Prunus domestica L.) is an ancient domesticated species cultivated in temperate areas worldwide whose genetic structure has been scarcely analyzed to date. In this study, a broad representation of Spanish European plum germplasm collected in Northeastern Spain and a representative set of reference cultivars were compared using nuclear and chloroplast markers. The number of alleles per locus detected with the SSR markers ranged from 8 to 39, with an average of 23.4 alleles, and 8 haplotypes were identified. Bayesian model-based clustering, minimum spanning networks, and the analysis of molecular variance showed the existence of a hierarchical structure. At the first level, two genetic groups were found, one containing ‘Reine Claude’ type reference cultivars altogether with ca. 25% of local genotypes, and a second one much more diverse. This latter group split in two groups, one containing most (ca. 70%) local genotypes and some old Spanish and French reference cultivars, whereas the other included 24 reference cultivars and only six local genotypes. A third partition level allowed a significant finer delineation into five groups. As a whole, the genetic structure of European plum from Northeastern Spain was shown to be complex and conditioned by a geographical proximity factor. This study not only contributes to genetic conservation and breeding for this species at the national level, but also supports the relevance of undertaking similar tasks of collection and characterization in other unexplored areas. Moreover, this kind of research could lead to future coordinated actions for the examination of the whole European plum diversity, to define conservation strategies, and could be used to better understand the genetic control of traits of horticultural interest through association mapping.European plum ( Prunus domestica L.), a hexaploid species (2n = 6x = 48), is still controversial and remains uncertain [P. spinosa (2n = 4x = 32) and P. cerasifera (2n = 2x = 16) [Prunus genus within the Prunoideae subfamily of Rosaceae [P. domestica L. was used by humans, at least, 6,000 years ago, and it is known to have been widely cultivated in Roman times. This long history of domestication has resulted in a rich diversity of morphological characteristics and crop aptitudes [The biological origin of European plum  and chloroplastic DNA (cpDNA) markers to elucidate the pattern of genetic diversity, structuration, and differentiation of P. domestica L. from the Northeast of the Iberian Peninsula.The specific aims of this study were to discern the extent of the genetic structure of the P. domestica accessions collected in areas from Northeastern Spain and an additional set of 46 cultivars used as a reference . This SSR set contains three markers per chromosome, except for chromosomes 1, 2 and 4, for which two were included , the number of observed alleles per locus (AO), the mean number of alleles per genotype (AM), the effective number of alleles (AE), and the number of rare alleles per locus (AR) were estimated for each SSR locus. In order to quantify the occurrence of rare alleles, two levels were considered, present in < 5% (A<5%) and < 1% (A<1%) of the genotypes.Since allele dosage determination in polyploid species is a complicated issue, we compared all multilocus genotypes scoring and recording the alleles as present/absent. Thus, for instance, AABBCC and AAAABC genotypes were both codified and included in the dataset as ABC. This way to codify the data is known as the ‘allelic phenotype’ approach , 43 and B) [A pairwise distance similarity matrix between all genotypes was calculated using Bruvo distance (DB)  through B)  on the RB) , and graB) . Bruvo’sPrunus species [As a complement to SSR diversity analysis, the polymorphism degree of the 18 possible cpDNA region/enzyme combinations was assessed. The study of chloroplast DNA variation using the PCR-RFLP has been shown to be useful and informative for studying chloroplast DNA diversity and phylogenetic relationships among  species , 52–55. K) and to quantify the membership probability of each genotype to the inferred subpopulations. Analysis was performed under the admixture model and correlated allelic frequencies, and run using the recessive allele approach [K values ranging from 1 to 10 inferred clusters, with 10 independent runs each, applying a burn-in period of 200,000 followed by 500,000 iterations. Structure Harvester ver. 0.6.93 [K method defined by Evanno et al. [K value. Each genotype was assigned to the group for which it had the highest assignation probability (qI), considering a strong membership coefficient of a genotype to a particular group whenever qI ≥ 0.80 [qI < 0.80 were considered as admixed genotypes. Placement of accessions on the inferred groups was determined using CLUMPP ver. 1.1 [The software STRUCTURE version 2.3.4  was usedapproach  codifyinapproach , 58–59. . 0.6.93 , which io et al. , was useI ≥ 0.80 –19, 21, ver. 1.1  and the ver. 1.1  to graphST for dominant binary data [The degree of differentiation between the genetic groups derived from STRUCTURE was estimated by performing analyses of molecular variance (AMOVA) using GenAlEx 6.5 , 65. Theary data , were caA), the number of exclusive alleles (NEA) and the mean number of alleles per genotype (AM).The mean pairwise distance (MPD), i.e., mean of the pairwise PhiPT per genetic group, an index of the group differentiation relative to other genetic groups, was calculated for each group. The within-group sum of squares divided by the number of individuals in the group was applied as a normalized intra-group variability index (nSSWG) . AdditioPrunus species [The study of amplification products showed that eight out of the 21 SSR markers used were problematic in terms of absence of amplification product, insufficient fluorescence signal, low level of polymorphism or complex scoring pattern and, as a consequence, were excluded from subsequent analyses . Further species , 67–68, Based on the remaining 11 SSR markers, 135 unique genotypes were found within the whole set of accessions evaluated , which corresponds to a 18% of duplication degree as a whole. Seventeen groups of SSR duplicates were identified. Two accessions had the same SSR profile than ‘Stanley’, whereas eight had the same profile than cultivars within the ‘Reine Claude’ group . The remaining groups of SSR duplicates were comprised exclusively by local accessions. It is worth noting that the latter SSR duplicates frequently included germplasm collected either in the Pyrenees or in the Iberian Cordillera, seldom including accessions collected from different areas. This fact may indicate that independent local selection processes could have been performed by the farmers from these areas in the past, seeking for adaptation to specific habitats across the heterogeneous Northeastern mountain landscape.The presence of slight allelic differences was quantified by the pairwise Bruvo distance coefficient between genotypes, showing that 46 pairs of genotypes involving as a whole 24 differed in less than 0.05, which corresponded to a very few allelic mismatches. Slight allelic differences may result from potential genotyping errors, but also from putative somatic mutations, relatively frequent in long-lived tree species for which vegetative propagation has been used since ancient times –70.M) ranged from 2.74 (UDP98-409) to 4.89 (CPSCT026), with a mean value of 4.01. In spite of the high number of alleles found, the mean number of effective alleles (AE) was low (9.21), indicating that an important number of alleles appeared in the population at very low frequency. Thus, the proportion of rare alleles was high, with a mean percentage of 35.4% and 10.3% alleles per locus found respectively in less than 5% and 1% of the genotypes, and 32 alleles were unique (i.e. identified only in one genotype). This fact highlights the richness and allelic singularity that still can be found in traditional material, thus representing an important unexplored source of genetic variation.The 11 SSR markers proved to be highly diverse in terms of their degree of polymorphism, ranging from 8 (UDP96-008) to 39 (BPPCT025) alleles per locus, leading to a total number of 257 alleles . The aveP corresponded, as expected, to those markers being the richest in detected alleles  and 34.09% (UDP96-008) of the genotypes.Genetic diversity was also quantified using the allelic phenotype approach, as this task is much more problematic in polyploids than in diploids, since more than two alleles per individual and locus are transmitted, potentially including multiple copies of a given allele . In this alleles . The mosChloroplast DNA (cpDNA) variation was assessed using PCR-RFLP and, seven out of the 18 primer-restriction enzyme combinations resolved polymorphic fragments in the reference set of cultivars. When analyzed with those seven primer-restriction enzyme combinations, 13 polymorphic sites were detected , and theP. domestica germplasm preserved at the French National Plum Collection [A low degree of cpDNA variation can be due to historical events , but also to human-mediated activities . The prellection . These aK over the range of K values evaluated in STRUCTURE revealed that the germplasm could be divided into two and three groups, with a less pronounced peak found at K = 5  and 98 (G2.2) genotypes . For K = 3, group size was 67, 38 and 30 for G3.2, G3.1 and G3.3 respectively, and for K = 5 it varied from 3 (G5.5) to 41 (G5.3)  . The facartifact .K value showing the highest ΔK, but also for some others showing relatively high values for this parameter in order to delineate further levels of substructure of the diversity. Therefore, the most likely divisions obtained for K = 2, K = 3 and K = 5 will be examined and compared throughout the subsequent sections. The consistency in the clustering of the genotypes between runs was examined to analyze the robustness of the divisions obtained at the three K values. The assignment of the genotypes was very consistent between runs, none shifting from genetic group when K = 2 and K = 3, and just 13 admixed genotypes showed slight discrepancy between runs when K = 5. These three partitioning levels were then compared in terms of their mean assignment probability of genotypes to the inferred groups for each of the three K values considered, as well as to the proportion of genotypes strongly assigned (qI ≥ 0.80) [K values was very high and almost identical (around 0.80). The proportion of genotypes strongly assigned differed between K values  based on Bruvo’s distance  . The meaK values , with 64distance  were conK = 2, ca. 25% of the local genotypes clustered together in G2.1 with all the reference cultivars of the ‘Reine Claude’ group  remained essentially unmodified (corresponding to G3.1 and G5.1), whereas higher K values allowed disentangling internal pattern of substructure of G2.2. The study of Prunus domestica L. germplasm maintained in the French National Plum Collection [For e’ group , a variee’ group . This mallection  also repllection  argued tK = 3 was considered, G2.2 was partitioned into G3.2 and G3.3 , whereas G5.5 was a very small group, consisting of only three genotypes , where representatives of either the ‘Reine Claude’ group or old Spanish and French cultivars were found. The geographical proximity and historic connections between Spain and France, and the existence of ancient commercial roads and pilgrim’s routes since the Middle Ages could have favored the exchanges of cultivars between regions from both countries. Conversely, just a few local genotypes (ca. 5%) clustered together with other reference cultivars, which could result from the introduction in the late 1960s of cultivars such as ‘Stanley’, ‘President’, or ‘Ruth Gerstetter’ by commercial nurseries . TherefoK = 2 was considered, G2.1 mostly comprised genotypes carrying haplotype H1 (ca. 95%), except for two admixed genotypes with haplotype H5, whereas G2.2 contained genotypes showing the eight haplotypes, including all those constituted just by one or two genotypes. With regards to the two most frequent haplotypes (H1 and H5), when K = 3 was considered, the frequency of occurrence of H1 was considerably lower in G3.3 (~67%) than those found for G3.1 and G3.2 (≥ 90%), whereas haplotype H5 was four times more frequent in G3.3 (20%) than in the other two groups (~5%). Similarly, the haplotype H5 was not equally distributed between groups when considering K = 5, being mainly concentrated in G5.2 and G5.4. Chi2 tests indicated significant differences in the distribution of the haplotypes between all pairs of groups except for the pairs G2.1-G2.2 (P = 0.120) and G3.1-G3.2 (P = 0.171).The analysis of the frequency distribution of the chloroplastic haplotypes into the genetic groups defined by STRUCTURE revealed noticeable differences . When K Biparentally and maternally inherited markers such as, respectively, nuclear and cpDNA markers, have different inheritance modes and evolutionary rates. It is widely accepted that cpDNA markers reflect a past change in population variation, such as a population expansion or decline, whereas nuclear markers infer recent events in the population –75. TherK value considered , below the range observed for the other groups (between 0.49 and 0.58), and its intragroup variability index (nSSWG) was lower than the remaining ones, also mirroring its lower intragroup genetic diversity. Heat maps represented in The study of the intragroup