Dactylis glomerata (orchardgrass or cocksfoot) is a forage crop of agronomic importance comprising high phenotypic plasticity and variability. Although the genus Dactylis has been studied quite well within the past century, little is known about the genetic diversity and population patterns of natural populations from geographically distinct grassland regions in Europe. The objectives of this study were to test the ploidy level of 59 natural and semi-natural populations of D. glomerata, to investigate genetic diversity, differentiation patterns within and among the three geographic regions, and to evaluate selected populations for their value as genetic resources.D. glomerata, exclusively tetraploid individuals were identified based on 29 SSR markers. The average expected heterozygosity  ranged from 0.44 to 0.59 and was highest in the Norwegian region. The total number of rare alleles was high, accounting for 59.9% of the amplified alleles. 80.82% of the investigated individuals could be assigned to their respective geographic region based on allele frequencies. Average genetic distances were low despite large geographic distances and ranged from D = 0.09 to 0.29 among populations.Among 1861 plants from 20 Swiss, 20 Bulgarian and 19 Norwegian populations of D. glomerata within selected populations and numerous rare and localized alleles which were geographically unique. The large, permanent grassland patches in Bulgaria provided a high genetic diversity, while fragmented, semi-natural grassland in the Norwegian region provided a high amount of rare, localized alleles, which have to be considered in conservation and breeding strategies. Therefore, the selected grassland populations investigated conserve a large pool of genetic resources and provide valuable sources for forage crop breeding programs.All three case study regions revealed high genetic variability of tetraploid  Dactylis glomerata L. (orchardgrass or cocksfoot), a long-lived and perennial grassland species is the fourth most important forage grass in the world [D. glomerata is used for hay or silage production and grazing worldwide. Continuous outcrossing by wind-pollination, natural selection and adaptation processes have resulted in a wide geographic range and large morphological variability [D. glomerata has a genome size of 4312 Mbp and comprises diploid (2n = 2× = 14), tetraploid (2n = 4× = 28) and hexaploid (2n = 6× = 42) accessions [D. glomerata are the most widespread [D. glomerata are rare and restricted to certain areas, e.g. Libya, Egypt or Spain [he world . Its ecohe world . Due to iability . D. glomcessions . Within oid 2n = × = 42 acdespread . Compareor Spain . Tetraplor Spain .D. glomerata has led to a widespread occurrence in natural and semi-natural grassland across Europe. Natural populations of D. glomerata are of major importance for forage crop breeding. In natural and semi-natural grasslands, those populations harbor high genetic diversity, which provides advantages for future breeding and conservation programs in particular with respect to climatic changes and an increasing demand for forage and food production [D. glomerata, which could be sources for genetically diverse material, is rather scarce. Investigation of genetic patterns in natural or semi-natural grassland populations may not only reveal fundamental knowledge on population genetic structures, but may also support the evaluation and utilization of natural resources with respect to forage crop improvement and in situ conservation. Our recent investigations in Switzerland suggest high genetic diversity within, but low genetic variability among populations in permanent grassland .The high plasticity and heterogeneity of the genome of oduction . Detailein situ protection of forage crop genotypes and populations from genetic erosion and provide new germplasm for forage crop breeding.Geographically distinct populations can differ in their level of genetic diversity or in the distribution of diversity within and among regions . The valSimple Sequence Repeats (SSRs) are genetic markers consisting of one to six nucleotides occurring in a repeated pattern (tandem repeats). Their high abundance across the genome, neutral and co-dominant inheritance, and highly polymorphic character qualify SSRs as multi-allelic genetic markers for a broad range of applications, e.g., in breeding and crop improvement as well as in population and ecological genetics .D. glomerata populations in Bulgaria, Norway and Switzerland, representing three grassland regions in Europe. The objectives were: (1) to investigate the ploidy level of D. glomerata individuals in three selected grassland regions, (2) to study the genetic diversity and patterns of differentiation within and among populations from different geographical regions, (3) to evaluate the use of geographically distinct regions for the in situ conservation of genetic resources of the grass species D. glomerata.The aim of this study was to investigate the population structure and genetic variability of natural and semi-natural 2, with distances of 0.18 to 47.36 km between sites . Management was characterized by low-input farming of permanent grassland for cattle and sheep based dairy production. The Swiss region (CH) was located in the canton Obwalden, in the Northern Swiss Alps. Ranging from 600 to 1100 m.a.s.l, the 20 selected sampling sites were distributed across an area of 12 km2, with distances of 0.09 to 6.03 km between sites . Farms were dominated by natural, permanent grassland for cattle-based dairy production . The Norwegian region (NO), located in Nord-Østerdal in the north of Hedmark County, covered an area of 4871 km2, ranging from 500 to 1600 m.a.s.l.. The 19 sampling sites, which had not been re-sown for at least 6 years, were located at distances of 0.06 to 46.69 km from one another . Management was characterized by sheep raising and hay production. Seed mixtures applied in the Norwegian sampling sites did not contain D. glomerata cultivars and occurring D. glomerata were considered natural populations. On-farm interviews and questionnaires were used to obtain information about farming systems, on-farm production, management and the potential application of commercial seed mixtures.Sampling sites were located in grassland regions of three European countries. The Bulgarian region (BG) was located in the Smoljan region in the Rhodope Mountains of South Central Bulgaria. At altitudes ranging from 900 to 1400 m.a.s.l, the 20 selected sampling sites were distributed across an area of 3193 kmD. glomerata plants from a total of 59 sampling sites  and 0.2 μM of fluorescently labeled forward primers  and unlabeled reverse primers. PCR was performed using an iCycler  under the following conditions: Initial denaturation at 94°C for 5 min, followed by 12 cycles of 'touchdown’ PCR consisting of 30 s denaturation at 94°C and 1 min annealing between 72°C and 60°C (decreased 1°C at each cycle), 1 min at 60°C, 1 min elongation at 72°C, followed by 25 cycles denaturation for 30 s at 94°C, 1 min at 60°C, 1 min elongation at 72°C and a final extension of 15 min at 72°C. Fragments were sized on a 48 capillary 3730×l DNA Analyzer using POP 7 polymer and the ROX HD400 standard . Fragment analysis was performed at the Genetic Diversity Centre . SSR alleles were automatically binned using GeneMarker® Version 1.95 . All binned peaks were checked for correct assignment to corresponding bands and corrected manually. Samples were randomly arranged for PCR and fragment analysis.Dried plant tissue (30 mg) was ground three times by metal beads at 30 hz using a mill . DNA was extracted using the NucleoSpin® 96 Plant II  extraction kit. Quantity and quality of DNA was assessed by photospectrometry using NanoDrop  and the ND-1000 software. SSR marker analysis was performed in multiplex reactions using 29 primer pairs  among and within regions was calculated based on six independently repeated runs of 100000 iterations (length of burn-in period) followed by 100000 Markov Chain Monte Carlo (MCMC) repetitions after burn-in applying the implemented admixture model and correlated frequencies [K was set from 1 to 8. Within regions, K was set from 1 to 20, (19 in the Norwegian region). The K-value revealing the highest maximum likelihood 'Ln P(D)’ after several independent runs was selected for the assignment of individuals to subpopulations based on their membership probability. Populations in which all individuals had membership probability of ≥ 0.8 were regarded as distinct populations, whereas populations containing individuals with membership probability < 0.8 were considered as admixed [The ploidy level of collected individuals was calculated based on the maximum and mean number of alleles per locus, for all samples and across all loci using the R package “polysat” ,19. As usize HE,C and alleram 1.3a . The totram 1.3a . A multiram 1.3a ,26. AMOVram 1.3a  based onram 1.3a ,28. Mantsu.edu/, ). Signifsu.edu/, . The optquencies . For the admixed . In orde admixed -35.D. glomerata plants, the 29 SSR primers detected 257 polymorphic alleles, varying in size from 73 to 260 bp  across all loci was high in all regions, ranging from 0.44 to 0.59  when compared to the Bulgarian region . There was no significant difference between Switzerland  and the two other regions . Among 1861 P < 0.05) , but not Bulgaria  Table . The mea Table . ia Table . HierarcM = 0.37, P = 0.01) and among the three regions  were identified by testing for isolation by distance  and D = 0.06 (NO-BG), to D = 0.09 (CH-BG). Genetic distances among populations within regions ranged from D = 0.01 to 0.02 (CH), D = 0.009 to 0.05 (NO) and D = 0.01 to 0.21 (BG). The greatest genetic distance between populations from different regions was D = 0.29 for the Swiss population CH04 and the Bulgarian population BG04. Significant correlations between pairwise genetic distances (D) and the corresponding geographical distances between populations within the Norwegian region ) among different tested K values than among repeated runs and was considered as the optimal number of populations  based on 1861 individuals and 257 SSR alleles Figure . The firs Figure a. In totK Figure b - d.D. glomerata sampled from 59 natural and semi-natural populations in three distinct regions of Europe. Although tetraploid and diploid populations can occur in sympatry [D. glomerata have been reported to be most abundant in cultivars and natural populations [Rorippa amphibian or Bromus species in European populations [D. glomerata indicated them to be valuable sources for germplasm collections.This study revealed exclusively tetraploid individuals of sympatry ,7, autotulations , which wulations ,38. BothE,C of D. glomerata within populations from different grassland regions in Europe was comparable with D. glomerata cultivars [Poa alpina, Festuca pratensis or Lolium multiflorum[Lolium perenne from different geographical regions worldwide [D. glomerata populations strongly depends on various life history traits, such as the outbreeding mating system and efficient pollen dispersal by wind [E,C and the lowest mean number of alleles per locus per population within selected populations was detected in the Norwegian region. Since the semi-natural grassland populations did not receive any seed mixtures that included D. glomerata varieties, genotypes from natural and commercial gene pools must have immigrated from outside populations [E,C was high for all populations. As revealed by previous studies on grassland genetic diversity in space and time, habitat age, connectivity and past use in a landscape and historical context have a major impact on current genetic diversity patterns [E,C[D. glomerata populations could then explain these fragmented grassland patches and the relatively high number of rare and localized alleles. Whereas in the Bulgarian region, some populations of D. glomerata revealed high HE,C and others indicated low HE,C-values, lowering the average HE,C across all population, but augmenting the range of HE within the region. There, the landscape provided some large grassland patches with high levels of gene flow and high HE,C as well as single, remote grassland patches with low gene flow due to low connectivity. A lower gene flow into more isolated populations has been revealed for grassland species such as Globularia bisnagarica[E,C and a lower average HE,C across all investigated populations as the Norwegian region. Here, permanent grassland has been established for a long time without sod disturbances, e.g. rotational forage crops, or the introduction of new genetic material, e.g. by re-sowing or extended seed-recruitment. Furthermore, the selected populations were located in a small geographic range  increasing the connectivity and gene flow among populations leading to constant intermixing and, therefore, high HE,C in all