrad Thermal Cycler  in 12.5 μL reactions containing 3 μL of DNA template, 2.25 μL sterile distilled water, 0.5 μL each primer, and 6.25 μL GoTaq mastermix using the following thermal profile: 2 min. at 95 °C, followed by 35 cycles of 30 s @ 95 °C, 30 s annealing @ 50 °C, and 2 min. extension @ 72 °C, with a final elongation step of 4 min at 72 °C. We verified successful amplification using ultraviolet visualization following gel electrophoresis with a 1 % agarose gel. We purified PCR product with Millipore MultiScreenMAFFT v6 . We aligMAFFT v6 . We alsoMAFFT v6 , 40. BecMAFFT v6 .ST values in Arlequin v3.5 [We calculated genetic diversity, and haplotype and nucleotide diversities in DnaSP v9. To estimate overall population structure, we calculated Φuin v3.5 . We useduin v3.5  to generWe performed tree reconstruction using a Maximum Likelihood (ML) approach in RAxML v7.2.6 . After ea priori groups according to major drainage basin as follows: Columbia, Colorado River, Great Basin, Rio Grande, Yukon, and Hudson Bay, and Kenai. We assumed for our null hypothesis that if hydrologic connectivity  is important in determining population structure, as it is with many freshwater obligates such as fish, the AMOVA should show that a large percentage of the mtDNA diversity is explained by differences among drainage basin [To test the importance of hydrologic connectivity in determining population structure, we performed AMOVA in Arlequin v3.5  and defige basin , 48–50. ge basin  to deterge basin . We usedge basin  implemenge basin  to test ge basin . We usedge basin , we discge basin .n = 5). Thus, we estimated gene flow between three localities, two having a direct hydrologic connection between them, and a third that lies in an adjacent drainage basin. This analysis pattern was repeated three times across our sampled area. One locality in the analysis, Salina Creek, had individuals from multiple clades (Old Rio Grande and Widespread). We removed the specimens from the Old Rio Grande clade prior to analysis such that gene flow was only estimated between specimens belonging to the Widespread clade. Gene flow estimates were calculated using MIGRATE-n v3.2.7 [We compared the importance of overland flight to dispersal through water connectivity by comparing gene flow estimates between localities with and without water connections. We selected localities to include in the gene flow analysis based on three criteria: having geographic proximity and direct hydrologic connectivity to an intra-basin locality, geographic proximity to an inter-basin locality , and having several sample replicates  in each of the major clades, using a Mantel T-test implemented in IBDWS v3.23 [To examine isolation by distance as a mechanism for determining population structure, we searched for a correlation between Slatkin’s  linearizWS v3.23 . The claTo test the effect of past climate oscillations on historical demography, we estimated changes in effective population size through time using a Bayesian skyline plot  implemenThe data set supporting the results of this article are available in GenBank (KU180827-KU182359 and KU182360) and the Dryad repository (doi:10.5061/dryad.c1sd4) . All field-work was conducted in compliance with local legislation. All specimens were collected on public lands for which special permits were not required. No endangered or regulated invertebrates were affected by this study.


Ocyurus chrysurus, found along the coast of the western South Atlantic. O. chrysurus is a lutjanid species that is commonly associated with coral reefs and exhibits an ample geographic distribution, and it can therefore be considered a good model for the investigation of phylogeographic patterns and genetic connectivity in marine environments. The results reflected a marked congruence between the mitochondrial and nuclear markers as well as intense gene flow among the analyzed populations, which represent a single genetic stock along the entire coast of Brazil between the states of Pará and Espírito Santo. Our data also showed high levels of genetic diversity in the species (mainly mtDNA), as well a major historic population expansion, which most likely coincided with the sea level oscillations at the end of the Pleistocene. In addition, this species is intensively exploited by commercial fisheries, and data on the genetic structure of its populations will be essential for the development of effective conservation and management plans.In the present study, five loci  were sequenced to determine the genetic diversity, population structure, and demographic history of populations of the yellowtail snapper,  Preservation of the biological diversity of any ecosystem is essential for evaluation of the distribution and connectivity of its populations  and the Ocyurus chrysurus (Bloch 1790), is a lutjanid fish found in tropical and subtropical coastal regions, where it is generally associated with sandy bottoms and coral reefs [O. chrysurus exhibits a pelagic larval development period of approximately 30 days [O. chrysurus in the region, despite not finding high levels of genetic divergence between populations. These results were attributed to a set of factors, particularly the influence of ocean currents and limitations on the movement of the post-larvae and adults [The yellowtail snapper, al reefs . This spal reefs ,11. Simi 30 days . Followi 30 days ,14, whic 30 days  that incd adults .Additional studies have provided evidence of the sub-structuring of yellowtail snapper stocks in the western Atlantic, including the Caribbean . VasconcO. chrysurus reported to date. Additionally, there was a large gap between the northernmost Brazilian population examined by