Gibbula divaricata from five coastal locations in the central-south Adriatic Sea  and one in the adjacent northern Ionian Sea, using 21 described polymorphic microsatellite loci. Observed and expected heterozygosity varied from 0.582 to 0.635 and 0.684 to 0.780, respectively. AMOVA analyses showed that 97% of genetic variation was observed within populations. Nevertheless, significant, although small, genetic differentiation was found among nearly all of the pairwise FST comparisons. Over a general pattern of panmixia, three groups of populations were identified: eastern Adriatic populations, western Adriatic populations, and a third group represented by the single northern Ionian Sea population. Nonetheless, migration and gene flow were significant between these groups. Gibbula divaricata is thought to have a limited dispersal capacity related to its lecithotrophic trochophore larval stage. Our results indicated high levels of self-recruitment and gene flow that is mainly driven through coastline dispersion, with populations separated by the lack of suitable habitats or deep waters. This stepping-stone mode of dispersion together with the high levels of self-recruitment could lead to higher levels of population structuring and differentiation along the Adriatic Sea. Large effective population sizes and episodic events of long-distance dispersal might be responsible for the weak differentiation observed in the analyzed populations. In summary, the circulation system operating in this region creates natural barriers for dispersion that, together with life-history traits and habitat requirements, certainly affect connectivity in G. divaricata. However, this scenario of potential differentiation seems to be overridden by sporadic events of long-distance dispersal across barriers and large effective population sizes.Genetic connectivity studies are essential to understand species diversity and genetic structure and to assess the role of potential factors affecting connectivity, thus enabling sound management and conservation strategies. Here, we analyzed the patterns of genetic variability in the marine snail  Connectivity among populations determines the dynamics of metapopulation systems, how genetic diversity arises and is maintained within species, and the adaptability and resilience of populations to environmental changes, among other factors . DefininGibbula divaricata, a common species of shallow-water trochid gastropods (commonly known as top shells). This species was recently reassigned to the genus Steromphala Gray, 1847  and high differences on the bathymetry, temperature, chlorophyll-like pigment concentration, among others factors that differentiate up to six different regions  potentially involved in such structuring. Genetic connectivity studies also provide valuable information for conservation and management plans in the area. For instance, coastal erosion and subsidence, among other factors such as the positive eustatism of the sea, will continue to affect the coastline for years to come  or Othonoi  islands, which, at least in the latter case, is likely due to the presence of Gibbula rarilineata, and its occupation of suitable habitats. The six remaining localities , according to the manufacturer’s protocol, including a RNase treatment. DNA was quantified with the Quant-iT dsDNA HS Assay using a Qubit fluorometer, according to the manufacturer’s instructions. Aliquots of 2 ng/μl were prepared for subsequent genotyping analyses. DNA quality was also checked on 1% agarose gels. All individuals were identified to the species level by molecular and (when possible) morphological determination. Molecular identification was made by DNA barcoding following G. divaricata  were estimated for each population with GenAlEx. We also tested the influence of null alleles and genotyping failures on FIS with INEST 2.2  for the six populations, using linkage disequilibrium, heterozygosity excess, and molecular co-ancestry methods, as implemented in NeEstimatorv2.1 . We estiFST) using Weir and Cockerham’s estimators with GENETIX v.4.03  were performed in GenAlEx with the obtained FST values. Furthermore, we used the function betas β in R with the Hierfstat package to calculate population-specific FST values  with the highest posterior probability. An admixture model was used under both the “popinfo” and the “popinfo” plus “location prior” functions with correlated allele frequencies. Twenty replicates per K were performed for K = 1–10 to calculate the mean log probability of the data [lnP(K)]. We exceed the number of analyzed populations to explore the potential existence of individuals belonging to locations outside the study area. MCMC iterations were set to 10,000 burn-in iterations and 100,000 sampled iterations. STRUCTURE Harvester  in the groups inferred by STRUCTURE. Clumpak ] and geographical distances (Ln distance), was assessed using the Mantel permutation test .Potential gene flow barriers were identified using BARRIER v.2.2 . PutativM) and mutation-scaled effective population sizes (theta). We then ran a full analysis under a Brownian motion microsatellite model, generating the initial theta and M values using the default FST calculations, and uniform priors of 0–50 and 0–2000 