D. glomerata populations.The genetic diversity in terms of average expected heterozygosity Hultivars . The genultivars . Howeverltiflorum,41. Simiorldwide . High ge by wind . On averulations . In the patterns . In the erns [E,C. When faerns [E,C. Relativsnagarica. Therefosnagarica. The SwiE,C represents a common measurement for genetic diversity based on allele frequencies, allelic diversity or allelic richness plays a more relevant role for genetic conservation [F. pratensis in Swiss ecotype populations by using SSR markers [in situ conservation and germplasm collections might comprise the potential to increase the quality of grassland cultivars in terms of resilience and persistence in currently unfavorable areas [Although, Hervation . The pre markers . In the  markers ,51. The le areas ,53.D. glomerata and L. multiflorum populations, which investigated populations less than 100 km apart [F. pratensis[D. glomerata populations in Turkey [Although the genetic diversity was high in selected populations and regions, the genetic distance of individuals among populations was low and did not indicate clear distinction of selected populations within regions. These results support previous studies on km apart ,36 (Lastkm apart . The higpratensis. The imppratensis,55. No in Turkey . In contD. glomerata worldwide [D. glomerata between distant regions reflects what has been found in previous studies on isolation-by-distance patterns of Festuca arundinacea investigated within large geographic ranges [The genetic distance that we found among populations from distinct and distant regions in Europe was slightly higher than the genetic distance between populations within regions, and reflects results from population genetic studies among natural populations and cultivars of orldwide . The incc ranges . Althougc ranges ,58. Althc ranges . Accordic ranges .D. glomerata, not only revealed exclusively tetraploid individuals, but high genetic diversity, a high number of rare and geographically unique alleles in geographically distinct populations. The three regions revealed genetically distinct patterns and were differentiated from each other. These populations of D. glomerata might contain valuable sources for plants adapted to specific, but differentiated environmental conditions. Especially the high amount of rare, localized alleles in Norway or the high amount of unique alleles located in Bulgaria may indicate valuable sources for breeding material adapted to climatic and environmental changes in certain regions. To conserve a high amount of genetic diversity large, permanent grassland patches with natural populations of D. glomerata, as represented in Bulgaria, should be considered. Fragmented and smaller grassland patches as represented in the Norwegian region on the other hand, can provide a high amount of rare, localized alleles of D. glomerata. In general, genetic material from distinct geographical regions and multiple populations should be considered for ex situ and in situ conservation.The investigation of 59 natural and semi-natural populations of The authors declare that they have no competing interests.LL collected the plant material for this study in Switzerland and Norway, carried out the molecular analysis using SSRs, performed the genetic data analysis and drafted the manuscript. FW discussed the results and participated in writing the manuscript. WF contributed to the sampling site selection and sample collection in Norway. SS participated in the initial discussion of this project, contributed to the sampling site selection and performed the plant material sampling in Bulgaria. WF and SS participated in writing the manuscript. RK conceived and supervised the project, assisted in the data analysis, discussed the results and contributed to draft the manuscript. All authors read and approved the final manuscript.


Odontesthes, from a non-sampled area in the SW Atlantic Ocean to address species discrimination and to define Managements Units for sustainable conservation. All phylogenetic analyses based on the COI mitochondrial gene were consistent to support the monophyly of the genus Odontesthes and to include O. argentinensis, O. perugiae-humensis and some O. bonariensis haplotypes in a basal polytomy conforming a major derivative clade. Microsatellites data revealed somewhat higher genetic variability values in the O. argentinensis-perugia populations than in O. bonariensis and O. perugia-humensis taxa. Contrasting population genetics structuring emerged from mitochondrial and microsatellites analyses in these taxa. Whereas mitochondrial data supported two major groups (O. argentinensis-perugia-humensis vs. O. bonariensis-perugiae-humensis populations), microsatellite data detected three major genetic entities represented by O. bonariensis, O. perugiae-humensis and an admixture of populations belonging to O. argentinensis-perugiae respectively. Therefore, the star COI polytomy in the tree topology involving these taxa could be interpreted by several hypothetic scenarios such as the existence of shared ancestral polymorphisms, incomplete lineage sorting in a radiating speciation process and/or reticulation events. Present findings support that promiscuous and recent contact between incipient species sharing asymmetric gene flow exchanges, blurs taxa boundaries yielding complicated taxonomy and Management Units delimitation in silverside genus Odontesthes from SW Atlantic Ocean basins.The present paper integrates phylogenetic and population genetics analyses based on mitochondrial and nuclear molecular markers in silversides, genus  The New World presents multiple examples of atherinid species flocks or adaptive radiations arising from habitat transitions Odontesthes includes 20 nominal species Odontesthes species co-occur in the same habitats and they are characterized by a great morphological homogeneity The silverside genus O. bonariensis and O. hatcheri are endemic of rivers and lakes located east of the Andes in subtropical and temperate areas O. hatcheri occuring in the South (Patagonia), whereas O. bonariensis occupying Central and Northern Argentina, South Brazil and Paraguay. The occurrence of the spontaneous hybridization between both species in a communal laboratory tank has been reported Among freshwater representative species, two of them Odontesthes are endemic of a chain of small shallow lakes spread along the South Western Atlantic Ocean coastal plain O. argentinensis and O. incisa, whereas in the freshwater habitats of Patos-Mirim lagoon system can be found O. bonariensis, O. humensis, O. retropinnis and O. aff. perugiae.On the other hand, marine silversides generally have similar life history strategies, occurring in large numbers in semi-isolated populations in estuaries and coastal lagoons Odontesthes species represent economically important resources for artisanal and recreational fisheries in South America and particularly O. bonariensis shows a great potential for aquaculture development Most O. perugiae species complex which comprises several allopatric and sympatric morphotypes found in the lakes and rivers of southern Brazil, Uruguay and northern Argentina O. perugiae species complex, and also reported some of the most rapid speciation rates for a vertebrate group.The identification of incipient ecological species represents an opportunity to investigate the current evolutionary process where adaptive divergence and reproductive isolation are associated Gene flow among hybridizing species with incomplete reproductive barriers blurs species boundaries, while selection under heterogeneous local ecological conditions or along strong gradients may counteract this tendency Odontesthes taxa from the SW Atlantic Ocean, the Río de la Plata estuary and in the Uruguay River basins. At the same time this information will contribute for a long-term success of Management Units for sustainable conservation of these taxa in fisheries and aquaculture.In this study we implement a phylogeographic analysis based on mtDNA coding sequences  and ten microsatellite loci to access in the species boundaries and to test possible reticulation and introgression events among All sampling protocols for this scientific study were approved by CNEA  from Uruguay.Odontesthes from 20 sampling sites through three major regions, the Río de la Plata (RP) estuary (N = 45), Lower Uruguay and Negro river (UNR) basins (N = 23) and Atlantic coast (AC) sites of SWA Ocean (N = 95) were included in the present study. All these samples were primarily ascribed to O. bonariensis (Ob), O. perugiae species complex (Op), O. humensis (Oh), O. argentinensis (Oa) and only two specimens from O. incisa (Oi) according to Dyer A total of 163 individuals of Genomic DNA of sacrificed specimens was isolated from muscle tissue (fixed in ethanol 95%) using sodium chloride protein precipitation, followed by ethanol precipitation modified from Medrano et al. 2, 0.2 mM of each dNTP (10 mM), 0.25 µM of each primer (10 µM), 0.1 units of Taq DNA polymerase (Invitrogen) and approximately 100 ng/ul of template DNA. Cycling conditions consisted of one initial denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 52°C for 30 s, 72°C for 1 min and a final extension of 72°C for 10 min.A fragment of 650 bp from the COI gene was amplified using FishF2 and FishR1 primers Amplified COI products were sequenced directly on both strands in a Perkin-Elmer ABI Prism 377 Automated Sequencer . Sequence alignments were performed using Clustal X 1.8 FsDCorrected estimates of pairwise sequence divergence were obtained using Kimura's Odontesthes populations from the sampled area were assessed by using two different methodologies. A non-model based method  was implemented in PAUP* 4.0b10 The phylogeographic relationships among mitochondrial COI haplotypes in On the other hand, two model based approaches were also used, i.e., maximum-likelihood (ML) and Bayesian inference (BI), implemented in PAUP* 4.0b10 http://atgc.lirmm.fr/phyml), according to the algorithm developed by Guindon et al. In ML and BI analyses, the best-fitted nucleotide substitution model for each data set was determined in Modeltest v.3.7 O. regia, O. incisa, O. smitti, O. hatchery and O. platensis and a more distantly taxon Atherina hepsetus retrieved from the GenBank.All trees were rooted by means of an outgroup criterion using sequences of For the data set, divergence time of nodes and the age of the most recent common ancestor (tMRCA) were estimated with the BEAST v.1.5.4 software Odontesthes samples the variance components among hierarchical partitions in the dataset were assessed by Analysis of Molecular Variance (AMOVA) CT), among populations within groups (ФSC), and among individuals within populations (ФST), after disregarding either their original populations or their groups. For both molecular markers, populations were ascribed to three major sampling areas such as Atlantic coast (AC), Rio de la Plata (RP) and Uruguay and Negro river basins (UNR), and different grouping hypotheses for the populations were tested. The significance of the observed Ф-statistics was tested using the null distribution generated from 3,000 non-parametric random permutations of the data matrix variables and P-values were adjusted with sequential Bonferroni corrections for multiple comparisons To determine the genetic structure of http://www.fluxus-engineering.com/sharenet.htm), which implements the median-joining method, in the absence of recombination Relationships and geographical distribution of the haplotypes were analysed in the haplotype network constructed with NETWORK v. 4.6.0.0  in relation to the expected under a sudden population expansion model ut u is the mutation rate of the sequence . If the sudden expansion model was not rejected, then τ was converted to time since expansion (t) in years before present as follows: [YBP (t = τ/2u)]. For Odontesthes silversides, the mtDNA substitution rate was estimated in 0.023 t) is measured in generations and the age at sexual maturity for Odontesthes was calculated as minimum population doubling time 1.4–4.4 years (http://www.fishbase.org), to convert to time since expansion in years, we have multiply by the generation time of a mean 2.9 years.To assess to the historical demography of Odontesthes were amplified: Odon02, Odon09, Odon27, Odon38, Odon39 A total of 120 individuals from 13 populations were analyzed using these nuclear markers . Ten polPCR amplifications were carried out in a reaction volume of 10 µl  each containing DNA extract (400 ng/ul); dNTPs (0.1 mM each); primers (10 µM each); MgCl2 Invitrogen (0.8–2.5 mM); Taq DNA Polymerase Invitrogen (0.04 U/µl); and Invitrogen buffer (1X). Amplification conditions were those proposed by Odontesthes data set, based on biogeographic criteria and to avoid statistical bias due to the low number of samples in some collecting