variability revealed that the ‘Reine Claude’ group contained as a whole a lower level of diversity than other groups. This fact was supported by a much lower percentage of total alleles captured in comparison to those found in the other groups, and especially by a much lower presence of exclusive alleles harbored in this group. This trend was maintained irrespectively of the nsidered . MoreoveP. domestica L. and other Prunus species, since the occurrence of interspecific hybridizations, either natural or human-mediated, is relatively common within the genus Prunus [The other group that presented distinct characteristics with this regard was G5.5, the genetic group integrated by just three genotypes. Despite its very small size, this group displayed seven exclusive alleles  and belos Prunus , 12, 76.P < 0.001) for all the three K levels studied. Intergroup variation accounted for 11.4%, 10.8% and 12.7% of the total variation at K = 2, 3 and 5, respectively (K = 3 resulted in moderate differentiation for the three pairs of genetic groups (0.10–0.14), whereas very high PhiPT estimates (up to 0.45) were found for some of the pairs of groups identified at K = 5 , respectively. This high level of differentiation from ‘Reine Claude’ group was an unexpected finding, since it is the varietal-group reported to be the prevailing one in the past in terms of abundance and geographical distribution in Northeastern Spain. Taking that prevalence into account, it appeared sensible to expect a higher degree of relatedness and, consequently, a less differentiation between this group and those containing most of the Spanish traditional germplasm. The contribution of ‘Reine Claude’ group cultivars to create new variability appears to have been relatively limited to generating a relatively closed set of local cultivars, as they cluster with ~25% of local genotypes, but they apparently played a much lesser role in the origin of local genotypes in other groups. ‘Reine Claude’ varietal-group, having been crucial in European horticulture, with representatives as ‘Reine Claude Verte’ grown for more than five centuries , 77–78, Prunus species, as well as of hybrids between P. domestica and other species.Finally, G5.5 showed the highest MPD (0.27)  and pairOur work demonstrates that, prospecting missions in unexplored areas may still be useful to recover an important source of diversity for this species, as local genotypes have been shown to enrich the genetic diversity held in varieties grown worldwide. Therefore, it would be advisable to perform similar tasks of collection and characterization, since understanding the extent and organization of diversity could promote efficient conservation actions, recovering ancient cultivars of potential interest, and ease their use into breeding programs in a near future. Nonetheless, the potential value of most local genotypes for the present-day market needs is largely unknown, since they have not been sufficiently characterized from an agronomic and consumer point of view. Hence, comprehensive phenotypic characterization based on standardized methods would be necessary in order to learn the commercial potentiality of these cultivars.Our approach, based on Bayesian model-based clustering, minimum spanning networks, and the analysis of molecular variance, revealed the existence of a hierarchical structure in European plum germplasm from Northeastern Spain. At the first level, two genetic groups were found, one containing ‘Reine Claude’ type reference cultivars altogether with ca. 25% of local genotypes, but the study of genetic structure at further levels evidenced the existence of an additional internal substructure, that yielded up to five genetic groups. The inferred groups were clearly differentiated and showed noticeable differences in the allelic composition at the group level. Additionally, the fact that ca. 70% of the European plum local genotypes clustered with old Spanish and French reference cultivars indicates that population structure has been deeply conditioned by a geographical proximity factor, and underlines that the genetic background of an important part of the Spanish germplasm may differ from genepools of other origin. The genetic characterization reported herein not only constitutes the most comprehensive study of population structure from Spain, but also puts into value collection and characterization actions. Moreover, this kind of research could lead to future coordinated actions for the examination of the whole European plum diversity, to define conservation strategies, and could be used for defining an European core collection, useful to better understand the genetic control of traits of horticultural interest through association mapping.S1 FigK) calculated according to [K. (B) Mean values of the log likelihood of the data given K is plotted against K for 10 simulations with a burn-in period of 200,000 followed by 500,000 iterations. The error bars show the standard deviations of the mean values.(A) Estimates of the rate of change of the slope of the log likelihood curve (Ärding to  are plot(PNG)Click here for additional data file.S2 FigK = 2 (A), K = 3 (B) and K = 5 (C).Minimum spanning networks (MSN) performed on Bruvo’s distances for all genotypes clustered in the groups defined by STRUCTURE at  Each node represents one genotype. Edge thickness and color are proportional to genetic distance, while edge lengths are arbitrary.(TIFF)Click here for additional data file.S3 FigK = 2 (A), K = 3 (B) and K = 5 (C). Color scales in the matrix indicate genetic distance between genotypes. Red indicates identical, while yellow indicates distantly related.Genetic groups inferred using STRUCTURE at (TIF)Click here for additional data file.S1 TableK = 2, K = 3 and K = 5 were considered) and the chloroplastic haplotypes.Collection information include name, collection site, specific longitude, latitude, approximate elevation, group placement by structure analysis with the mean qI values (when (XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TablePolymorphic sites are indicated in bold letters.(DOCX)Click here for additional data file.S4 TableK = 2 (A), K = 3 (B) and K = 5 (C).Frequency distribution of the chloroplastic haplotypes within the genetic groups defined by STRUCTURE at (DOCX)Click here for additional data file.


The ability of perennial species to adapt their phenology to present and future temperature conditions is important for their ability to retain high fitness compared to other competing plant species, pests, and pathogens. Many transplanting studies with forest tree species have previously reported substantial genetic differentiation among populations within their native range. However, the question of “how local is local” is still highly debated in conservation biology because studies on genetic patterns of variation within and among populations at the local scale are limited and scattered. In this study, we compare the level of genetic differentiation among populations of six different perennial plant species based on their variation in spring flushing. We assess the level of additive genetic variation present within the local population. For all six species, we find significant differentiation among populations from sites with mean annual temperature ranging between 7.4°C and 8.4°C. The observed variation can only be partly explained by the climate at the site of origin. Most clear relationship between early flushing and higher average spring temperature is observed for the three wind‐pollinated species in the study, while the relations are much less clear for the three insect‐pollinated species. This supports that pollination system can influence the balance between genetic drift and natural selection and thereby influence the level of local adaptation in long‐lived species. On the positive side, we find that the native populations of woody plant species have maintained high levels of additive genetic variation in spring phenology, although this also differs substantially among the six studied species. Higher spring temperatures are expected to prolong the growing seasons due to earlier bud burst  populations, and test whether differences (if any) influence the fitness revealing signs of local adaptation.QST) and additive genetic variation (VA) among species.Estimate levels of genetic variation within and among populations in spring phenology and compare the levels of population differentiation , European Crab Apple  Mill.) and Glaucous Dog Rose  Boulay), while three are wind pollinated—Downy birch (Betula pubescens Ehrh.), Common Hazel (Corylus avellana L.) and Sessile oak (Quercus petraea (Matt.) Liebl.). We used data from progeny trials with offspring from open pollinated trees based on seed collected from putative natural populations across Denmark . This was to ensure maximum variation among individuals in their bud burst ranking. Bud burst for All analyzes were made per species, because they were tested in different common garden trials. The variation among trees in their phenological stage was separated into variance and covariance components reflecting genetic differences among and within populations as implemented in the software ASReml , and genetic variance component corresponding to each of the random effects in the model was estimated from the analysis; Yijkl is the trait measured for tree l, μ is the overall mean of the trait, Bi is the fixed block effect, Pj is the fixed population effect, λij is the fixed population by block interaction, fk(j) is the random effect of family within population, τijk is the random effect of plot k, and εijkl is the residual. QST values were estimated from model (1) as well having population effects as random.where The significance of the genetic variances of traits within sites and genotype by environment interaction across sites was tested using the log likelihood ratio  as 4σf2 and narrow sense heritability within sites as; ⌢h2=4σf2/(σf2+στ2+σε2), where σf2 is the estimated family variance, στ2 is the estimated plot variance, and σε2 is the estimated within plot variance. The phenotypic variance was estimated as ⌢VP=σf2+στ2+σε2. Families were considered to represent groups of half‐sibs. This assumption will overestimate the additive genetic variance, heritability, and expected response to selection, if the offspring within progeny groups on average are more related than half‐sibs  using the software ASReml  covariance between trait x and y, σfx2 is the family (within population) variance for trait x, and σfy2 is the family (within population) variance for trait y.Genetic correlations between bud burst and height growth were estimated based on individual tree data to assess whether the height as a proxy for realized fitness in the present climate varied with the phenology. Genetic correlations could be estimated between survival and phenology based on plot means, because the plants were grown in family plots in all trials (see Appendix 33.1VA) thus ranged from 0.07 in C. sanguinea to 0.34 in Q. petraea (Table QST) ranged from 0.04 in M. sylvestris to 0.27 in C. sanguinea , or close to significant (p = .05) for B. pubescens and C. avellana  for Q. petraea .The regression between bud burst of populations and population site minimum temperatures in March were significant  were characterized by having high levels of genetic variation within populations  and counteract loss of genetic variation (maintaining high h2) within populations  , and for these species, it therefore also seems reasonable to assume that populations can evolve in spring phenology with a speed that can match the predicted increase in temperature. The response will only be realized if early bud burst has substantial influence on fitness  or because of directional selection implemented in domestication programmes. The estimates in the present study refer to the latter situation, because the heritability was estimated in managed progeny test and the realized heritability in natural population may be substantially lower due to higher environmental and developmental heterogeneity. In Denmark, all major woody plant species are included in domestication programs based on breeding seed orchards in order to support effective and rapid selection for continued fitness  and genetic diversity may therefore be lower compared to populations closer to the refugial areas. But we still observed substantial level of genetic variation in the fitness‐related trait. For species occurring in extremely small fragmented populations assisted migration at a more localized landscape level may still be desirable to counteract inbreeding within these populations due to random drift. Adaptive potential of a species is not only determined by the presence of genetic variation within and among populations as studied here, but also by the presence of phenotypic plasticity in adaptive traits .None declared.Albin Lobo was responsible for the design of study, analysis and interpretation of data, and writing of the manuscript. Jon Kehlet Hansen was responsible for supervision of the work, design of study, and writing and approving of manuscript. Lars Nørgaard Hansen was responsible for field data collection, and revising and approving the final version of the manuscript before submission. Erik Dahl Kjær was responsible for overall supervision of the study, conception and design of work, data interpretation, and writing and final approval of the manuscript. Click here for additional data file.