for the two parameters, respectively. We ran one long MCMC chain with 105 steps recorded and an increment of 100, excluding 104 steps as burn-in. We also checked for the adequacy of this full population and migration model by comparing its probability  with that of three other demographic scenarios: panmixia [a single population (KAP+BOK+KOR+TOG+OTR+POC)], two populations [Adriatic (KAP+BOK+KOR) vs. Apulian (TOG+OTR) + Ionian (POC) sides], and three populations [Adriatic (KAP+BOK+KOR) vs. Apulian (TOG+OTR) vs. Ionian (POC) sides]. To estimate the marginal likelihood for each of these four scenarios, we ran new analyses with the same priors as above and four MCMC chains with a static heating scheme . We also ran two analyses with asymmetrical models of gene flow, the first one favoring counter-clockwise dispersal, following the sea surface currents in the Adriatic, and the second one that goes against sea surface currents. These analyses were run in the Cipres Science Gateway v3.3 : Migrate-n estimates long-term or historical migration, BayesAss estimates recent migration, and GENCLASS2 identifies first generation migrants. For the first approach, implemented in Migrate-n 3.6 , we ran way v3.3 . We thenway v3.3 .m) between populations with the following parameters: number of iterations, 10,000,000; sampling frequency, 100; length of burn-in 1,000,000; delta allele, 0.1; delta migration, 0.2; and delta F, 0.1. Probabilities of exclusion and inclusion were also calculated. If the probability of exclusion was greater than 95%, an individual was excluded from its sampling site; it was re-assigned to one of the other sampled populations if the probability of inclusion was greater than 10% , indicating a heterozygosity deficit. When correcting the effect of null alleles on this parameter, the values decreased (avg. Fi = 0.016–0.078), and all highest posterior density intervals (HPDI = 0–0.143) included the zero value. Almost all populations showed nb as the best-fit model, thus reducing the role of inbreeding in the differences between observed and expected heterozygosities. For BOK and KOR, nfb was the best-fit model, although differences in DIC values between the two models were less than 1 /2]. However, in order to interpret the resulting HWE χ2 tests accurately, the sample size and the expected number for all genotypes should be more than 50 and 5, respectively  alleles among populations.Linkage disequilibrium among loci was not observed in any of the populations, implying that the 23 loci can be considered statistically independent. Even after Benjamini and Hochberg corrections, all localities had significant deviations from HWE for different loci, usually due to heterozygote deficits, which may be accounted for by the presence of null alleles. Two loci (Gd-L22 and Gd-L42) presented high frequencies for all studied locations and were, thus, removed from subsequent analyses. Problems associated with amplification, scoring or mutations in the regions where primers were designed may account for the presence of null alleles at these two loci. Other reasons may account for the deviation from HWE for the other loci. For example, 31 alleles were found for the locus Gd-5 in KOR, where 30 specimens were analyzed. These 31 alleles can be combined to give 496 different genotypes [ectively . The corFST values than neutral expectations, indicating positive selection. BAYESCAN showed four loci  as putative outliers under negative selection. Given the lack of agreement between the two tests of neutrality, all loci were considered for downstream analyses.Despite being considered neutral markers, LOSITAN identified two outlier loci: Gd-L3 and Gd-L32 showed significantly higher Estimates of pairwise relatedness among the samples ranged from -0.079 to 0.471; the mean value was -0.003. Plotting the indices, a unimodal distribution around zero was obtained (data not shown), indicating no relatedness among most specimens.Effective population sizes for the six populations were, in general, very large . Either FST revealed a low but significant level of genetic differentiation . Pairwise FST values ranged from negative values (that should be considered as zero) for KOR vs. KAP and TOG vs. OTR to 0.0460 (0.1738 for the standardized FST) for OTR vs. KAP  and geographic distance was revealed when using Euclidean distances between locations across the sea . In contrast, a higher correlation was found when geographic distances were measured following the coastline between sampled locations .No significant association between genetic differentiation  , a result that was supported by the highest ΔK value (ΔK = 7.80)  . The twoctively) . Since t = 7.80) . In factAMOVA analysis of the eastern and western groups revealed that 96.64% of the genetic variation originated within populations and only 2.31% among populations. The lowest percentage of variation (1.05%) was among populations within the two groups .FST values among populations represented in the PCoA analysis showed that 96.81% of variation could be explained by the first two axes. The first axis of the PCoA clearly separated OTR, TOG and POC from the other Adriatic localities  barrier between the east and the west coasts of the Adriatic Sea, dividing KAP, BOK, and