sites, the populations were first ascribed to different taxa as follows: Oa including population from GL collecting site; Ob belonging to populations from CAL, SL and CL; Oph populations from CAB, BAD, RBD; finally Oap embracing populations from BP, PNB, PAS, SCS, SGS, PRB and RL. The number of alleles, the allelic richness, the expected heterozygosity corrected for sampling bias, the observed heterozygosity, the polymorphic information content and the estimated null allele frequency were calculated for each locus in the whole population per taxon using CERVUS version 3.0.3 F-statistics .An analysis of population subdivision and clustering of individual genotypes was implemented with STRUCTURE v. 2.2 ST values for pairwise comparisons of the 13 Odontesthes populations and their significance level for genetic differentiation (P = 0.05) and Rst were tested additionally with FSTAT Different groups of hypotheses and populations as sources of variation were assessed in the AMOVA considering all ten loci using ARLEQUIN 3.1 software package Ob as a freshwater sister taxon of Oa and Oph, sharing a common freshwater ancestor with these taxa.To discriminate between the relative effects of divergence and gene flow on the speciation process, we analyzed our data set under the Isolation with Migration model θ = 4Nu), splitting time  and migration rates (2NM = 4Nu×m/2). The priors were finally set as follows: the upper bound of population sizes q = 10, splitting times t = 4 and migration rates m = 2, respectively. We run the MCMC simulations with 100,000 burn-in steps and 10,000.000 sampled genealogies. The posterior distributions of migration rates and population sizes are derived analytically from the sampled genealogies.The method estimates posterior probability distributions for both ancestral and actual population sizes, directional migration rates between the two populations, and the time elapsed since population splitting. An MCMC approach is used to draw a sample from the posterior distribution of genealogies and to estimate three types of population parameters: population size  were retrieved from the GenBank and included for both the pairwise distance comparisons and the phylogenetic analyses.This study includes a data set of 655 bp of mitochondrial COI gene from 156 individuals belonging to populations of O. argentinensis, 30% of them were shared with O. perugiae and 25% with O. humensis respectivelly. Among 7 COI haplotypes initially grouping O. bonariensis sequences, 58% of them were shared with O. perugiae and 8% with O. humensis. Therefore we partitioned the statistical analysis in two different data sets: O. argentinensis-perugiae-humensis (Oaph) and O. bonariensis-perugiae-humensis (Obph).Among 36 COI haplotypes initially assigned to Oaph populations showed higher haplotype diversity (h) and nucleotide diversity (π) than Obph (Oaph populations whereas only seven in Obph taxa. Except for the three most common haplotypes (H_1 and H_2 in Obph and H_6 in Oaph), most haplotypes represented rare variants that explained the observed haplotype diversity in each taxa  was 0.047±0.011 (mean ± SE), whereas the divergence between the former and O. incisa was 0.075±0.018. The average divergence between the ingroup and the outgroup A. hepsetus was 0.611±0.153.In  in Obph . The aveOaph and Obph populations. All performed phylogenetic analyses (ML and beast) conducted using the HKY+Г model of sequence evolution, clearly identified a major monophyletic and recently derivate clade with a high posterior probability of occurrence, including minor monophyletic clades which collapsed in a basal polytomy joining most of the 41 haplotypes of Oaph and a well supported clade of Obph  were reciprocally monophyletic in relation to the minor clade of O. bonariensis and the major derivate clade, whereas O. incisa was the most basal taxon of the genus Odontesthes. Simultaneously we used a reference calibration time for nodes by assuming a substitution rate of conventional rate for mitochondrial genome of 0.023 mutations/site/per million years Odontesthes clades showed a high probability to have diverged between 0.1 and 2.5 Mya (Quaternary). The divergence between the genus Atherina and Odontesthes occurred in the Miocene.Present phylogenetic analyses included 43 haplotypes from  of Obph . Other mST values of the COI data set among the 15 collecting sites analyzed with this marker. Low population genetic structure was detected among localities ascribed to Oap from RP estuary and AC areas respectively. Nevertheless, these localities appeared divergent to those ascribed to Obph from some RP estuary sites, and UNR basins. Remarkably the CAL collecting site seems to be the most divergent from all the remaining ones. The most plausible population structuring based on COI data set among tested hypotheses in the AMOVA was addressed following two different grouping criteria: (a) assigning all populations to two-group of samples; (b) forming three groups of populations (CT), suggesting a remarkably higher level of genetic structure when samples from marine and estuarine morphs ascribed to Oaph populations was considered as a separate group from the other including freshwater samples from Obph taxa.ulations . The twoOaph is present in 11 sampling sites and is shortly interconnected by one to three step-mutations to most haplotypes of the network, belonging to Oaph populations. A single step mutation separated H_6 from the other most frequent haplotype (H_2), which included samples belonging to Obph taxa from 6 sampling sites. Remarkably, the network topology showed some loops involving the central H_6 and H_2 haplotypes and their respective derivate ones. Therefore these alternative links may be representing equally good connections due to homoplasy or perhaps the existence of peripheral reticulation events among them.The haplotype network based on COI gene  showed aOdontesthes.Taking into account all collecting sites, negative values in Mantel test were observed , showing a negative correlation between genetic and geographic distances and excluding the isolation by distance model of population differentiation in Oaph populations. The sum of squares deviations was SSD = 0.148 (P>0.06) and Harpending's Raggedness index was 0.486. The estimate parameter under the model was τ = 3.218. The time of expansion-decline in Oaph based on a substitution for this marker was estimated to have started around 227,000 YBP. In the Obph populations data set the sum of squares deviation value was SSD = 0.180 (P>0.05) and Harpending's Raggedness index was 0.492, therefore population growth-decline hypothesis was accepted for this taxon. The estimate parameter under the model was τ = 7.843 and the time of expansion-decline in Obph populations were estimated to have started around 497,000 YBP.Oap (Odon27 and Obo54); Oph ; Ob . This could be due to a Wahlund effect, with a reduction of heterozygosity caused by any subpopulation structure. The null hypothesis of equilibrium was accepted across all loci in Oa population and when a global test across loci and populations was performed .The measures of microsatellite genetic variation including total number of alleles, allelic richness, heterozygosity and HWE deviation for each locus and the corresponding average across all loci per taxon are showed in  p<0.05, . Based oOap populations. The number of alleles per locus varied from 11 (Odon27) to 71 (Obo01) in the total sample. Average number of alleles ranged from 25 (Oap) to 5.8 (Oa)  and that the deficit of heterozygotes in these loci, were not due to stuttering or large allele dropout but it might be the result of null alleles, such as in the cases of Obo01 and Obo 54 in the 5.8 (Oa) . AllelicExact test of genotypic linkage disequilibrium were not significant (p<0.05) after Bonferroni correction in the global approach, except for two pairs of loci (Odont 39-Odont27 and Obo01-Obo54).Oap taxa, as well as among those belonging to Oph, whereas the populations from O. bonariensis presented little to moderate genetic differentiation (Oap and Oph populations whereas large differentiation (FST>0.15) was evident between Oph and Ob taxa pairwise comparisons.Pairwise Fst values among 13 populations showed little differentiation for ntiation . ModeratAmong all grouping hypotheses tested, AMOVA results showed that three groups rendered the most plausible hypothesis with the largest percentage of variance within individuals, followed by among groups, whereas remaining components of variance value were very low .L, CL and CAL ascribed to Ob were assigned mainly to one genetically homogeneous cluster, whereas those from eight localities ascribed to Oap populations were mostly assigned separately, and finally three other localities  were clustered as a separated and genetically homogeneous group belonging to Oph taxa (Both STRUCTURE  and NeigOph taxa .The NJ tree based on Nei's genetic distance matrix detected the same three separated clusters belonging to the same aforementioned taxa .Oaph populations (based on both molecular markers) it was considered as single unit in the IMa2 under the two-population analyses (Oaph vs. Obph. The peaks of the six parameter estimate were confined to narrow ranges with the corresponding posterior distribution (Oaph) under two markers was two-fold higher than that of the ancestral population (q2), indicating a possible population expansion events (Obph), whereas minor signals of bottleneck followed by population expansion appeared . Two of them including genetically homogeneous groups such as  and CAB . The nucOa and the freshwater Oph taxa. This group showed a gradual variation in its nuclear genomic structure from the hypothetical shared ancestors. The variation is yet evident in the same marine-estuarine environment  irrespective of its geographic distance and excluding the isolation by distance model of differentiation.On the other hand, a second cluster of individuals belonging to the remaining collecting sites represent a highly heterogeneous group of mixed ancestry between the marine-estuarine taxon Obph populations and other integrated by Oaph samples. Consistent with genetic distances, haplotype network showed a strikingly star-shaped topology including two most frequent haplotypes shortly interconnected by branch lengths of one step mutation, one of them ascribed to Oaphs and the other one belonging to Obph taxa.In contrast to microsatellite data, the AMOVA based on mitochondrial COI gene straightforward supports the existence of two groups: one including Oaph taxa seems to have occurred toward O. bonariensis matrilineal genome. Contrasting IMa2 results from both molecular markers were consistent with a hypothetic hybridization and introgression scenarios between these taxa.Taking into account all present data, the existence of hybridization events could have occurred among these four highly related taxa in sympatric estuarine areas. In particular introgression from O. bonariensis vs. O. argentinensis) in the Salada Grande Lagoon from Argentina.Previously, it has been reported In the area under study, Pleistocene and Post-Pleistocene marine transgression produced habitat modifications and fragmentations in particular in coastal and shelf regions from South America Odontesthes populations in the study area. The Río de la Plata represents a continuous estuarine environment linking marine (SWA Ocean) to freshwater populations of Odontesthes inhabiting the Uruguay-Negro River basin.Nevertheless there is absence of absolute geographical and physiological barriers to gene flow that separate these Oaph group of populations would be explained by alternatively shared ancestral polymorphism, incomplete lineage sorting in radiating taxa and/or past recent process of reticulation events. In this sense, Beheregaray et al. O. argentinensis-perugiae populations share common mitochondrial haplotypes perhaps representing the matrilineal ancestor of them. These authors hypothesized that a fundamental issue is whether the O. perugiae morphotypes endemic to coastal lakes are the outcome of an evolutionary radiation or merely reflect phenotypic plasticity within a single species.Remarkably, the nuclear genomic heterogeneity detected in the Oaph populations. We note that the inferred IMa2 parameter of the effective population size in Oaph is larger than the supposed common ancestor and the Obph. This scenario could be consistent with secondary contacts between incipient species showing different combinations of nearly interspecific hybridizations and recent mixing populations.Mismatch distribution and neutrality tests based in the COI data set yielded to different historical demographic scenarios in which population expansion could be proposed for Obph suggest that these populations have undergone past recent of more or less dramatic bottlenecks and founder effect episodes of population reductions, perhaps associated to freshwater Pampean mainland lakes and lagoons environments changes since later Pleistocene.Nevertheless, historical demographic parameters in Odontesthes model of speciation. Present findings pointed out that promiscuous and recent contact between incipient species sharing high level of asymmetric gene flow blurs species boundaries yielding to complicated taxonomy and species delimitation among silverside genus Odontesthes.Beheregaray and Sunnuck Oaph populations from the SW Atlantic Ocean, RP and UNR basins could be considered a metapopulation system for fishery policies and conservation purposes.Based on present data we propose that O. bonariensis, within the aquaculture programs, regarding the moderate to relatively low genetic diversity found in this taxon, despite potential past/present reticulation events among its highly related sister species.Present nuclear and mitochondrial data alert us about the sustainability in native and captive populations of Appendix S1Odontesthes individuals from lower Uruguay and Negro river (UNR) basins, the Río de la Plata (RP) estuary, and associated coastal lagoons and sites from SW Atlantic Ocean (AC). Samples included in the microsatellite population analyses are indicated with an X.Catalog number, collecting sites and areas, GenBank accession numbers and haplotype of (DOC)Click here for additional data file.