Pyura chilensis along the southeast Pacific coast (24°–42°S). This species is sessile as an adult, has a short-lived larval stage, and may also be dispersed by artificial transport as biofouling. We found that the main signal in neutral loci was a highly divergent lineage present at 39°S, and a subjacent signal that indicated a separation at 30°S (north/south), widely reported in the area. North/south separation was the main signal in outlier loci, and the linage divergence at 39°S was subjacent. We conclude that the geographic structure of the genetic diversity of outlier and neutral loci was established by different strengths of environmental, historical and anthropogenic factors.Marine benthic organisms inhabit a heterogeneous environment in which connectivity between populations occurs mainly through dispersive larval stages, while local selective pressures acting on early life history stages lead to non-random mortality, shaping adaptive genetic structure. In order to test the influence of local adaptation and neutral processes in a marine benthic species with low dispersal, in this study we used Genotyping by Sequencing technology to compare the neutral and putatively selected signals  in SNPs scattered throughout the genome in six local populations of the commercially exploited ascidian  Marine benthic species that inhabit patchy and heterogeneous substrates such as rock beds or sandy beaches are likely to be structured as metapopulations135658The consequences of local adaptation in marine benthic species have been assessed with experimental methods such as reciprocal transplants and common garden experiments. These studies have shown there is differential survival between organisms of different origins in different environmental conditions53One experimental approach to analyze neutral and putatively adaptive signals is the use of genome scans to detect thousands of Single Nucleotide Polymorphism (SNP) loci scattered throughout the genome, some of which can be identified as having signals of natural selection by analyzing several individuals from different environments141617191922Pyura chilensis Molina 1972 along the Humboldt Current System (HCS) located in the southeast Pacific Ocean. The HCS is characterized by high productivity and regions with strong upwelling2524In this study we analyze neutral and adaptive spatial genetic structure of the benthic ascidian Pyura chilensis is a conspicuous inhabitant of the HCS widely distributed in the intertidal and shallow sub-tidal from 10°S to 44°S in the southeast Pacific coastPyura chilensis has short-lived (12–24 hr) lecithotrophic tadpole-shaped larvae that provides a very low capacity to disperse between geographic areas along the HCS. In contrast to what has been reported for other sympatric benthic species of the HCS with low larval dispersal capacityP. chilensis did not show a genetic discontinuity at 30°S when analyzed with sequences of the mitochondrial gene Cytochrome Oxidase I (COI) and the nuclear gene Elongation Factor 1 alphaP. chilensis it may be possible to detect variation at 30°S, as would be expected in a benthic species with a low dispersal potential.P. chilensis is transport in artificial structures such as ship and boat hulls, where the species can grow attached3132P. chilensis, the species has often been seen on the hulls of boats of artisanal fisheries, on buoys, ropes and on any other marine facility that provides substratum as an important part of the fouling community34303132An alternative means of dispersal for ascidians such as P. chilensis is the genetic structure of the species based on COI sequences. The COI phylogeographic structure of the species was characterized by three mitochondrial haplogroups, two of which were sympatric and widespread, and a third that was highly divergent from the other two and geographically restricted, predominantly found between 39°S and 42°S, being most abundant at 39°S in Los Molinos (LM)P. chilensis using neutral and putatively adaptive SNP loci, and explore if natural selection follows the same signal as neutral sequence divergence, i.e. if LM is also highly differentiated with putatively adaptive loci. Even though physical transport of a few individuals per generation between local populations should be enough to maintain genetic homogeneity in the whole genome at both individual and population levels3Another relevant aspect about P. chilensis covering from 24°S to 42°S, and across the 30°S biogeographic discontinuity in the southeast Pacific. We aim to assess the influence of contrasting evolutionary forces, including natural selection , isolation and drift (lineage divergence at 39°S), and gene flow , on the spatial genetic structure of the benthic ascidian P. chilensis in the HCS.Here we evaluate the spatial genetic structure of neutral and outlier SNP loci in six populations of P. chilensis  of 36 and a mean quality score of 33.12. After applying the trimming and demultiplexing approach, 163,692 tags were produced with a mean of 4.38 reads per individual. The UNEAK pipeline detected a total of 81,837 SNP. After filtering with a minor allele frequency (MAF) of 4.4% and a minimum call rate (mnC) of 90%, the resulting dataset consisted of 2,332 SNPs.Extracted DNA was of high quality, with average 260/280 and 260/230 ratios of 1.96 ± 0.01 and 2.15 ± 0.03, respectively. Genotyping by Sequencing (GBS) of 90 individuals of q-values of 0.05, 81 of the 2,332 loci uncovered showed FST values greater than expected under a neutral distribution   correction of  < 0.05) . These 8E and HO, respectively) were mostly similar between neutral and outlier loci. HE mean values ranged from 0.290 to 0.294 and 0.250 to 0.358 for neutral and outlier loci, respectively. Mean HO values ranged from 0.169 to 0.304 and 0.143 to 0.345 for neutral and outlier loci, respectively (r) within population ranged from 1.560 to 1.662 for neutral loci and 1.511 to 1.626 for outlier loci. Northern populations generally showed lower values of Ar in neutral loci and similar values between all sampled populations in outlier loci; LH and LM were the sites with the lowest values. The exact test for HWE deviations showed that after FDR correction, only the LM site showed significant deviation  with the exception of Los Molinos (LM) that was highly differentiated with values ranging between 0.393 and 0.417, which are an order of magnitude greater than all the rest of the values. In contrast, all FST values calculated with outlier loci were, as expected, relatively high; remarkably, in neutral loci LM did not show higher levels of differentiation than the other population pairwise comparisons. In the outlier loci population-pairwise differentiation matrix, the values between pairs from the northern  versus southern , i.e. north/south of 30°S, showed the highest differentiation values  (P = 0.048) (P = 0.064)  . Excludi= 0.048) . In the = 0.064) .In order to detect spatial population genetic structure we used the GENELAND software, which revealed the presence of three groups in the neutral loci dataset. One corresponded to the three northern localities PA, CP and LH; the second included TH and AC and the third was LM . Five grCluster analyses confirmed the disparate signals of neutral and outlier loci. In the clustering approach of the neutral loci analysis using DAPC (considering the sampling site as a prior), the first principal component axis  separated LM from all the rest of the populations . The secAs with GENELAND analyses, DAPC performed without considering the information of the geographic origin of the samples, and based on the BIC, detected three and five groups as the optimal separation for neutral and outlier loci, respectively . For neuDendrograms constructed with Identity-by-Site (IBS) without incorporating geographic information detected three and four clusters for neutral and outlier loci, respectively, providing a similar pattern of differentiation as previous analyses . With neutral loci LM appeared as a distant separate cluster, while the two other clusters were closer together. These are the three localities north of 30°S and the southern localities TH and AC . Again, K = 2 was determined as the optimal separation for both data sets in analyses based on STRUCTURE . When LM was excluded from the analyses, the optimal separation also corresponded to K = 2  (39°S), and had a subjacent weaker signal of genetic structure across the 30°S biogeographic boundary. Inversely, outlier loci showed a stronger signal across 30°S and a weaker subjacent signal of lineage divergence at 39°S (LM). The two sets of loci showed the same two phylogeographic patterns, structure across 30°S and 39°S, but their strength was in reverse order. Additionally, the degree of neutral genetic differentiation detected was lower than expected from the short larval duration (2 days) and did not adjust to a pattern of isolation by distance (IBD), suggesting that artificial transport also contributes to the neutral genetic structure of P. chilensis along the Humboldt Current System (HCS). SNP sampling and detection of putatively adaptive loci allowed the evaluation of the contributions of several evolutionary mechanisms in the phylogeographic structure of P. chilensis. We detected a strong adaptive signal of differentiation at the 30°S biogeographic break, a strong neutral signal associated with the divergent lineage present at 39°S, and the influence of artificial anthropogenic transport.The use of putative selected markers (outlier loci) in marine benthic invertebrates has usually increased the strength of detected phylogeographic structure with respect to neutral loci, as is expected, albeit the general patterns remain the same192122LM had pairwise genetic differentiation values with neutral loci one order of magnitude higher than the rest. This is explained by 12 of the 15 individuals from LM that were highly differentiated from all other individuals of all other sites, independent of whether the analytical approximation used was frequency or distance-based. These differentiated individuals belonged to the divergent mitochondrial DNA haplogroup previously identified in LMK = 4) in STRUCTURE  is restricted, despite the genetic evidence that there is transport of individuals between the two areasP. chilensis showed phylogeographic structure across 30°S as expected, although it had less differentiation across 30°S than other species with similar dispersal potential based on larval duration.Phylogeographic patterns of marine invertebrates along the HCS have shown that the biogeographic boundary at 30°S is a historical discontinuity and that species with low dispersal potential retain the genetic signal and have a concordant phylogeographic breakP. chilensis30313233Differentiation was greater than had been previously reported for P. chilensis after periods of isolation during the PleistoceneDiplosoma sp.), a slower growth rateet al.P. chilensis is a good example of a strong competitor with relatively low colonizing ability.Physical transport as biofouling may have allowed the latitudinal expansion of Shipping routes  may alsoAlthough the results are robust and allow inferring the spatial genetic structure of loci affected by contrasting evolutionary forces, it is important to consider alternative explanations associated with the quality of information provided by the data. For example, identified outlier SNP