these authors, in the state of Ceará, and Belize. In others words, the northern limit of the Brazilian stock—or how many stocks exist—remained unclear, considering the enormous extent of the northern sector of this country’s coastline. Distinct stocks display independent evolutionary dynamics and can respond in different ways to intense fishing pressure [In spite of the economic and ecological relevance of this species as a fishery resource, Vasconcellos et al.  conductepressure . TherefoLutjanus purpureus [Lutjanus campechanus [O. chrysurus populations distributed along more than 3,000 km of the coast of the western South Atlantic in Brazil (i. g. representing most of the species distribution in the Brazilian coast) and provides robust data on the population structure, genetic variability, and demographic history of this species.Phylogeographic research in the western Atlantic  has revealed a lack of effective barriers to gene flow in some fish species, such as two demersal lutjanids, the Southern and Northern red snappers, urpureus  and Lutjpechanus , respectpechanus , the preO. chrysurus adults were collected from eight localities on the coast of Brazil, between 2007 and 2012, in the states of Pará, Maranhão, Ceará, Rio Grande do Norte, Paraíba, Pernambuco, Bahia, and Espírito Santo  were extracted from each specimen and frozen until analysis. All of the specimens were included in the Lutjanidae tissue bank of the Applied Genetics Laboratory at the Bragança, Campus of the Federal University of Pará (UFPA). Their genomic material was obtained using a phenol-chloroform and enzymatic extraction protocol and was precipitated with sodium acetate, isopropanol and ethanol .For this study, we analyzed two mitochondrial markers, Cytochrome b (Cytb) and the control region (CR) ,21, and The amplicons were purified with PEG 8000 (polyethylene glycol) following the protocol  and sequInitially, only one of the strands of each genomic region was sequenced see . When amhttp://imperialis.inhs.illinois.edu/dmitriev/indel.asp) [The DNA sequences were edited and aligned in BIOEDIT v. 7.1.3.0 . In the del.asp) .The gametic phase of each nuclear marker was defined using the PHASE algorithm , availabW test [For the nuclear data, the minimum number of recombination events was estimated via the Rm method , availabW test , availabW test . LinkageW test , which wDetermination of the number of polymorphic sites and the identification of possible stop codons (in the case of codifying regions) were performed in MEGA 6 . The ideThe haplotype network was generated using HAPLOVIEWER  based onO. chrysurus populations was initially evaluated through an analysis of molecular variance (AMOVA) [st values [p values using the False Discovery Rate test [st/(1-Fst)) and geographic (km converted to Ln) distances [http://ibdws.sdsu.edu/∼ibdws) [The genetic homogeneity of the  (AMOVA)  for eacht values  were uset values , with a ate test . To veriistances . Negativ/∼ibdws) , with 10For comparison between Brazilian and Caribbean populations, the control region sequences used by Vasconcellos et al.  (accessihttp://taylor0.biology.ucla.edu/structureHarvester/) [Bayesian methods, using STRUCTURE v. 2.3.4 , were apvester/) .Cluster analyses were also conducted in STRUCTURAMA . For thiTo check the fit of the historical population dynamics to a model of exponential growth, we used a mismatch distribution  togetherO. chrysurus were visualized using a Bayesian Skyline Plot (BSP) [Historic fluctuations in the demography of ot (BSP)  and an eot (BSP) . These pot (BSP) , based oot (BSP) ,52 (HKY ot (BSP) ,54.Two runs were performed using different random seeds, including 200 million generations for each BSP run and 400 million for each EBSP run, with samples taken at intervals of 10,000 generations, 10% of which were discarded as burn-in. The convergence and mixing of the chains were inspected visually in TRACER v.1.5 . The conD [Fs [Tajima’s D  and Fu’sD [Fs  were alsD [Fs , with thO. chrysurus specimens, and 645 bp of the Cytochrome b gene was sequenced in 170 specimens  was sequenced from the Control Region in 152 pecimens . ConsideST values being obtained  and lower for Cytb (h = 0.653±0.002), and the same pattern was observed in the case of nucleotide diversity, with π = 1.7% for the CR, but only π = 0.15% for Cytb . AMOVA iobtained , and thiulations . Howeverulations . The Fstulations , indicatThe genetic homogeneity of the populations from the Brazilian coast was also emphasized by the distribution of the haplotypes, given the lack of any clear geographic pattern in the network . This geFs values were significant in some populations, or when the specimens were grouped in a single population for both mitochondrial markers . For this intron, an indel region of approximately 140 bp was identified. In the case of the Adenine Nucleotide Transporter (ANT) gene, 320 bp of intron 1 (together with a portion of the exons) was sequenced in 142 specimens, and 176 bp of intron 5 (together with a portion of the exon) of the Growth Hormone (GH) gene was sequenced in 150 specimens. The Фw test demonstrated the existence of recombination events only in IGF 2 . When the indel region was removed, recombination was not detected (p = 0. 