Repeated recolonization of freshwater environments following Pleistocene glaciations has played a major role in the evolution and adaptation of anadromous taxa. Located at the western fringe of Europe, Ireland and Britain were likely recolonized rapidly by anadromous fishes from the North Atlantic following the last glacial maximum (LGM). While the presence of unique mitochondrial haplotypes in Ireland suggests that a cryptic northern refugium may have played a role in recolonization, no explicit test of this hypothesis has been conducted. The three-spined stickleback is native and ubiquitous to aquatic ecosystems throughout Ireland, making it an excellent model species with which to examine the biogeographical history of anadromous fishes in the region. We used mitochondrial and microsatellite markers to examine the presence of divergent evolutionary lineages and to assess broad-scale patterns of geographical clustering among postglacially isolated populations. Our results confirm that Ireland is a region of secondary contact for divergent mitochondrial lineages and that endemic haplotypes occur in populations in Central and Southern Ireland. To test whether a putative Irish lineage arose from a cryptic Irish refugium, we used approximate Bayesian computation (ABC). However, we found no support for this hypothesis. Instead, the Irish lineage likely diverged from the European lineage as a result of postglacial isolation of freshwater populations by rising sea levels. These findings emphasize the need to rigorously test biogeographical hypothesis and contribute further evidence that postglacial processes may have shaped genetic diversity in temperate fauna. Salmo trutta L., McKeown et al. Gammarus duebeni, Krebes et al. Repeated recolonization of freshwater environments after Pleistocene glaciations has played a major role in shaping the evolution and distribution of numerous anadromous taxa Hewitt . LocatedCoregonus pollan – suggests a novel recolonization history within Europe  is one of the few native fish species in Ireland and is present in both marine and freshwater habitats throughout the region  framework. Specifically, we tested whether divergence and the presence of a third evolutionary lineage unique to Ireland was consistent with the hypothesis of a spread from a cryptic Irish refugium based on divergence times and effective population size.n = 730) were captured using minnow traps, beach seines, dip nets, and electrofishing, euthanized using an overdose of MS-222 or clove oil, and then placed immediately in 100% ethanol. An additional sample of marine sticklebacks from the North Atlantic Ocean was obtained during a research trawl by the Irish Marine Institute research vessel the R.V. Celtic Voyager in order represent a purely marine population , representing the 68 sample sites, was amplified for two mitochondrial regions, cytochrome B , and a partial control region sequence , 200 μM dNTPs, 1 μL of each primer (10 picomoles), 2U Taq polymerase (Invitrogen), and approximately 20 ng stock DNA. For cyt B, thermocycling conditions consisted of one denaturation step at 94°C for 3 min, followed by 35 cycles of 30 s at 94°C, 30 s at 62°C, 60 s at 72°C, and then a final extension step at 72°C for 45 min. Similar conditions were used for control region, although the annealing temperature was set to 52°C. Products were purified and then sequenced using the Big Dye v3.1 cycle sequencing kit  prior to analysis on a 96 capillary ABI 3730XL DNA Analyzer (Applied Biosystems Inc). Sequence base calls were obtained using SEQUENCE ANALYZER v5.4 (Applied Biosystems Inc.) and were manually checked and edited using ChromasPro v1.42 and aligned with BioEdit v7.0.5.3.Deoxyribonucleic acid was extracted from caudal fin clips using Qiagen DNeasy tissue kits or following a salt extraction method . Raw fragment profiles for each individual were then manually genotyped using GENEMAPPER v4.1 (Applied Biosystems Inc.).Seven hundred and thirty samples from across all 69 sampling locations were amplified for nine microsatellite loci in two multiplex PCR cocktails after Kalbe et al. . Additional sequences from three UK populations  from Mäkinen and Merilä  were alsG. aculeatus and three sequences from Gasterosteus wheatlandi .Our first aim was to use mitochondrial DNA to examine relationships between populations in Ireland and Britain and to examine how these grouped with previously described European mitochondrial lineages. Therefore, we constructed two mitochondrial datasets: The first included all unique haplotypes found in Ireland and Britain, and the second included these haplotypes alongside all of those from the European and Trans-Atlantic lineages described by Mäkinen and Merilä . To resolve ambiguities and reticulations in the network, the network was edited by hand following Pfenniger and Posada  using the correlated allele frequencies model  Ireland was colonized from two marine sources (European and Transatlantic lineages) and a third (Irish) lineage has subsequently arisen due to postglacial isolation. This scenario is consistent with Mäkinen and Merilä  Ired Merilä  in that d Merilä . The pre6 datasets were simulated and 30 summary statistics generated for each. Following this, posterior probabilities for each scenario were calculated using the logistic regression step  from a comparatively smaller region. While resampling our dataset using a sample size similar to that of Mäkinen and Merilä , this still represents 42% of reported diversity. Basic genetic indices further reflect the high diversity of stickleback populations in the region (Table P < 0.05), indicating recent population expansion.A total of 364 individuals from Britain and Ireland were successfully amplified and sequenced for both mitochondrial regions. All resulting sequences were found to be of excellent quality, with very few ambiguous base calls. On the few instances where these were observed, the relevant samples were excluded from subsequent analyses. In total, 117 composite haplotypes  were observed in the complete dataset. As we did not use the nested primer approach of Mäkinen and Merilä , we perfd Merilä  to the ld Merilä  reveals G. aculeatus sequences formed a monophyletic group relative to G. wheatlandi, and a split between Pacific Ocean and Atlantic clades was also highly supported (posterior probabilities = 1.0), consistent with the findings of Ortí et al.  varied in reliability. The majority of estimates produced unimodal posterior probability distributions Fig.  and 95% K values estimated from STRUCTURE analyses provided the highest support for K = 19 (see S7), revealing a pattern of clustering among populations located in the same geographical regions (S8). Delta K values typically support hierarchical levels of structure  was also apparent for multiple K values including K = 2, 3, 5 and 12 , structuring largely occurred among populations in close geographical proximity. However, a large number of samples could not be clearly assigned and were instead considered admixed compared with K = 19, the highest value of K which likely also reflected greater population-level structure.Delta  19 see S, reveali 19 see S, revealiP < 0.001). Instead, the AMOVA indicated that population groupings based on geographical proximity explained the greatest proportion of variance. For example, when populations were grouped by marine basin and region (see Table S1), these factors explained 2 and 3% of the genetic variance, respectively , although higher values accounted for the greatest percentages . Regardless, across all hypotheses, the largest significant partitioning of variance occurred within (83.5–85.0%) and among populations within groups (12.2–15.5%).AMOVA indicated that grouping samples by mitochondrial lineage explained a negligible proportion of the total variance (<1%), suggesting low concordance between the mtDNA and microsatellite datasets and divergence since colonization. Similarly, habitat explained a very low but significant percentage of genetic variance  and combined microsatellite and mtDNA data Fig. . ABC anaE for the Irish lineage  compared with both the European  and Transatlantic lineages and when scenario 1 was excluded . This kind of geographical relatedness has been also observed in other European stickleback studies, although it is typically weak in comparison with the among-population component . In contrast, microsatellites appear to reflect hierarchical structuring from broad geographical groupings to individual populations. For example, at K cannot be determined as the number of markers and samples used are insufficient to resolve separate populations effectively  and AMOVA indicated that genetic variance was greatest at the population level . Thus, for three-spined stickleback, it seems unlikely that isolation in an Irish refugium during the LGM can explain unique genetic diversity in the region.Assuming these haplotypes do represent a putatively Irish lineage, our results suggest that at face value, a cryptic Irish refugium could have played role in postglacial recolonization of sticklebacks in the region. A cryptic Irish refugium has previously been hypothesized as the cause of unique diversity in aquatic populations in Ireland (Ferguson et al. Nonetheless, unique Irish diversity in sticklebacks and other anadromous species suggests that Ireland's glacial history has shaped genetic structure in some way. On the basis of our findings, we suggest that further consideration should be given to the nature of a cryptic refugium in Ireland. A major complication for reconstructing colonization history in Ireland has been uncertainty over the glacial history of the region. Using the traditional model of LGM ice extent in Ireland, several studies have suggested that a refugium may have occurred for both terrestrial and aquatic fauna in the Kerry Peninsula to the south-west (Sinclair et al. It is possible then that exposed coastline to the south of Ireland could have acted as a refugium for some anadromous and marine species. However, exposed coastline at the southern margin of the BIIS was unlikely to have been isolated from the rest of the Atlantic Basin Fig. ; therefoRegional variation in glacial history has shaped the distribution of genetic diversity across Northern Europe as a result of postglacial recolonization. Patterns of unique genetic diversity in multiple taxa from Ireland suggest the possibility of recolonization from a cryptic refugium. However, in light of recently updated glaciological models, it is essential to test the hypothesis that isolation in a refugium close to Ireland occurred during the LGM. Our study shows that while a unique lineage found largely in Ireland may appear to be evidence of the role of an Irish refugium for three-spined sticklebacks, observed divergence time parameters do not support this hypothesis. Instead, rapid deglaciation at the southern margin of the British–Irish Ice Sheet may have facilitated rapid recolonization to some parts of Ireland well before colonization occurred in the rest of the British Isles. Further analysis is now needed to explicitly test the question of an Irish refugium in other species and to see whether our updated hypothesis of recolonization can explain unique haplotypes in Irish populations of these taxa. With an increasingly well-resolved glacial history, and rapid postglacial isolation, Britain and Ireland represent an excellent testing ground for these questions.