loci may be linked to loci that are the direct target of natural selection instead of natural selection directly operating on them46P. chilensis is a non-model organism, information on candidate genes is not currently available. In spite of the possible caveats, 3.5% (81) of the total retained SNPs  showed low and significant q-values and higher levels of FST for 90 individuals, allowing us to distinguish individuals genetically between sampling locations at a relatively small geographic scale, and suggesting that significant differences could be due to positive selective pressures19222350Demonstrating that local adaptation has occurred involves not only the detection of variation at outlier loci, but also the understanding of the functional differences between alleles of individual candidate genesPyura chilensis. The comparison allowed us to conclude that both outlier and neutral loci were diverse and variable in space, and that both have shaped the genetic diversity of P. chilensis. The geographic structure of the genetic diversity of outlier and neutral loci was established by different strengths of environmental, historical and anthropogenic factors.In this study, we obtained several SNPs scattered throughout the genome and analyzed and compared the genetic structure of neutral and putatively adaptive outlier loci in Pyura chilensis were obtained by local fisherman from six sites along the Humboldt Current System in the Southeast Pacific Ocean  from each individual was used to extract DNA using the DNeasy Blood® & Tissue Kit  according to the manufacturer’s instructions. Quantity/purity of DNA was measured in Nanodrop 2,000 .Samples of ic Ocean . FishermPstI, PstI/TaqI) and finally ApeKISamples were sequenced in the Biotechnology Center of the University of Wisconsin, USA using Genotyping by Sequencing (GBS)Libraries were prepared for the DNA of 15 individuals of each population. Following the restriction enzyme digestion, DNA fragments were ligated to a unique barcode adaptor for each individual. The library was prepared in a 96-well plate with 6 wells as blank, using a Hiseq2000  platform, which sequences reads of approximately 100 base pairs (bp). The reads were visualized in FastQC version 0.10.1de novo locus identification2 (α = 0.05) based on the null hypothesis that the counts of two paired tags of a SNP are equal in all heterozygous individualsAll the data were prepared and analyzed using the pipeline Universal Network-Enabled Analysis Kit UNEAK using TAAdditionally, a minor allele frequency (MAF) of 0.044 was used to filter the number of SNP loci whose variability was well represented in the 90 analyzed individuals. This value was determined empirically based on the probability that at least 8 of the 180 possible allele calls  correspond to the minor allele. Values of minimum call rate (mnC) from 75 to 90% were tested previous to the final SNP calling, finally choosing the most exigent value of mnC (90%) in order to remove all SNPs containing 90% or more “N” values, which designate those individuals for which no allele was assigned for the locus or that were covered by no more than one tag and because of the amount of missing data detected, preserving a reduced but robust set of SNPs with reliable genotypes without excluding individuals from the analysis. These restrictive filters were performed because all loci were sampled at a mean coverage of ≥10x per individual. Under this condition, in theory each individual had in average 10 or more copies of a locus.ST outlier approach, which is based on the estimation of those loci with greater values of FST (outliers) with respect to the expected values under a neutral distribution. We used the software BAYESCAN 2.1ST outlier detection, because it has been reported to have lower rates of false positives with respect to other similar softwareq-values of 0.05 was applied in BAYESCAN to avoid the occurrence of false positives. The data set was then separated into two parts, one with the outlier SNPs, which are loci putatively under diversifying selectionST values that match the expected under a neutral distribution.To identify the SNPs putatively under diversifying selection we used the FFrom here on, all analyses were performed separately for each of the data sets .To evaluate the differences in genetic diversity across the study area in each data set, we calculated the expected heterozygosity, observed heterozygosity and allelic richness with rarefied allele counts, using the HIERFSTAT package version 0.04–22ST and their significance were determined using a permutation test  in ARLEQUIN Version 3.5a priori groupings. Additionally, a Mantel test with 10,000 permutations was carried in out in ARLEQUIN 3.5 to test if there is a pattern of isolation by distance, using the relationship between the geographic distance (Km) and Slatkin’s linearized genetic distance (FST).Pairwise FK clusters. An independent model of allele frequencies was carried out for both data sets, with 10 independent runs with 1eK from 2 to 6. The optimum K was inferred from the posterior individual membership probability of each genetic group. Posterior probabilities of membership were plotted with the shapefiles of the coastline available in the database of GEOdas (NOAA), filtering in the study area using GEOdas Coastline Extractor v 1.1.3.1 (https://www.ngdc.noaa.gov/mgg/geodas/geodas.html).Spatial population genetic structure was assessed using the Bayesian clustering algorithm implemented in GENELAND v 3.1.4adagenetotal/3), using all the 6 discriminant functions. We determined the optimal number of PCs using 1,000 simulations to execute the final DAPC.A clustering approach was used with a discriminant analysis of principal components (DAPC) in the package To infer SNP-based relatedness structure within populations, an analysis based on pairwise IBS (identity-by-state) was used. This approach uses the information of the genotypes to calculate the probability that two alleles in the same segment have the same ancestor. A cluster analysis of the matrix generated with the IBS pairwise coefficients between all possible pairs of individuals was performed using a permutation score of 10,000 to determine how many groups were present in each data set. This was performed in the package SNPRelateK) varied from one to seven, corresponding to the number of analyzed locations plus one. Ten replicate runs, each with 500,000 MCMC with a 10% burn-in period, were performed under the admixture and correlated frequency model. The most probable K value was inferred based on the delta K method proposed by Evanno et al.To determine the number of genetic groups we used STRUCTURE 2.3.4SNP calls for all data set  are available in a VCF file that has been deposited in DataDryad (doi:10.5061/dryad.0s87v).How to cite this article: Segovia, N. I. et al. Lineage divergence, local adaptation across a biogeographic break, and artificial transport, shape the genetic structure in the ascidian Pyura chilensis. Sci. Rep.7, 44559; doi: 10.1038/srep44559 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Ne) and genetic divergence due to differences in migration timing and to estimate whether these quantities have changed over time. We predicted that temporal trends toward earlier migration timing and a corresponding loss of phenotypic variation would decrease genetic divergence based on migration timing and Ne. We observed significant genetic divergence based on migration timing and genetic heterogeneity between early‐ and late‐migrating fish. There was also some evidence for divergent selection between early‐ and late‐migrating fish at circadian rhythm genes, but results varied over time. Estimates of Ne from multiple methods were large (>1200) and Ne/Nc generally exceeded 0.2. Despite shifts in migration timing and loss of phenotypic variation, there was no evidence for changes in within‐population genetic divergence or Ne over the course of this study. These results suggest that in instances of population stability, genetic diversity may be resistant to climate‐induced changes in migration timing.Though genetic diversity is necessary for population persistence in rapidly changing environments, little is known about how climate‐warming influences patterns of intra‐population genetic variation. For a pink salmon population experiencing increasing temperatures, we used temporal genetic data  to quantify the genetic effective population size ( Describing and understanding the distribution of genetic variation within populations is fundamental to the management of species, particularly in a rapidly changing world (Allendorf and Luikart Oncorhynchus gorbuscha) population in rapidly warming . The Ne of a population is one of the most important parameters in evolutionary and conservation biology (Waples Ne provides important information about population viability (Frankham Ne to be less than the census population size (Nc) including natural selection, uneven sex ratios, temporal variation in population size, that exceed Poisson variance in reproductive success, and population age structuring  because competition for spawning areas  can lead to redd superimposition .Little is known about h2 = 0.51, Carlson and Seamons Describing genetic population structure within‐ and between‐populations is another way to quantify genetic diversity. Understanding within‐ and between‐population genetic structure is critical to understanding the evolutionary and demographic forces influencing a population and for making informed management decisions Waples . Whereas. Finally, changes in migration timing could increase variance in reproductive success as a result of natural selection against late‐migrating fish and by increasing density dependence, both of which may act to decrease Ne over time.We hypothesized that changes in migration timing in the Auke Creek pink salmon population could influence intra‐population genetic diversity through several mechanisms. First, loss of divergence between early‐ and late‐migrating fish could arise via genetic admixture as a result of a compressed spawning distribution, and/or as a result of decreased genetic variation due to a strong reduction in the late‐migrating phenotype (i.e. truncation of the migration timing distribution). Alternatively, increases in divergence are possible if genetic drift was increased among late‐migrating fish because of a decrease in the abundance of this phenotypeNe and the Ne to Nc ratio, and (iv); test if Ne and genetic divergence based on migration timing have changed over time. These objectives clarify how genetic diversity is distributed in salmonid populations and directly test our hypothesis that shifts in migration timing can alter patterns of intra‐population genetic diversity.Specifically, we addressed four objectives related to the genetic diversity of Aukc Creek pink salmon: (i) describe patterns of genetic divergence and temporal autocorrelation in allele frequencies due to variation in migration timing, (ii) determine if circadian rhythm genes appear to be related to migratory timing, (iii) estimate Nc = 1548 (1995) to Nc = 28 127 (1999), but the population is stable and population growth rate is at the replacement level  are located within the Clock and Cryptochrome circadian rhythm genes that that are correlated with migration timing and development rate in several salmonid species and populations  or from recent (<24 