25), and all of the analyses for this marker were performed excluding this region.A fragment of 1,048 bp was sequenced for the nuclear regions, 552 bp of which corresponded to intron 2 of the Insulin-like Growth Factor (IGF) gene, which was sequenced in 130 specimens , with intermediate values  being recorded for GH 5 and considerably lower values being obtained for ANT 1  .The results of the AMOVA revealed that most of the genetic variance was present within, rather than between the populations, which is consistent with high levels of genetic similarity between populations . This fiO. chrysurus populations  that could allow some connectivity between the locations analyzed. Thus, all of these features are consistent with high genetic similarity along the Brazilian coast (coast Pará to Espírito Santo), as demonstrated in this study, and they may be the main factors regulating the genetic connectivity between the Brazilian populations of O. chrysurus, as observed in another lutjanid, L. purpureus [However, the larvae of  ability , and the ability . The dyn ability . Some st ability ,69, furturpureus .L. purpureus [L. campechanus [O. chrysurus [The results of the present analysis revealed high levels of genetic variation in the investigated yellowtail snapper populations, especially in the mitochondrial markers, which presented a large number of haplotypes, similar to that recorded for urpureus  and L. cpechanus , with sipechanus . The higpechanus ,53,71,72hrysurus .Pleuronectes platissa covering almost 100 years, even though this species has been heavily exploited since the XIX century. Additionally, Pinsky & Palumbi [Lutjanus, even for species that are considered to be overfished.This high genetic variability cannot be interpreted as an absence of an impact of fishing on these populations because the commercial exploitation of this species is relatively recent (approximately 50 years and less than 20 generations). Examples from the literature reveal several situations where overfishing apparently shows no direct relationship with genetic diversity indices. For example, Hoarau et al.  did not  Palumbi  recentlyCentropomus [Distinct levels of genetic diversity were observed at the three nuclear loci analyzed in the present study. The most variable nuclear locus was the intron of the IGF 2 gene, which presented a degree of polymorphism comparable to that observed in the mitochondrial Control Region, indicating that it is a potentially useful marker for population-level and phylogeographic studies in lutjanids, as observed for tropomus .The yellowtail snapper inhabits coastal waters  and is tChromis genus. O. chrysurus generally prefers water at temperatures between 24–30°C, with temperatures above 34°C being lethal for this species [O. chrysurus populations.The temperature fluctuations that occurred in this period may have had a great influence on the historical demography of a number of marine species from this region. For example, Rocha et al.  lists te species . Thus, tCynoscion guatucupa [Chromis multilineata [L. purpureus [L. campechanus [Historic events of population expansion are commonly identified in demographic studies on marine fish species. In the western Atlantic, events of this type have been recorded in uatucupa  and Chroilineata  as well urpureus  and L. cpechanus .O. chrysurus stocks analyzed in the present study clearly indicate population expansion along the Brazilian coast. This process is similar to that observed in Caribbean O. chrysurus, although Vasconcellos et al. [Fs test, was significant only for the total population for the mitochondrial markers examined in the present study. Fu's test shows greater statistical power and sensitivity for detecting events of geographical expansion [The results obtained for the s et al.  concludexpansion  and is tO. chrysurus is also clear from the BSP and EBSP analyses, which identified an abrupt increase in population size approximately 25,000 years ago, which would coincide with the last glacial maximum in this region  [O. chrysurus as well as other marine organismsThe expansion of the Brazilian populations of  et al.) . In fact et al.) ,86, and Ocyurus chrysurus undertaken to date. The results clearly revealed the existence of a single panmictic population along the Brazilian coast [The present study is the most comprehensive investigation of the Brazilian populations of e et al.), and as e et al.), there iOcyurus chrysurus along the Brazilian coast.In relation to the high levels of genetic diversity observed, mainly among mitochondrial markers, these findings cannot be interpreted as a lack of any impact of fisheries on these populations, given that the commercial exploitation of this species is relatively recent, and the markers used in the present study would not be appropriate for the detection of alterations on this time scale . FurtheO. chrysurus, the formation of shoals for spawning is reported to occur at a number of locations [The data presented herein suggest that the populations of yellowtail snapper along the Brazilian coast may represent a single genetic stock, and if so, they should be managed as a unit. This conclusion has strong implications for the management of this species. For example, for various snappers, including ocations . Thus, tO. chrysurus populations.Further studies involving other classes of molecular markers  as well as studies tracking adults and larval dispersal should be conducted to obtain a better understanding of the structure of S1 Fig(TIF)Click here for additional data file.S2 FigAbove Mean Ln prob values ± standard deviation for each value of K. Each individual is represented by a bar. The height of the bar is proportional to the probability of the individual belonging to a given cluster.(TIF)Click here for additional data file.S3 FigEach individual analyzed is represented by a square, and that the probability values are represented by color . Numbers: 1- Pará, 2- Maranhão, 3- Ceará, 4- R. G. do Norte, 5- Paraíba, 6- Pernambuco, 7- Bahia, 8- Espírito Santo.(TIF)Click here for additional data file.S1 Supporting Information(XLS)Click here for additional data file.