Crassostrea gigas), three sixth-generation selected lines and four wild populations were assessed using 103 single nucleotide polymorphism (SNP) markers. The genetic diversity data indicated that the selected lines exhibited a significant reduction in the observed heterozygosity and observed number of alleles per locus compared with the wild populations (P≤0.05), indicating the selected lines tended to lose genetic diversity contrasted with the wild populations. The unweighted pair-group method with arithmetic mean (UPGMA) analysis showed that the wild populations and selected lines were not separated into two groups. Using four outlier tests, a total of 17 loci were found under selection at two levels. The global outlier detection suggested that 4 common outlier loci were subject to selection using both the hierarchical island model and Bayesian likelihood approaches. At regional level, 3 SNPs were detected as outlier using at least two outlier tests and one outlier SNP (CgSNP309) was overlapped in the two wild-selected population comparisons. The candidate outlier SNPs provide valuable resources for future association studies in C. gigas.In breeding industries, a challenging problem is how to keep genetic diversity over generations. To investigate genetic variation and identify breeding signatures in mass selected lines of Pacific oyster ( Crassotrea gigas), naturally distributed around Japan, China and Korea, is now an important cultivated oyster species worldwide [C. gigas since 1940s, mainly because of its rapid growth rate, high disease resistance and strong environmental adaptability [C. gigas is one of the most popular oyster species in China, and its main places of production are Shandong and Liaoning provinces [C. gigas as one of the most dominant species [C. gigas has gained little from heredity improvement by selective breeding [The Pacific oyster  was 10% [Crassostrea virginica). Moreover, Cruz et al. [Litopenaeus vannamei). The reduction of genetic diversity in a population may possibly reduce disease resistance and decrease adaptability to environmental changes [C. gigas.Several selective breeding projects have been launched in couple of countries and obtained encouraging results –9. With  was 10% . An incr was 10% . Yu and  was 10%  detectedz et al.  found se changes , 14. TheIn recent years, the single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) developed from EST sequences have been extensively used in population genetic studies. Microsatellites have been chosen in many studies for their codominant inheritance and high allelic variability . HoweverC. gigas. The main goal is to demonstrate the effects of cultivation on genetic diversity in the selective breeding program of C. gigas, and to identify candidate SNPs under selection through the use of outlier analysis methods.In the present study, we used 103 SNP markers to evaluate the genetic diversity level, assess the genetic differentiation, and identify putative loci under selection in three sixth-generation selected lines and four wild populations of C. gigas does not belong to endangered species and the sample locations are not in protection.The C. gigas were surveyed in our study. Collection details for the 7 C. gigas populations were shown in 7 oocytes for each female [Three selected lines and four wild populations of h female , 19. Oysh female .2, 5 μM SYTO9  and 10 ng DNA [A total of 103 SNP markers (from CgSNP4 to CgSNP457) were used in the study , 22. SNP0 ng DNA . The PCR0 ng DNA .Na), effect number of alleles (Ne), shannon's information index (I), observed heterozygosity (Ho), expected heterozygosity (He), minor allele frequency (MAF), inbreeding coefficients (Fis) and chi-square analysis of Hardy–Weinberg equilibrium (HWE) were evaluated by POPGENE 1.32 [Na, Ne, I, Ho and He) were calculated between wild populations and selected lines using Mann-Whitney U-Test [The observed number of alleles  was set from 1 to 10 with three replicates. The most probable K value was evaluated by STRUCTURE HARVESTER [The population genetic structure analysis was estimated by Structure 2.3.4 with a model-based Bayesian procedure , 30. TheARVESTER , 32.Fst-based outlier tests were conducted using two island models including a finite island model (FDIST approach) and a hierarchical island model implemented in Arlequin 3.5.1.3 [To explore candidate SNPs under selection,  3.5.1.3 , 33, and 3.5.1.3 . To deteFst versus heterozygosity. Loci which were outside the 99% confidence intervals were treated as outliers potentially under selection.The outlier analysis was implemented at two levels. (1) An overall analysis encompassed all populations under the hierarchical island models. Three groups were set (CS6+RS+DY+RC vs. JS6+MG vs. KS6) based on the Structure result. The analysis was conducted using 50000 coalescent simulations with 10 groups of 100 demes. (2) An analysis was conducted between selected lines and wild populations from which the selected lines originated  under a finite island-model. We conducted 50000 coalescent simulations with 100 demes to generate the joint distribution of 10 values of the posterior odds (PO) >0.5 and 2.0 were regarded as candidate SNPs under selection with substantial and decisive evidence [The outlier tests at the two levels were also calculated with the BayeScan software. The analyses were estimated using a pilot run of 20, a burn-in of 50000 with 100000 iterations each, a sample size of 5000, a thinning interval of 10, and an FDR of 0.05. Loci with logevidence .pop1 and Hpop2 denote expected heterozygosity for population 1 and population 2 [The lnRH test was applied to estimate the ratio of gene diversity (heterozygosity) of all loci in the population pairwise comparisons as follows:lation 2 . The lnRlation 2 . Those Shttp://www.ncbi.nlm.nih.gov/) and OysterBase (http://www.oysterdb.com), and the critical E value was set as 1.0×10−6. The NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) was used to distinguish synonymous SNPs, non-synonymous SNPs or SNPs from untranslated regions (UTRs). The putative function of genes was identified by using the Gene Ontology (GO) annotation by mining the Swiss-Prot database (http://us.expasy.org/sprot/sprot-top.html) and OysterBase.Sequence annotation was conducted by BLASTx software in NCBI database  than the wild populations, but no significant loss of Ne, I or He were detected (P > 0.05). The observed heterozygosities ranged from 0.2703 to 0.2939 with a mean of 0.2806 in the wild populations, and ranged from 0.2479 to 0.2733 with a mean of 0.2599 in the selected lines. There is a significant reduction of observed heterozygosities in the selected lines in contrast with the wild populations (P = 0.05). The observed number of alleles per locus varied from 1.9417 to 1.9709 in the selected lines, and varied from 1.9806 to 2.000 in the wild populations. There was a significant reduction of the observed number of alleles per locus in the selected lines compared with the wild populations (P < 0.05).Genetic variability indices for the four wild populations and three selected lines of HWE after sequential Bonferroni correction (P < 0.05/103), and all loci showed heterozygote deficiencies except for CgSNP261 in CS6 and JS6, CgSNP36 in KS6 and MG, and CgSNP402 in RS.Information of the 103 SNPs evaluated from the three selected lines and four wild populations were summarized in Fst values were significant (P<0.001), except for that estimated between wild populations RS and RC. The lowest value was observed between RS and RC (Fst = 0.00689), however the highest value was detected between JS6 and KS6 (Fst = 0.11737) (Most pairwise 0.11737) . Within C. gigas populations. Moreover, this cluster was further divided into 2 subgroups; the first subgroup contained JS6 and MG originated from Japan, and the second subgroup contained CS6, RS and DY originated from China, and RC from Korea.The pairwise Nei's genetic distances among populations were also shown in The genetic structure test indicated that the most likely number of genetic groups was K = 3. The first group consisted of CS6, RS and DY originated from China, and RC from Korea. The second group consisted of JS6 and MG originated from Japan. Moreover, the selected line KS6 originated from Korea constituted the third genetic group .Both the hierarchical island model and Bayesian likelihood approach revealed 4 loci  under selection across all populations ; Fig 3. Salmo salar) and Pacific abalone  populations [Maintenance of genetic variation is known to be important for long-term survival of populations because the level of variation determines their adaptability to environmental changes , 39. Hetulations , 43.C. virginica. Wang et al. [Argopecten irradians). Compared with wild populations, 20% to 48% fewer alleles were detected in farmed salmon strains from Ireland and Norway [P<0.05). Moreover, 0.98% (2/205) and 2.43% (5/206) fewer alleles were found in selected lines CS6 and JS6 than their wild progenitors (RS and MG), respectively. The CS6 stock seemed to be missing two rare alleles at 2 loci (CgSNP252 and CgSNP265) compared with RS. No individuals with the rare allele of the CgSNP265 found rarely in RS, were sampled from CS6. There was a possibility that this rare allele existed in CS6, but was not sampled in the population. As this rare allele frequency was 0.0521 in RS, the probability of completely missing this rare allele in the CS6 (48 samples) should be estimated as 0.947996 = 0.0059. Therefore, it is most likely that the CS6 has indeed lost this rare allele at the CgSNP265. Similarly, the JS6 may have indeed lost rare alleles at 4 loci . Taken together, we can make a conclusion that the selected lines through breeding programs tended to lose genetic diversity compared with the wild populations. The decrease of genetic diversity may result from genetic drift, bottlenecks, and inbreeding caused by a reduced effective population size.Many researches have indicated loss of alleles is often more easily observed than decrease of heterozygosity in cultivated or selected populations , 11, 44.g et al.  found thd Norway . In our HWE were detected both in selected lines and wild populations. In selected lines, heterozygote deficiency may be mainly because of a limited number of founders and artificial selection [Significant deviations from election . In the The UPGMA analysis indicated that the selected lines and wild populations were not separated into two groups and even the wild populations and selected lines from the same origin country were culstered into the same subgroup except for KS6 and RC, suggesting there was no clear division between the wild populations and selected lines. The result is also supported by the Structure analysis.Fst<0.15) was detected. The UPGMA analysis also demonstrated the three selected lines fell into three groups. The significant genetic differentiation among the three selected lines may result from three different founder populations, thus providing the genetic basis for the establishment of selected lines for faster growth using three C. gigas stocks from China (Rushan), Japan (Miyagi Prefecture) and Korea (Pusan).Within the selected lines, moderate genetic differentiation (0.05<Fst<0.05) were detected among the three populations RS, DY and RC. The Nm values among these populations ranged from 7.98723 (RC and DY) to 36.03447 (RS and RC), which appeared to be sufficiently large to swamp potential for large genetic differences. Since oyster