h) carcasses (1993). Genetic samples were collected from 10 fish every other day so that 170–192 fish were genotyped in each year  for the allozymes, we calculated G”ST manually and obtained 95% confidence intervals by bootstrapping over loci in the ‘boot’ package in Program R  within the overall migration timing distribution. For each year we used the admixture model, no prior information about population of origin, 100 000 iterations of burn‐in, and 500 000 samples from the posterior distribution to estimate the likelihood of K given the data. We considered K = 1–6 and averaged the log‐likelihood based on four iterations of the MCMC chain.Population genetic structure of pink salmon within Auke Creek may exist along a gradient of time (isolation by time) or bimodally/multimodally (i.e. an early and late migrating population). To test for distinct population groupings we used the program STRUCTURE  between early‐ and late‐migrating fish at any particular locus differed from a null distribution of FST generated from the empirical data assuming an island model of gene flow between early‐ and late‐migrating fish.To determine if the circadian rhythm loci Ne was estimated from the temporal variance in allele frequencies across samples (FTEMP and MLNe), linkage disequilibrium within a sample (LDNe), and approximate Bayesian computation based on summary statistics estimated from single samples (ONeSAMP). We used multiple approaches because each method makes different use of the data and in some cases estimates conceptually different values (inbreeding versus variance effective population sizes), thereby providing a more robust understanding of Ne and the Ne/Nc ratio . NEESTIMATOR 1.3 was used to estimate Ne with the FTEMP approach , except MLNe also makes use of the 2001 sample for the 1993–2009 estimate of Ne. The upper bound for Ne was 10 000.The s Waples . SamplesNe. We used the program LDNe , 182 (2001), and 178 (2009). This filtered data set was used to estimate Ne for each method .Two single sample approaches were used to estimate h Waples  and repoh Waples . We alsoNe/Nc based on results from ONeSAMP and LDNe, we used the Nc value from the generation prior to the Ne estimate , except for two circadian rhythm genes  that had elevated FIS values in one or more years. Nonetheless, we retained these loci in analyses because we a priori predicted there might be some divergent selection at these genes. Given 23 loci, there were 253 pair‐wise tests for linkage disequilibrium in each year, and by chance we expected to observe 13 significant values (at α < 0.05). In each year, the number of significant estimates was ≤13 . No pairs of loci exhibited significant linkage in all 3 years.We genotyped Auke Creek odd‐year pink salmon at 23 microsatellite loci in 1993, 2001, and 2009. All of the loci conformed to Hardy–Weinberg expectations and/or had G”ST between the earliest and latest migrating fish ranged from −0.002 to 0.011 for data from 1979 to 2009, but bootstrap 95% confidence intervals included 0 in each year for which we had data (Table P < 0.05) positive autocorrelation  between individuals migrating within 4 days of one another  was for the maximum distance class (40 day separation) in 2009. Weak negative autocorrelation was significant (P < 0.05) in 14 of the 20 estimates before a sequential Bonferroni correction for multiple tests, and significant in 7 of 20 after correction (each data set was corrected independently).Intra‐generational estimates of K = 1). Similarly, G‐tests for homogeneity failed to detect genetic divergence (P > 0.05) between quartiles of the migration timing distribution in 1993 (Table G‐tests based on data from 2001 showed significant (P < 0.05) divergence between fish sampled in the first and third quartile of the migration timing distribution into Auke Creek, and between fish sampled in the first and fourth quartiles. In 2009, there was significant genetic divergence between fish from the first and fourth quartiles of the migration distribution, and between fish from the second and fourth quartiles.There was no evidence of population clustering or substructure revealed by STRUCTURE , and in each instance it was one of the loci associated with circadian rhythm genes. However, the outlier loci apparently under directional selection between early‐ and late‐migrating individuals differed between temporal samples  in 1993 and Cry2b (FST = 0.010) and Cry3 (FST = 0.019) in 2009, while no locus was found in this category in 2001. Interestingly, the largest FST value in 1993 was for OtsClock1b (FST = 0.017), but this value did not exceed neutral expectation. With 69 FST estimates , we would anticipate approximately three false positives (69 × 0.05) at α = 0.05, so these results should be interpreted with caution. Nevertheless, it is notable that the only loci that demonstrated outlier behavior were the circadian rhythm genes that we considered a priori to be candidates for natural selection.Across all years, allelic richness for G‐test for divergence between the first and fourth quartile in 2009 became non‐significant (P = 0.19). All other results were qualitatively identical.Because there were no consistent signals for selection at the circadian rhythm genes, nor evidence that allele lengths were related to migratory timing, estimates of genetic divergence used data at the candidate loci. However, removing the candidate loci resulted in just one fewer significant autocorrelation value , and the Ne based on FTEMP ranged from 1079 to 3788 depending on the time period of interest, and all lower confidence/credible intervals exceeded 788  but confidence intervals in all years included infinity. ONeSAMP estimated Ne based on data only from 1993 (Ne = 1256 CI: 788–2644). Across all methods, all Ne estimates exceeded 1000, and 5 of the 9 non‐infinite estimates were between 1256 and 2006. Ne/Nc ratios obtained from ONeSAMP, LDNe, MLNe and FTEMP varied widely from 0.09 to 1.35 across the time periods considered  than that for 2001 (r = 0.0125 CI: 0.0095–0.0154), and the 95% confidence intervals for 2009 barely overlapped (r = 0.0125 CI: 0.0091–0.0160) with the estimate from 1993, suggesting that positive autocorrelation for fish migrating within 4 days of one another was weaker in 1993. Additionally, three of the non‐overlapping estimates were higher than expected positive values of the autocorrelation coefficient  for which we have no biological explanation. The weaker patterns of divergence in 1993 were also evident from the G‐tests for differences in allele frequencies between groups of individuals sampled in different quartiles of the migration timing distribution .The 95% confidence intervals for Ne and the Ne/Nc were quite large and generally greater than values observed in other populations, but were hampered with imprecision. Despite rapid changes toward earlier migration timing and loss of phenotypic variation, patterns of within‐population genetic divergence based on allozyme  and microsatellite data  have remained relatively stable. Similarly, the genetic effective population size appears stable from 1993 to 2009. Below, we provide potential explanations for these observations and discuss their implication for the management and conservation of genetic diversity.In this study we used temporal genetic data for a pink salmon population to test for genetic differences between early‐ and late‐migrating fish, to determine whether circadian rhythm genes appear to be related to migration timing, and to estimate the genetic effective population size and the ratio between the genetic effective population size and abundance. We used our temporal data to test our hypothesis that rapid changes in migration timing in this population have altered patterns of genetic diversity. Similar to what has been found in other salmonid populations, we observed that Auke Creek pink salmon demonstrate genetic differences between fish that migrate into freshwater at different times. However, the magnitude of divergence was small and did not result in any distinct population grouping based on allele frequencies. Circadian rhythm genes were the only loci that showed any evidence of divergent selection between early‐ and late‐migrating fish, but patterns of selection were inconsistent across years. Across all years, our estimates of Kovach et al.  noted thpersonal observation).Alternatively, it may be more difficult to detect subtle divergence in some years than in others. For example, intra‐annual environmental variation (e.g. stream flow) may cause individuals from different portions of the migration timing distribution to migrate earlier or later, resulting in overlaps in migration timing and a mixture of early‐ and late‐spawning fish in the genetic samples collected. This possibility is supported by the fact the number of days over which fish migrated into Auke Creek in 1993 was the lowest on record for this population. Alternatively, biological phenomena such as strong assortative mating, and/or reduced fitness of progeny from mating events between individuals with different migration timing are acting within this population. Therefore, sampling multiple generations may be required to detect genetic divergence based on variation in migration/reproductive timing. Another explanation is that migration from outside populations has helped re‐establish the genetic differentiation between early‐ and late‐migrating fish in this population (i.e. late‐migrating fish are from different populations). This possibility seems unlikely because gene flow and migration between Auke Creek and other nearby populations is relatively low  and the allozyme data in previous studies . Therefore, it is possible that we failed to detect very subtle temporal changes in population structure because we failed to detect any genetic divergence with G”ST. Unfortunately, this was the best method available to compare divergence between the allozyme allele frequency data from 1979, 1981, and 1983, and the microsatellite data we collected from samples in 1993, 2001, and 2009.Interestingly, no temporal changes were detected in estimates of Cryptochrome gene (Cry2b) exceeded neutral expectation in two of the 3 years for which we had data, indicating that this gene may be associated with or linked to genes that influence migration timing. Importantly, genetic variation was extremely low in the polyQ repeat region of the Clock gene (OtsClock1b) and Cry2b  of any of these loci . While this strongly suggests that Ne has not rapidly decreased in this population (Antao et al. Ne for this population is limited by low statistical power at these effective sizes (i.e. >1000; Palstra et al. Ne may be due to the decline in the late‐migrating phenotype and therefore a decrease in the number of redds of early‐spawning fish that are destroyed by late‐spawning fish. Alternatively, genetic compensation (e.g. Ardren and Kapuscinski Ne; though there are fewer late spawning fish, the variability in their reproductive variance may be diminished because of reduced competition during spawning. Indeed, genetic compensation is common in salmonid fish (Palstra and Ruzzante Contrary to our prediction that Understanding the factors that limit or decrease genetic diversity within populations will improve our understanding of adaptive potential and, therefore, persistence in the face of climate change Frankham . In salm10.5061/dryad.p2m22.Data deposited in the Dryad repository: doi:


Poa pratensis is one of the most common species of meadow grass in Europe. Most cultivars of the species found in Poland were originally derived from its ecotypes. We compared the effectiveness of the RAPD and ISSR methods in assessing the genetic diversity of the selected populations of P. pratensis. We examined whether these methods could be useful for detecting a possible link between the geographical origin of a given population and its assessed genetic variation.The molecular markers RAPD and ISSR were used and their efficiency compared using, inter alia, statistical multivariate methods (UPGMA and PCA).The low value of Dice’s coefficient (0.369) along with the significantly high percentage of polymorphic products indicates a substantial degree of genetic diversity among the studied populations. Our results found a correlation between the geographical origin of the studied populations and their genetic variations. For ISSR, which proved to be the more effective method in that respect, we selected primers with the greatest differentiating powers correlating to geographical origin.P. pratensis originating from different regions of Central Europe with different terrain structures and habitat conditions can be a source of great genetic variability.The populations evaluated in this study were characterized by a high genetic diversity. This seems to confirm the hypothesis that ecotypes of  Poa pratensis L. is one of the most common species of meadow grass in Europe. It occurs throughout the country and is highly valued as a grass cultivar that can be used for various purposes  were included in our research and subjected to the same analyses. All the plant material was evaluated for genetic diversity using two systems of markers based on DNA amplification (RAPD and ISSR). In the present study, the effectiveness of the methods in assessing the genetic diversity of the selected forms of P. pratensis was compared. Furthermore, we investigated whether these methods could be useful in establishing a link between the geographical origin of a given population and their assessed genetic variability. Primers with the greatest differentiating powers correlating with geographical distance were selected for ISSR, the more effective method in that respect. Principal Component Analysis (PCA) was used for this procedure, which was performed on the chosen values of DNA amplification products obtained in the presence of those selected ISSR markers with the highest genetic differentiating power with respect to the studied forms of P. pratensis.purposes . Due to ifficult . In addiifficult . As a reifficult . This imecotypes . The popP. pratensis was carried out in 2014. The study involved 3 cultivars and 15 ecotypes selected from locations as distant as possible from each other. The seeds used in the study came from the national collection of the Botanical Garden of the Institute of Plant Breeding and Acclimatization in Bydgoszcz (IHAR); the caryopses used for establishing this collection were collected during field trips organized by the staff of the Botanical Garden and originated from different geographical regions of Poland, including 8 different voivodships (provinces) (This assessment of the genetic diversity of 18 selected forms of ovinces) . The maiP. pratensis 100–150 mg of material was taken from 80 random etiolated seedlings. The DNA was isolated in two repetitions.Genomic DNA was isolated from the plant tissue using magnetic beads (Novabeads Plant DNA STANDARD Purification KIT), following the procedure for monocotyledons and the manufacturer’s instructions . For each form of 2+ , 1.5 mM MgCl2, 0.2 mM dNTPs, 1.0 µM primer, 1.25 U RedAllegro Taq Polymerase  and 125 ng of DNA.RAPD analysis, based on random amplification of polymorphic DNA, was performed according to the modified method described by 2+ , 1.5 mM MgCl2, 0.25 mM dNTP, 0.4 µM of primer 1 U RedAllegro Taq Polymerase  and 50 ng of DNA.ISSR analysis was performed following the modified method described by The amplification reactions were carried out in a T100™ Thermal Cycler (Bio-Rad Polska) for 40 cycles. The ISSR PCR reaction was as follows: initial denaturation at 94 °C for 4 min, then 94 °C—30 s, 52 °C—45 s, 72 °C—2 min, with a final elongation carried out at 72 °C for 7 min.P. pratensis was determined in accordance with Dice’s coefficient ; then the determined genetic similarity matrix was used to construct UPGMA dendrograms in FreeTree and TreeView 1.6.6 software  in a TAE buffer for 4–5 h. Visualization, documentation and analysis of the results were carried out using Gel Doc™ XR+ and Quantity One 4.6.5 software manufactured by Bio-Rad. The Polymorphism Information Content (PIC) index for dominant primer systems was calculated for both RAPD and ISSR . The Assfficient  followinsoftware . To analP. pratensis. Of these, the largest number of DNA fragments (16) which had a unique composition were obtained in reaction with the M14 primer  was obtained in reaction with primer 808, while in the presence of primer 807 only one product with a unique composition (649 bp) for ecotype lbs05 170 was obtained  up to 3,105 bp (for 808) were obtained . In reacobtained . In totaP. pratensis. Independently of each other, the RAPD and ISSR methods revealed little genetic relationship between the studied populations and cultivars , which was carried out on the geographical coordinates of the habitats from which the surveyed populations had come. P. pratensis in the presence of ISSR microsatellites 807, 808, 834 and 840; these were the dinucleotide repeat sequences (AG)n (GA)n, anchored at the end of 3′. The studied populations were distributed on both sides of the axis of coordinates and distinctly away from its center. They formed five separate groups, which indicates their considerable diversity. Three populations from Podlasie , and ecotypes from West Pomerania (2) and Kujawy regions (1) together with the lawn cultivar ‘Limousine’ (17), formed two clearly distinguishable groups ) which came from the borderlands of the Lower Silesian and Lubuskie voivodships respectively. Populations from Silesia (10) and Lesser Poland (11) were also contained in this group. On the same side of the horizontal axis we find another group containing the fodder cultivar ‘Eska 46’ (18) and three ecotypes  from the Lubuskie, Lower Silesian and Silesian voivodships respectively. In the course of the principal component analysis, two forms of P. pratensis were identified (12 and 16 (‘Alicia’)), which were not included in any of the groups. As with the UPGMA dendrograms, the population from Podkarpacie (bie04 189) differed distinctly from the other ecotypes and constituted a distinct genotype 2 and (AC)2, anchored at the end of 3’. As suggested by n, (CTC)n and (GA)n and (CA)n are particularly abundant. In the genus Poa, ISSR primers with dinucleotide (AC)n (CA)n (GA)n and (AG)n repeats turned out to be particularly useful for the identification and evaluation of genetic relationships between various species, including P. angustifolia, P. trivialis, P. arachnifera, P. pratensis, P. compressa, P. ligularis, and P. secunda and their hybrids , for which they determined correlations. In the case of RAPD and ISSR, the Spearman’s coefficient (0.461) calculated by Budak was similar to ours, but their results turned out to be statistically insignificant. Both RAPD and ISSR markers revealed a small genetic similarity among the studied ecotypes and cultivars of P. pratensis. The average value of Dice’s coefficient for both methods combined (RAPD + ISSR) was low (0.369), which together with the high number of polymorphic products may indicate substantial genetic differences between the studied populations and their low level of relatedness. It can be speculated that natural spatial barriers might have inhibited and minimized the transfer of genes among these populations; this would certainly have contributed to their diversity. At the same time, we can surmise that there were populations among the studied ecotypes in which apomixis was the main mode of reproduction. The ecotypes originating from three voivodships ), were determined to be those genetically most distant from the other forms. These populations had the lowest similarity index and included the most uncommon genotypes, for which the largest number of specific amplification products were identified. In comparison to the other cultivars, ‘Eska 46’ was clearly different in terms of its genetic structure, and, overall, few amplification products were generated for it. It should be stressed that in previous studies aimed at evaluating RAPD polymorphism in different populations of P. pratensis, such a high genetic diversity among ecotypes had never been found, while the average Dice’s coefficient was about 0.7  or from regions located not far from one another were depicted similarly in two-dimensional space as in the UPGMA dendrogram constructed for the ISSR process. Those populations from eastern Poland and from West Pomerania and Kujawy clearly differed from the ecotypes originating from the western and southern parts of the country and were located on the opposite side of the horizontal axis of coordinates. They also differed from one another, which can be seen in their arrangement on both sides of the vertical axis. Some discrepancies were found between the UPGMA and PCA clusters of the studied forms, which were related, inter alia, to the distribution of the cultivars. Only a modest relationship between genetic divergence and geographical origin in P. pratensis was reported by several authors , ISSR markers seem to be a more useful and reliable tool for this kind of analysis. The plant material evaluated in this study was characterized by a high genetic variability, which seems to confirm the hypothesis that ecotypes of P. pratensis originating from different parts of Central Europe, each with different terrain structures and habitat conditions, can provide great genetic variability and a large collection of alleles. Microsatellites with dinucleotide repeat sequences (AG)n, (GA)n anchored at the end of 3’ proved particularly useful for assessing the genetic diversity of these ecotypes and determining their main directions of variability. PCA helped to establish a correlation between the geographical origin of the studied ecotypes and their genetic variation.Our results suggest that both the systems of dominant RAPD and ISSR markers that were used in our research can be used to assess the genetic variability of selected ecotypes and cultivars of 10.7717/peerj.2489/supp-1Dats S1P. pratensis; 1—band, 0—no band (b) The distribution of amplification products for ISSR primers; 1—band, 0—no band (c) The distribution of amplification products for RAPD primers; 1—band, 0—no band (d) Genetic Similarity Index (Si) of the studied populations of P. pratensis for ISSR markers. (e) Genetic Similarity Index (Si) of the studied populations of P. pratensis for RAPD markers. (f) Genetic Similarity Index (Si) of the studied populations of P. pratensis for RAPD and ISSR methods. (g) The number and size of the selected amplification products obtained in the presence of each ISSR primer, was used as the initial data for PCA.(a) The distribution of amplification products for RAPD and ISSR primers, differing of the studied ecotypes and cultivars of Click here for additional data file.


Orientia tsutsugamushi, which is endemic to several Asia-Pacific Rim countries, including Japan. Although micro-indirect immunofluorescent assay (micro-IFA) is the standard method for the serological diagnosis of scrub typhus, enzyme-linked immunosorbent assay (ELISA) is considered to be more objective, by providing digitized results as opposed to being subject to the judgment of the evaluator as in micro-IFA. Therefore, the aim of this study was to develop a broad-ranging ELISA using the five major prevalent serotypes of O. tsutsugamushi in Japan as the antigens. Furthermore, in contrast to previous studies that used purified microorganisms via ultracentrifugation, we directly used the infected cells, and evaluated the diagnostic accuracy of this simplified method to that of micro-IFA.Scrub typhus is a mite-borne rickettsiosis caused by infection of Evaluation of paired patient sera against the five serotypes showed that the accuracy of ELISA relative to micro-IFA was 87.4 and 79.5% for immunoglobulin (Ig)M and IgG assays, respectively, at the optimized cut-off value. Further evaluation of patient sera against the expected serotype of the infecting strain showed that the accuracy of ELISA compared to micro-IFA increased to 100 and 97.4% in the IgM and IgG assays, respectively. This suggests that use of the five prevalent serotypes contributed to the increase of the accuracy of ELISA. When applying the criteria of serological diagnosis for paired sera samples to ELISA, all 19 patients were diagnosed as positive; a ≥4-fold elevation of the antibody titer was observed in 15 of 19 patients that were positive, and very high antibody titers were observed in both paired sera samples of the remaining four patients. In addition, all samples of healthy subjects and patients with other types of rickettsiosis were diagnosed as negative using these criteria.Our results suggest the excellent performance of the new broad-ranging and convenient ELISA, which appears to be applicable for the diagnosis of scrub typhus patients infected with the wide variety of prevalent strains in Japan. Furthermore, the ELISA is more objective than the micro-IFA, and can therefore provide more accurate diagnoses in Japan.The online version of this article (doi:10.1186/s12866-016-0910-5) contains supplementary material, which is available to authorized users. Orientia tsutsugamushi [Scrub typhus is a mite-borne rickettsiosis caused by the intracellular bacterium ugamushi . The micugamushi , where augamushi .O. tsutsugamushi in Japan, such as the Kato, Karp, Gilliam, Kuroki [Serological diagnosis is the main method for confirmation of scrub typhus, because direct detection of the microorganisms is a generally difficult and cumbersome process. For example, isolation of microorganisms requires biosafety level-3 facilities and is a lengthy procedure, and bacterial DNA detection from the blood using polymerase chain reaction (PCR)-based methods is unreliable, as negative results are common, especially once antibiotic treatments have been initiated. Recently, DNA detection from the eschar was shown to be useful ; however, Kuroki , and Kaw, Kuroki  types, w, Kuroki . That is, Kuroki , 9.The micro-indirect immunofluorescent assay (micro-IFA) , 11 is cHowever, micro-IFA is associated with a major disadvantage, in that the end point of interpretation of the antibody titer can vary for the same sample depending on the evaluation of the individual conducting of the serological test according to their ability and/or experience , 13. TheO. tsutsugamushi in Japan. In addition to the objectivity advantage, this method has additional convenience, since previous methods for ELISA for rickettsia have involved a purification step of the sample by ultracentrifugation as the antigen [In this study, we developed a broad-ranging and simplified ELISA directly using infected cells of the five major prevalent serotypes of  antigen , 9, 14. O. tsutsugamushi, Kato, Karp, Gilliam, Kuroki, and Kawasaki type, were used. Kato, Karp, and Gilliam are international standard strains, whereas Kuroki and Kawasaki are unique isolates in Japan. Diversity of the five strains that was based on the amino acid sequences of the type specific antigens (56 kDa major outer membrane proteins) was shown in Additional file 7 cells from a 75-cm2 flask/ml). The cell lysate was mixed well by vortexing, and then the supernatant was collected after spinning at 10,000 rpm  at 4 °C. The numbers of microorganisms in each cell lysate were quantified by real-time PCR as previously described [w/v) (PBS-TM) was added to each well, and the plate was incubated for 4 h at room temperature for blocking. After washing with 200 μl of PBS-T three more times, the antigen plate was stored at −80 °C until use.Five different serotypes of escribed . AccordiN = 38) and 18 paired sera samples of healthy subjects (N = 36) were used as the positive and negative sera samples, respectively, to determine the cut-off value of ELISA. In addition, 12 paired sera samples of patients with other types of rickettsiosis, including 9 pairs of sera samples of patients with Japanese spotted fever (N = 18) and three pairs of sera samples of patients with murine typhus (N = 6) were used to evaluate the specificity of ELISA. These serum samples were collected from 2000 to 2012 and sent to our laboratory for serological tests of scrub typhus and other rickettsiosis. This study was approved by the Ethics Committee for Medical and Health Research involving Human Subjects of the National Institute of Infectious Disease .Nineteen paired sera samples of scrub typhus patients (O. tsutsugamushi and mock (the lysate of non-infected cells)  of each well was determined by measuring absorbance in a spectrophotometer . The ELISA value of each serum sample was calculated by subtracting the OD405 against the mock well from the OD405 against each serotype.The serum was diluted 4-fold serially from 1:100 to 1:6400 with PBS-TM, and 100 μl of each diluted serum sample was applied to two wells (for IgM and IgG) of each serotype of A previously reported micro-IFA method  was modiThe mean ELISA value + 2, 3, and 4 standard deviations (SD) of the 18 pairs of sera from healthy subjects were applied as cut-off values to find the most suitable value, whereas for micro-IFA, the common cut off value of ≥1:80 that is routinely used in Japan was applied. Serological diagnosis was judged using the temporal criteria of ELISA of an IgM value > the mean + 3 SD for a single serum sample and/or ≥4-fold elevation of ELISA antibody titers for the paired sera . Micro-IFA results were judged according to the standard criteria of IgM ≥1:80 for a single serum sample and/or ≥4-fold elevation of micro-IFA titers for paired sera. The results of ELISA and micro-IFA were compared with non-linear regression analysis. All statistical analyses were conducted with Excel software .To evaluate the performance of the ELISA developed in this study, the ELISA value at the serum dilution of 1:100 was compared with the determined micro-IFA titer  values of the healthy sera for the IgM and IgG assays were 0.0010 (0.0593) and 0.0279 (0.0614), respectively. The ELISA values and micro-IFA titers of the 19 paired sera samples of scrub typhus patients against the five serotypes, and their concordance are presented in Fig. The expected serotype of the strain that infected the patients in this study was defined as the serotype against which the sera of each patient showed the highest titer. ELISA values and micro-IFA titers of the 19 paired serum samples of scrub typhus patients against only the expected infecting strain are shown in Fig. The specificity of ELISA was examined by comparing the ELISA values and micro-IFA titers from paired serum samples of Japanese patients with other types of rickettsiosis, and the results are shown in Fig. The serological diagnoses of the 19 patients according to the above-mentioned criteria are shown in Table Furthermore, the second serum samples of one healthy subject and one patient with Japanese spotted fever, and the first serum sample of a patient with murine typhus were positive according to the criteria of a single serum sample. Their IgM ELISA values were very close to the cut-off value, although these cases were all diagnosed as negative according to the criteria of paired sera (data not shown).In Japan, micro-IFA is the standard serological diagnostic method for scrub typhus. However, the interpretation of this assay is subjective and requires a highly trained person to accurately determine the precise end point. Therefore, the end point for the same sample can be discordant depending on the individual conducting the test, especially when the fluorescent background is high. This fact sometimes leads to false-positive results in serological diagnosis. In addition, the antigen slide used for micro-IFA is generally hand-made with a pen cap , 11, andO. tsutsugamushi in Japan  was established. The digitized ELISA value was obtained by subtracting the absorbance of the mock well (lysate of non-infected cells) from the absorbance of the well with each serotype. Therefore, even when the background is high, only the specific absorbance of each serotype can be effectively evaluated with this method. For evaluation of the ELISA cut-off value, the reaction of each serum sample against the strains comprising the five serotypes was examined. Even when using the optimized cut-off value, the mean + 3 SD, some discrepancies in the results of ELISA and micro-IFA were observed. These discrepancies were most likely due to differences in the extent of serological cross-reactions against the five serotypes among patients. These differences can be caused by variations in the serotype of the infecting strain and individual immune reactions, as well as the different nature of the adopted antigens, since the infected cells themselves were used for micro-IFA, whereas the lysates of the infected cells were used for ELISA. This suggests that the conformational epitope of rickettsia may be slightly but not drastically changed in the lysate of infected cells.One of the most important factors for establishment of a serological diagnostic method is to confirm the match between the antigen used and the expected infecting strain. In the case of scrub typhus, our previous report showed that the serum antibody level of some patients was only elevated against limited serotypes, whereas that of most patients was elevated against all serotypes . In someTo solve these discrepancies, we also evaluated the results against only the expected serotype of the infecting strain, which was that showing the highest ELISA value in each patient. Non-linear regression analysis