adults are sedentary, larval dispersal becomes a decisive factor affecting gene flow among wild populations. Therefore, the long pelagic larval phase (14–21 days) and high fecundity  of the Pacific oyster may account for the relatively large gene flow detected among RS, DY and RC [Mactra chinensis and Crassostrea ariakensis [Within the wild populations, slight genetic differentiations  and brook charr  [In order to improve the productivity traits of e change , 53. Oure change . Flori ee change  detectedtinalis) , 57. HowFour different tests  were used to detect outlier SNPs potentially under selection during the selective breeding program. These approaches rely on the rationale that selection will lead to increased genetic differentiation between populations and should exhibit a reduction or elevation in genetic variation contrasted with neutral genes. Actually, selection is not the only cause of variation changes at particular loci, reduced variation or increased differentiation can also arise from genetic drift, bottlenecks or founder events . The effThe highly consistent results from the hierarchical island model and Bayesian likelihood approach for global outlier analyses strongly suggested that the identified outlier SNPs were subject to selection. Moreover, the same three SNPs  were also detected as outliers using selected lines and wild populations from which the selected lines originated  (data not shown). At regional level, 3 SNPs  was detected as outlier using at least two analysis methods. Those outlier SNPs found in only one test should be considered with caution. The inconsistent results obtained by the three methods  were probably due to the different measures of variability and different assumptions . AlthougC. gigas. The genetic diversity analysis showed that the selected lines tended to lose genetic diversity contrasted with the wild populations during the successive six-generation selection. Moreover, the UPGMA results suggested there was no clear division between the wild populations and selected lines. In addition, a total of 17 loci were found under selection at two levels using four outlier tests. Further functional studies are needed to confirm the role of the candidate outlier SNPs during the domestication process in C. gigas.In this study, SNPs has been demonstrated to be a good marker of choice for monitoring the genetic variation in wild populations and selected lines of S1 Fig(a) Genetic clusters obtained with three groups. Each individual is represented by one vertical line with 3 segments colored proportionally according to their belonging to a genetic group. Black lines separate individuals from different populations. (b) Graph of delta K.(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.


Plasmodium falciparum differentiation that could affect fitness and adaptation to drugs and target antigens for vaccine development. This study describes the genetic structure of P. falciparum populations in urban and rural sites from southwestern Nigeria.Genetic diversity studies provide evidence of P. falciparum infections from three localities: Aramoko-Ekiti, a rural community; Lekki, an urban location and Badagry, a peri-urban border settlement. Analysis was performed on the genetic diversity, linkage disequilibrium, population structure and inter-population differentiation.Ten neutral microsatellite loci were genotyped in 196 E) values between 0.65 and 0.79. No matching multilocus haplotypes were found and analysis of multilocus LD showed no significant index of association. Genetic differentiation between populations was low (ΦPT = 0.017).Allelic diversity values were similar across all populations, with mean expected heterozygosity (HP. falciparum in southwestern Nigeria is evident in the lack of significant differentiation between populations separated by about 200 km. This implies that a fairly uniform malaria control strategy may be effective over a wide geographic range in this highly endemic region. However, more wide-scale survey across the country will be required to inform malaria control in this large and densely populated endemic region.The absence of detectable population structure of  Plasmodium falciparum which maintains population fitness against targeted interventions such as drugs [The incidence of malaria infections and malaria related mortality has reduced in many countries in Africa –3. Howevas drugs , 7. Infoas drugs . This isP. falciparum populations in Nigeria.There are various approaches to molecular determination of population structure including typing for polymorphic repeats in merozoite surface proteins (MSP 1 and 2) and glutamate rich proteins (GLURP) , 10. UpoP. falciparum strains showed contrasting dominance between subtypes of GLURP [Earlier studies on Nigerian of GLURP , and MSPof GLURP –20 polymof GLURP –23, thisP. falciparum isolates from rural, urban and semi-urban settings in southwestern Nigeria where interventions are being intensified. Neutral microsatellite loci of P. falciparum isolates from one inland and two coastal communities in southwestern Nigeria were analysed.To provide further insight into current patterns in parasite population, this study determined the extent of genetic diversity of Plasmodium falciparum-positive samples were spotted on 3 mm Whatmann filter paper . Genomic DNA was extracted from punched-out disc from each filter paper dried blood spot using the QIAmp DNA blood midi kit  followed by molecular analyses at The Medical Research Council, Gambia Unit.Participants presenting with symptoms of malaria at three health facilities each representing three localities in southwestern Nigeria: Aramoko-Ekiti (AMK), a rural community in Ekiti State; Lekki (LEK), an urban community and Badagry (BDG), a peri-urban border community in Lagos State Figure , were reA two-round hemi-nested PCR was used to amplify 12 microsatellite loci from parasite DNA following described procedures and primers . The locE provides an indication of the probability that two individuals will be different. It has a potential range from 0  to 1 . To understand the potential for multilocus haplotypes to spread through the populations, multilocus linkage disequilibrium (LD) was calculated for the entire population as a whole, and separately for each subpopulation using the standardized index of association, (D/(VE) – 1), where VE is the expected variance of n - the number of loci for which two individuals differ. The observed variance is given by VD. To test whether the ratio of VD/VE was significantly greater than one, we employed a randomization test as previously described [The allele frequencies, numbers of alleles per locus, allelic diversity within each population, and allele frequencies per locus per population were calculated using GENALEX 6 . Allelicnterface  and the escribed , 27.Between population and within population variance was determined with the analogue of Wright’s Fst, AMOVA (ΦPT), as it is flexible enough to accommodate different types of assumptions about the evolution of microsatellites . ΦPT = 0P. falciparum infections only were reported. Of the 12 microsatellite loci genotyped, 2 (TA87 and TA1) gave less efficient PCR amplification and were therefore excluded from subsequent analyses. The allelic frequencies at each of the ten loci in each of the three parasite populations are presented in Figure E values across all loci between 0.65 (for LEK) and 0.79 (for AMK) . Although the mean number of genotypes detected per isolate was highest in AMK , were 0.164, 0.175 and 0.074 respectively. The relationship between genetic distance and the natural log of the geographical distance for each pair of parasite population studied is presented in Figure Forty-three isolates (~22%) had complete genotype data for all loci from which analysis of multilocus haplotypes was examined. No matching multilocus haplotypes were found. Comparisons of populations using AMOVA showed that genetic differentiation was low with ΦPT = 0.017 (P = 0.772). Pairwise genetic distances between LEK and BDG, LEK and AMK and BDG and AMK parasite populations, calculated as Nei unbiased genetic distance (P. falciparum populations from southwestern Nigeria across an area spanning over 200 kilometres. Samples were recruited from both urban and rural settings to explore parasite population differentiation, given the variation in access to drugs and other interventions [Molecular typing of parasite isolates provides vital information about the epidemiological patterns in a population following the implementation of intervention strategies or existence of barriers that could limit gene flow between populations. This study employed microsatellites to determine the structure of ventions . NigeriaP. falciparum populations analysed. The high expected heterozygosity values were similar to those reported in some other African countries with high levels of malaria transmission [There was high allelic diversity of 10 microsatellite markers that gave reliable amplification in all the three smission –15.Balloux and Lugo-Moulin  have putnd versus 3rd principal coordinates. Lack of sub-structuring suggests that gene flow precludes local natural selection and genetic drift. This is expected as vector species distribution in southwestern Nigeria is also largely homogenous for Anopheles gambiae s.s. negating any possibility of local selection by the vector species [The low among population variance and the existence of an inverse relationship in the genetic and geographic distances between BDG and AMK may imply a relatively free gene flow across southwestern Nigeria. Population structure by site of sampling was also not evident by principal coordinate analysis though there was an insignificant sub-grouping distinguishable at the 2 species , 35.In agreement with previous reports from other high malaria transmission areas , there wfections . High MOfections  who consfections , 16. Thoast Asia , 32, it P. falciparum in this region is diverse with an absence of detectable population structure indicating panmixia. The limited differentiation in parasite populations is likely to be a consequence of high and continuous transmission of randomly mating P. falciparum isolates facilitated by indiscriminate vector and human migration. A country-wide study of diversity will be needed to support these findings and inform the current drive to deploy interventions towards the elimination of malaria.This report suggests the population of