showed that the correlations between ELISA values and micro-IFA titers were higher when using the expected infecting serotype compared to those obtained against all five serotypes in the IgG assays, whereas the correlations were similar with the IgM assays. Furthermore, using the optimized cut-off value, the specificity and sensitivity of the ELISA results were relatively consistent with those of the micro-IFA results for both the IgM and IgG assays. These findings strongly suggest that the performance of ELISA was as high as that of micro-IFA. These results also suggested that the serotype that is most similar to the actual infecting strain should be used to obtain high specificity and sensitivity, and that adoption of the most prevalent plural serotypes for antigens is important to obtain the best serotype match between the antigen and infecting strain.In general, for the criteria of serological diagnosis, it is recommended that an elevation of ≥4-fold between paired sera should be observed, especially when the antibody titer is close to the cut-off value used as the criterion for a single serum sample. As similar criteria used for micro-IFA were applied to ELISA, all 19 of the scrub typhus patients were diagnosed as positive. Furthermore, all of the healthy subjects and patients with other types of rickettsiosis reported in Japan , 16–18 wO. tsutsugamushi were used, which is in contrast to previous studies that generally used purified microorganisms as the rickettsial antigens [Another advantage of this study is that cells infected with antigens , 19. An In this study, we established a broad-ranging and convenient ELISA for the diagnosis of scrub typhus in Japan using five different serotypes of antigens, which appears to be suitable for the diagnosis of scrub typhus patients infected with the wide variety of strains circulating in Japan. The specificity, sensitivity, and practicality of the broad-ranging ELISA were as excellent as the gold-standard serological diagnostic method, micro-IFA. In addition, judgment of the results in ELISA is more objective than that of IFA, mainly because a homogeneous and stable quality of the antigen is coated on the plates and digitized results are obtained in ELISA. These facts clearly suggest that the ELISA developed in this study will be more accurate than micro-IFA for the serological diagnosis of scrub typhus in Japan. Furthermore, the ELISA has a great advantage for serological screening in general epidemiological research owing to its ability to handle several sera samples simultaneously.


Lepus timidus) and brown hare (Lepus europaeus) hybridize and produce fertile offspring, resulting in gene flow across the species barrier. Analyses of maternally inherited mitochondrial DNA (mtDNA) show that introgression occur frequently, but unavailability of appropriate nuclear DNA markers has made it difficult to evaluate the scale- and significance for the species. The extent of introgression has become important as the brown hare is continuously expanding its range northward, at the apparent expense of the mountain hare, raising concerns about possible competition. We report here, based on analysis of 6833 SNP markers, that the introgression is highly asymmetrical in the direction of gene flow from mountain hare to brown hare, and that the levels of nuclear gene introgression are independent of mtDNA introgression. While it is possible that brown hares obtain locally adapted alleles from the resident mountain hares, the low levels of mountain hare alleles among allopatric brown hares suggest that hybridization is driven by stochastic processes. Interspecific geneflow with the brown hare is unlikely to have major impacts on mountain hare in Fennoscandia, but direct competition may.In Fennoscandia, mountain hare ( Species boundaries are frequently challenged by lineage divergence and hybridization . DivergeAs the genetic variation provided by hybridization is exposed to selection, hybrids experience an increased, decreased or neutral selection effect, and the effect of this selection may vary in direction and strength across the genome and context ,2,10. ThFrom a conservation perspective, introgression create challenges for management efforts, typically by blurring the species barrier , affectiLepus europaeus Pallas) and mountain hare (Lepus timidus L.). Benefitted by translocations and, potentially, climate change, the brown hare has been extending its distribution northwards since the 19th century. In contrast, mountain hare populations have been in decline in Finland and southern Sweden [Lepus is driven by demographic processes [In Europe, northern Fennoscandia represents the northernmost contact zone between brown hare  at the expense of a resident species (mountain hare). As ecological change forces species to adapt by changes in distribution or behavior, the demographic and genetic consequences of increased levels of sympatry and hybridization are important to consider for management.Here we analyze 6833 SNP markers across the genome to quantify introgression among Finnish- and Swedish mountain hares and brown hares. We show introgression of mountain hare markers into brown hare, but not ® 100 (Bio-Rad) method [For the genome-wide SNP analysis, DNA was isolated from 22 mountain hares and 27 brown hares (muscle from the base of the ear), including specimens with introgressed mtDNA and representing allopatric as well as sympatric populations almost throughout the full range of the species’ distribution in Sweden and Finland , Fig 1. ) method , followiCytb) fragment using polymerase chain reaction (PCR) and LCYTBF and LCYTBR primers [AluI restriction enzyme and run over a 3% high resolution agarose gel electrophoresis to reveal restriction fragment lengths and the subsequent genotype [Mitochondrial DNA was genotyped from all samples by amplifying a 669 bp cytochrome b . Analyses of population structure and -admixture were performed using 6833 polymorphic loci informative for both hare species (10.5061/dryad.n70q6). Basic population genetic analyses, such as computing allele frequencies and testing for Hardy-Weinberg equilibrium, were performed using Arlequin 3.5. [https://bioconductor.org/biocLite.R. Genetic diversity in the hare species was assessed using STRUCTURE 2.3.4 [K), three iterations with 1 million MCMC repetitions for burnin period of 500,000 [K or the best number of populations was obtained using STRUCTURE HARVESTER [199,693 SNPs were analyzed using the commercially available GeneChip species . The genuin 3.5. . The PCARE 2.3.4 ,36, usin 500,000 . Samplin 500,000 . K or thARVESTER , implemeL. timidus to L. europaeus at average level of 2% in regions of sympatry but being almost absent in allopatric populations. In total, 62,730 SNP genotypes had 100% call rate for the 49 samples and 6833 of these were polymorphic  generations to make the diverging species monophyletic at more than 95% of the loci during allopatric speciation [The genotyping data enabled us to detect highly asymmetrical introgression of nuclear genes from ymorphic  Files. Aand rats , which sand rats . Althougeciation . We idenAlthough the small number of polymorphic loci, ambiguity of chromosomal locations and relatively low levels of minor alleles as well as mean heterozygosity, resulting from using across species SNP array, disallowe more detailed population genetic analyses, the 6833 loci do pick up differences between the two hare species and work as a proxy to compare genetic diversity in Sweden and Finland. Interestingly, the Swedish brown hares show higher heterozygosity levels and minor allele frequencies than their Finnish counterparts, whereas the mountain hares from the two countries do not differ . The higth century and subsequently expanded to southern and central Sweden [It is likely that the overall higher heterozygosity in the brown hares compared to mountain hares does not only result from the biased gene flow from mountain hares to brown hares. For example, the country-specific differences in the genetic diversity among brown hares can be explained by the differential colonization histories in the two countries. Brown hares were first introduced to Sweden in 1858 to the island of Ven in the Öresund straight between Sweden and Denmark . After tl Sweden . The brol Sweden .th century in association with a general, northeastward expansion of brown hares noted by Thenius [Swedish brown hares show high mtDNA haplotype diversity, likely due to admixture due to repeated introductions of brown hares from several geographical areas of the native  range of brown hares . This hi Thenius . In FinlThus, the genotypic differences in SNPs observed between Swedish and Finnish brown hares in the current study, may reflect the differences in the population history of the recurrent populations. The composition of the Swedish brown hare gene pool is a result of admixture of hares from different areas in continental Europe  along wiL. europaeus background cannot be reliably estimated as retention of ancestral polymorphism because of incomplete lineage sorting (ILS) and secondary gene flow through introgression, produce very similar patterns of shared genetic diversity between two species [Population structure analysis enabled us to identify species-specific genetic markers and confirm introgression between mountain hares and brown hares. Based on the NEWHYBRID analysis of the species-specific markers, introgression was highly asymmetric, with brown hares being classed as backcrosses whereas all mountain hares included in the study were ranked as purebred . The ave species ,51. For  species ,29,33,52L. europaeus mtDNA represented 0.56 timidus and 0.44 europaeus share of ancestral populations in STRUCTURE analysis  and, more specifically, it is the first such study focused on mountain hares and brown hares in Fennoscandia. While it is clear that the next generation methods, such as Double Digest Restriction Associated DNA (ddRAD) sequencing , provideL. timidus to L. europaeus in Fennoscandia, as previously reported for mtDNA [L. granatensis specimens, assessing the patterns of the historical introgression events [ad hoc comparisons of genomes become the standard practice in population genetics in the near future.Our results provide support for the preferential introgression of nuclear genes from or mtDNA ,22,49. Bor mtDNA ,49. Contn events , our stun events , it is pn events ,56, muscn events , diet spn events  and immun events . At presAlthough brown hare may outcompete mountain hare under certain conditions ,60, the S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 FileSpecies on separate spreadsheets.(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.