Picea glauca × P. engelmannii hybrid zone across a broad latitudinal (40–60 degrees) and elevational (350–3500 m) range in western North America. Our results revealed a wide and complex hybrid zone with broad ancestry levels and low interspecific heterozygosity, shaped by asymmetric advanced-generation introgression, and low reproductive barriers between parental species. The clinal variation based on geographic variables, lack of concordance in clines among loci and the width of the hybrid zone points towards the maintenance of species integrity through environmental selection. Congruency between geographic and genomic clines suggests that loci with narrow clines are under strong selection, favoring either one parental species  or their hybrids (overdominance) as a result of strong associations with climatic variables such as precipitation as snow and mean annual temperature. Cline movement due to past demographic events  may explain the asymmetry in introgression and predominance of P. engelmannii found in this study. These results provide insights into the genetic architecture and fine-scale patterns of admixture, and identify loci that may be involved in reproductive barriers between the species.Hybrid zones provide an opportunity to study the effects of selection and gene flow in natural settings. We employed nuclear microsatellites (single sequence repeat (SSR)) and candidate gene single-nucleotide polymorphism markers (SNPs) to characterize the genetic architecture and patterns of interspecific gene flow in the  The genetic architecture of hybrid zones provides the key to understand the sequence of genetic changes that accompany or facilitate speciation . In addiCline theory provides a rich and powerful conceptual framework for understanding the balance between selection and gene flow in the maintenance of hybrid zones, based on estimates of cline width and linkage disequilibrium  may resuWith the advent of genomic tools to analyze large numbers of loci, novel methods of clinal analysis (genomic clines) have been developed to analyze introgression of individual alleles and genotypes against average levels of genome-wide admixture , 2010. TPicea glauca Moench  is a transcontinental, boreal species that occurs naturally in almost all forested regions in Canada, with the exception of the Pacific Coast. It generally occurs at low elevations, although in Alaska it ranges from low elevations to alpine treeline. P. engelmannii Parry ex. Engelm (Engelmann spruce) extends from British Columbia and Alberta in the north, to New Mexico and Arizona in the south. Engelmann spruce grows at high elevations and is restricted to cold, humid habitats. It has a low tolerance of high temperatures and summer drought, but high tolerance of short growing seasons and deep snowpacks  of white spruce, Engelmann spruce and their hybrids were sampled for DNA extraction , Table 1M dNTP, 1 μl 10 × Paq5000 Reaction Buffer, 1 U Paq5000 DNA Polymerase, 0.5 pmol of M13 Infrared Label Primer and 1 pmol each of forward and reverse tailed primers. Samples were amplified using a modified version of Rungis' (2004) protocol, in which the annealing temperature varied according to each primer  in conjunction with the GoldenGate allele-specific assay in a 96-well, 768 SNP format. White and Engelmann spruce samples were tested using 1536 SNPs from a large panel of candidate genes putatively involved in cold hardiness (Q) was estimated from nuclear SSR data using the Bayesian clustering approach implemented in STRUCTURE version 2.2 (K) from 1 to 10 were tested using 50 000 iterations for the pre- and post-burn periods using the admixture model. Using a larger number of post-burn iterations (100 000) did not change the results obtained. The degree of admixture, α, was inferred from the data. Each run was repeated 20 times in order to estimate K using the method developed by Q>0.9 from STRUCTURE results for either species were used as reference for ‘pure species' in hybrid index calculations using the INTROGRESS 1.1  to evaluate which Kmax provided the best fit with the genetic data. On the basis of the results of the Evanno test, alignments of clusters for K=2 for STRUCTURE and TESS were optimized using the program CLUMPP 1.1.2b  in GENALEX. Principal coordinates 1 and 2 were plotted using R 3.0.3 . To test for isolation by distance, Pearson's product–moment correlations were estimated between populations using genetic and geographical distances in Mantel tests based on 1000 permutations, using the Vegan package in R 3.0.3 . Genetic distance was expressed as Fst/(1−Fst).Global and pairwise population F GENALEX . DiffereHybrid zones are often well described by a sigmoid curve showing the expected allele frequencies along geographic axes. Cline analysis can be used to estimate the shape, location (center) and width of the cline, and to test for concordance and coincidence in these parameters between markers (INTROGRESS 1.1 (P<0.0001). Evidence for directional selection for homozygous genotypes corresponds to an increase (+) or decrease (−) in the observed probability density in comparison with neutral expectations, resulting in genotypes EE−, EE+, WW+ or WW−. In the same way, an increase (+) or decrease (−) in the observed probability density of heterozygous genotypes will be observed as ‘overdominance' (WE+), or ‘underdominance' (WE−), respectively. A combination of increased density of one homozygous genotype, whereas the other homozygous and heterozygous genotypes remain neutral may be indicative of epistasis . The trait was assumed to have a constant heritability of 0.5 and fitness W for an individual with phenotype P in each local population; and it was modeled as stabilizing selection around the local optima as ω is the intensity of stabilizing selection. All simulations used ω=10. In addition to the quantitative traits, we also simulated data for 40 bi-allelic neutral loci. Each simulation was initiated with three populations fixed for alleles representative of each parental species  and the three remaining populations were colonized through migration from neighboring populations. After simulations had finished, the data were read into R and analyzed using the package INTROGRESS 1.1 , and between elevation and genetic distance ; however, the other parental species and hybrids showed no significant differences . Neither parentals nor hybrids showed differences in interspecific heterozygosity . When loci under selection represent 30–50% of the loci under study, the interspecific heterozygosity decreases  and the hybrid index  decreases in range. After 3000 generations following initial admixture, with 50% of loci under selection, the differentiation between parentals and hybrids is lost, and the hybrid zone becomes a hybrid swarm  and 1000  generations to those of our SNP data.In a second scenario, we simulated variation in ancestry and interspecific heterozygosity over time . In the In the third scenario, we ran simulations to assess the impact of the island model of migration on the levels of ancestry and interspecific heterozygosity. Migration was set to occur at the same rate as in the stepping stone simulations; however, migrant individuals could enter any patch regardless of spatial location in all simulations. When migration occurred only between adjacent populations (stepping stone model), the patterns of ancestry and interspecific heterozygosity varied little even after 3000 generations following initial contact. On the other hand, with migration under the island model, the hybrid zone tends to merge into a big hybrid swarm as early as 100 generations . When coOur results indicate the presence of an extensive hybrid zone between white and Engelmann spruce between 50 and 56°N and 600–1800 m in elevation in Western Canada and the United States. Both SSR and SNP clinal analyses revealed a complex hybrid zone, in which different loci introgress in different directions, with the center of individual clines shifted either toward Engelmann or white spruce. SSR clinal analysis pointed toward a unimodal hybrid zone distribution, whereas the SNP clinal analysis showed differentiation between parental and hybrids' habitats, pointing toward a trimodal hybrid zone distribution .SSR cline width varied from 50.1 to 401 km, with seven of the ten loci having cline widths between 50 and 59.82 km, and the remaining three loci with variable widths over 100 km . The cliP>0.0001). Overdominance  and directional selection  were the main types of selection explaining deviation of genomic clines away from neutral expectations  followed a bell-shaped distribution, with the more central Quesnel population having the highest SSR and SNP diversity. The peak in Na is shifted slightly toward Engelmann spruce (south from the average center of the hybrid zone) in both SSR and SNP clinal analyses  in the hybrid populations. Somewhat surprisingly, private alleles were only found in hybrid populations originating between 600 and 1600 m of elevation, and not in parental populations.Whereas all populations were highly diverse for the microsatellite and SNP markers assayed, those from the introgression zone at intermediate elevations and latitudes had, on average, more alleles and tended to have higher levels of heterozygosity and allelic richness . The numanalyses . The numHe=0.592) when analyzing SSR diversity, and in nearby Mount Robson (He=0.307) when analyzing SNP diversity. Showing a similar pattern to Na, the peak in heterozygosity was also shifted toward Engelmann spruce (south of average center of the hybrid zone) for SNP markers compared with SSRs .Heterozygosity was highest in the McGregor population .In most hybrid zones, clines are characterized by their narrow width, consistent shape and close concordance, suggesting that clines are maintained by dispersal and selection against heterozygotes (tension zones). In our study, however, the lack of concordance between markers, the width of the clines and the abundance of advanced-generation hybrids suggest that the Engelmann × white spruce hybrid zone is not a tension zone, and supports our recent studies suggesting the Engelmann × white spruce hybrid zone is maintained by a bounded hybrid superiority model  gene, part of the molecular circadian clock that provides the timing mechanism required for the response to seasonal changes in photoperiod. In Arabidopsis, mutation of this gene caused disruption of the circadian clock regulation of gene expression and caused flowering to occur independently of photoperiod  were associated with various important functions including carbohydrate and lipid metabolism, calcium signaling and transcription regulation . Most ofMorus  × white spruce hybrid zone is also asymmetric and appears to have moved from Sitka toward white spruce, in part due to the postglacial colonization sequence of the region (Lepus granatensis × L. europaeus (Thomomys actuosus × T.b ruidosae (Poecile carolinensis × P. atricapillus; Although temporal isolation and differences in population density cannot be ruled out as causes of asymmetric introgression, the ancient nature of the hybrid zone suggests that the explanation for asymmetry may come from past changes in demography of the hybridizing species. The shift in allelic richness and heterozygosity toward Engelmann spruce suggests that the asymmetry in introgression has likely been caused by past cline movement . Pollen e region . SimilarP. glauca × P. engelmannii hybrid zone in western North America. Our study has revealed the permeability of spruce genomes to introgression in this hybrid zone, in which different alleles are favored in different environments or genetic backgrounds and maintained by selection. By identifying populations with high-diversity levels and adaptations to particular environments, we set the ground for future studies aiming to predict the response of populations to climate change. This knowledge can then be translated into climate-based seed transfer recommendations to improve reforestation success, forest health and productivity for new climates.The work described here is the last in a series of studies exploring the genetic and genomic basis of local adaptation to climate in the http://doi.org/10.5061/dryad.473n5. SNP data in available at the Dryad Digital Repository: http://doi.org/10.5061/dryad.7h65f.Microsatellite data are available at the Dryad Digital Repository:


Corylus mandshurica, also known as pilose hazelnut, is an economically and ecologically important species in China. In this study, ten polymorphic simple sequence repeat (SSR) markers were applied to evaluate the genetic diversity and population structure of 348 C. mandshurica individuals among 12 populations in China. The SSR markers expressed a relatively high level of genetic diversity . According to the coefficient of genetic differentiation (Fst = 0.1215), genetic variation within the populations (87.85%) were remarkably higher than among populations (12.15%). The average gene flow (Nm = 1.8080) significantly impacts the genetic structure of C. mandshurica populations. The relatively high gene flow (Nm = 1.8080) among wild C. mandshurica may be caused by wind-pollinated flowers, highly nutritious seeds and self-incompatible mating system. The UPGMA (unweighted pair group method of arithmetic averages) dendrogram was divided into two main clusters. Moreover, the results of STRUCTURE analysis suggested that C. mandshurica populations fell into two main clusters. Comparison of the UPGMA dendrogram and the Bayesian STRUCTURE analysis showed general agreement between the population subdivisions and the genetic relationships among populations of C. mandshurica. Group I accessions were located in Northeast China, while Group II accessions were in North China. It is worth noting that a number of genetically similar populations were located in the same geographic region. The results further showed that there was obvious genetic differentiation among populations from Northeast China to North China. Results from the Mantel test showed a weak but still significant positive correlation between Nei’s genetic distance and geographic distance (km) among populations , suggesting that genetic differentiation in the 12 C. mandshurica populations might be related to geographic distance. These data provide comprehensive information for the development of conservation strategies of these valuable hazelnut resources. Corylus mandshurica Maxim. et Rupr (synonym to C. sieboldiana), belongs to the family Betulaceae and is an important species, both economically and ecologically, among nut trees. C. mandshurica is a deciduous shrub of about 2 to 6 m in height with bracts forming a tubular husk [C. mandshurica is widely distributed in Japan, Korea, and Northeast to North China around Beijing. Most of C. mandshurica survived in the mountainous forest belts and deep valleys at high altitude [C. avellana, the nuts have better taste and fragrance. Its nuts are not only suitable for fresh eating, but also suitable for baking. Its nuts are an important ingredient in cake, and are a favorite of consumers [Hazelnut, lar husk –3.The lealtitude . The shealtitude , and theonsumers . Moreoveonsumers .In recent years, molecular markers have shown promise for assessment of genetic diversity, owing to their high discriminatory power and comparatively low cost. In particular, the superiority of molecular markers for the characterization of plants is well recognized. DNA based molecular marker technologies, such as simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP), sequence-related amplified polymorphism (SRAP) and single nucleotide polymorphisms (SNPs), have several advantages including abundant, independent from the environment, suitability for early and rapid evaluation, and having non-tissue specific characteristics. Among them, SSRs, also known as microsatellites or short tandem repeats (STR), are widely present in eukaryotic genomes  and veryC. mandshurica[C. mandshurica resources in China, and its genetic variation and population genetic structure are still unknown.SSRs have been developed for European hazelnut –14, and ndshurica. Besidesndshurica–21, compndshurica. Evaluatndshurica. HoweverC. mandshurica) in China, with a particular focus on population structure using SSR markers. A total of 12 C. mandshurica populations throughout its present main Chinese distribution range were studied. The aims of this study were to: (1) evaluate the genetic diversity and population structure of C. mandshurica, (2) estimate variance components and partition the within- and among-population variance, (3) obtain comprehensive information for the development of conservation strategies for these valuable C. mandshurica resources.In this study, we present the first investigation of genetic diversity of wide-ranging hazelnut   , Table 12, 50 mM Tris–HCl, 0.2 mM of each dNTP, 1 unit of Taq DNA polymerase  and accompanying buffer. PCR amplification was performed with the following cycling parameters: first a denaturation step at 94°C for 5min, followed by 35 cycles of 94°C for 30 s, annealing at 48°C~55°C for 40 s , effective number of alleles (Ne) [I), observed heterozygosity (Ho), expected heterozygosity (He), genetic differentiation coefficient (Fst), gene flow (Nm) and Nei’s genetic distance (Gd) [Nm was estimated as: Nm = 0.25(1−Fst) / Fst. Cluster analysis was carried out based upon the Nei’s genetic distance matrix with UPGMA (unweighted pair group method of arithmetic averages) and a dendrogram was constructed using MEGA (version 5) [The presence of null alleles of each locus was estimated by software MICRO-CHECKER 2.2.3 . The numles (Ne) , Shannonnce (Gd) S1 TableNa, effecrsion 5) .Genetic structure analysis was performed with the program STRUCTURE 2.3.4 with 10 runs and 100,000 Markov Chain Monte Carlo (MCMC) repetitions after a burn-in period of 100,000 interactions for each group number K. The optimum value of K was obtained by calculating the ∆k value to determine the most likely number of groups . AnalysiC. mandshurica.In this study, MICRO-CHECKER analysis indicated the presence of null alleles at a few loci in some populations. At CAC-B114, CAC-C003 and CAC-B028, null alleles were detected in five populations, at CAC-B105, CAC-A040, CAC-B020 and CAC-B005, null alleles were detected in two populations, and at CAC-C028, null alleles were detected in population TL, while at CAC-B001 and CAC-B113, no null alleles were detected. Thus, these 10 SSR loci are a proper set to assess genetic diversity of Na) per locus varied from 7 (CAC-C028) to 28 (CAC-B020) with a mean of 15.3 per locus. At the same time, effective numbers of alleles (Ne) varied from 2.0804 (CAC-C028) to 12.1102 (CAC-B020) with an average of 5.6604 per locus. Shannon's Information index (I) averaged 1.8853 and ranged from 0.9193 (CAC-C028) to 2.7178 (CAC-B020). Observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.4339 (CAC-C028) and 0.5201 (CAC-C028) to 0.8928 (CAC-B001) and 0.9188 (CAC-B020) respectively.A total of 153 alleles were found at the ten microsatellite loci in the 348 individuals . The numFis) per locus ranged from -0.1167 (CAC-B001) to 0.1143 (CAC-B114), with an average of 0.0215 alleles per locus. Furthermore, genetic differentiation (Fst) of individual loci ranged from 0.0689 at CAC-A040 to 0.2016 at CAC-B114, and averaged at a value of 0.1215 alleles per individual locus, suggesting low genetic differentiation among the populations. Moreover, the genetic diversity within populations (87.85%) was significantly higher than that between populations (12.15%). In addition, gene flow (Nm) ranged from 0.9903 at CAC-B114 to 3.3798 at CAC-A040 and averaged 1.8080 (Nm = 1.8080) between C. mandshurica populations.The inbreeding coefficient  ranging from 1 to 12. The ΔK showed a clear maximum for K = 2 (ΔK = 185.0287), indicating among 12 populations the existence of two groups . Group IP = 0.005; The Mantel test revealed a weak but positive correlation between Nei’s genetic distance and geographic distance (km)  was significantly higher than other Corylus species such as C. heterophylla (4.18) [C. avellana   and C. ana  . MoreoveDi et al , who anaJuglans mandshurica) [P = 0.005). Therefore, it’s speculated that the genetic structure of C. mandshurica population in China can be affected by geographic distance.Comparison of these results with the UPGMA dendrogram and the Bayesian STRUCTURE (K = 2) bar plot showed general agreement between the population subdivisions and the genetic relationships among the populations. The populations were divided into two main clusters. Similar results were previously reported on Chinese walnut (shurica) , two refNm>1, the gene exchange among population can prevent the impact of genetic drift and reduce the genetic variance among populations. In this study, the average of gene flow of C. mandshurica at each locus was 1.8080, indicating that the gene flow was one of important factors influencing the genetic structure of C. mandshurica populations. The relatively high level of gene flow likely prevents genetic differentiation, which is the reason for the observed low genetic differentiation. That is the reason why the variance within populations was significantly higher than that between populations. Therefore, the lack of differentiation is attributed to gene flow.Gene flow is defined as the transfer of alleles from one population of a species to another. Study of gene flow is critical for the understanding of population processes within and among species . The carC. mandshurica is wind-pollinated and self-incompatible; its pollen is easily dispersed over medium and long distances. In addition, the nuts of C. mandshurica are nutritious and thus serve as an important food source for small mammals and birds, who can transport them in small numbers over long distances. All of these factors can contribute to the observed high gene flow between different populations.Gene flow among populations was influenced by a number of factors including the mating system, geographic distribution, mechanisms of seed dispersal, and the stage of succession and colonization , 49. An C. mandshurica populations can provide a theoretical basis for the conservationist to formulate future conservation strategies of C. mandshurica.In recent years, it has become increasingly important to adopt a holistic view of biodiversity, which includes agricultural biodiversity, conservation and sustainable development. An in-depth understanding of hazelnut genetic diversity and ecological distribution is indispensable for the protection and utilization of this species . AnalyziC. mandshurica populations have a relatively high level of genetic diversity. However, in the past several decades, excessive deforestation and over-exploitation have damaged the ecological environment of many wild C. mandshurica living on natural hills. And local farmers have devastated the natural habitats of some populations in order to obtain the economic value. If not properly preserved, this species will likely become endangered because of both high demand and adverse climatic conditions, and even become extinct in the future. We suggest in situ conservation method: conservation to prevent the decrease in population sizes and loss of genetic diversity. Moreover, in order to achieve effective conservation of C. mandshurica germplasm resources, efforts are needed to carefully plan and construct pollen banks and gene banks for hazelnut. In the meantime, protected areas can be established to conserve and restore the habitat and the populations. In addition, it is important to develop a core collection of C. mandshurica in greater breadth and depth, which would not only mitigate the pressure of excessive use of wild resources, but also help achieve more effective management and use of hazelnut germplasm.In this work, the results based on SSR techniques discovered that wild S1 FigDeltaK = mean (|L''(K)|) / sd(L(K)).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.