KOR from TOG, OTR, and POC. The presence of a second barrier, separating POC from TOG and OTR, was also indicated but with lower support.M) were generally found between populations on either the eastern or western sides of the Adriatic Sea [populations KAP, BOK, KOR or TOG, OTR, POC, respectively  seems to have been historically constrained, as evidenced by the low M estimated for these two populations. However, when the marginal likelihood of the full migration model among the six populations was compared with those of the other demographic and gene flow scenarios , the results favored panmixia as the most likely scenario . The highest mutation-scaled migration rates  were found between populations from either the eastern or western sides   . Recent ctively) . RemarkaGibbula divaricata in the central and southern Adriatic Sea to infer its population structure and patterns of dispersal and gene flow. We found that, overall, populations seem well-connected and panmixia characterizes the species in the area. However, the presence of physical barriers to dispersal were also indicated in the area, and our results suggest that dispersion is mainly following the coastlines with help from the currents. One hypothesis is that this would eventually lead to high levels of genetic structuring and differentiation. However, this apparently is not the case in G. divaricata: we found evidence of weak though statistically significant differentiation among some of the populations, but overall panmixia is the rule. Large effective populations sizes and sporadic long-distance migration events among populations, as shown by the coalescence-based migration analyses, could explain this scenario. A large-scale study, nevertheless, would be necessary to estimate with more accuracy the patterns of gene flow, the demographic history of the species, and the precise physical environmental barriers influencing connectivity patterns in the species.Connectivity among populations, and patterns of dispersal and gene flow, are primarily determined by the physical characteristics of the landscape occupied by a species and the biological life-history traits of that species. Connectivity and gene flow, in turn, shape the patterns of genetic structuring of a species, which are of profound ecological and evolutionary importance. In this study, we analyzed the genetic structure of the topshell Overall, all of our analyses and tests consistently showed that connectivity was limited to some degree between the eastern and the western sites of the South Adriatic and, to a lesser extent, between Porto Cesareo in the Ionian Sea and the other two Italian localities along the Adriatic coast of the Apulian Peninsula. Taken together, our results indicate an Adriatic population  structured in two groups: one comprised of eastern populations and the other of western ones, although this last group showed a certain level of admixture see . FurtherMullus barbatus) (Scorpaena porcus)  can determine the capacity of larval exchange among populations and, hence, the extent and direction of dispersal (Genetic studies of the bentho-pelagic red mullet (arbatus)  and the  porcus)  have alsispersal . Likewisispersal .We found no relationship between genetic and geographic distances when using Euclidean distances between locations; however, an association was found when geographic distances were measured following the coastline distances between sampled locations. Therefore, factors such as the current system, coastline layout, small-scale local hydrodynamic processes or favorable habitat availability are likely to have greater influence on the degree of connectivity between different local populations than distance itself.G. divaricata, although thought to have limited dispersal abilities, can eventually reach and settle in distant populations. The precise timing of larval development in G. divaricata is still unknown; however, other species of the genus take no longer than 10 days to reach complete metamorphosis (G. divaricata) for particles to drift between TOG and OTR or POC , together with the coastal currents, likely favors a degree of dispersal along the coastline. The differentiation observed in the Ionian population may be due to, among other factors, the absence of sheltered habitats at intermediate points between OTR and POC. The southern area of the Apulian Peninsula consists of a high rocky coast exposed to intense wave action  and Ionian (Porto Cesareo) seas  seas . These fs sargus . Some aus sargus . NeverthGenetic homogeneity may be explained by a continuous and significant coastal marine current that connects different areas, thus promoting larval exchange. Surface drifter tracks and modeled particle trajectories indicate that the Adriatic circulation patterns sustain physical corridors as connection pathways within a large-scale functional cell of connectivity in the southern Adriatic . LikewisG. divaricata, may use patches of its specific habitat as stepping stones, thus allowing them to disperse over a wider range. As a result of stepping stone dispersion, ubiquitous shore species that have access to relatively continuous habitats would show high genetic connectivity, while species inhabiting patchy dispersed habitats, or isolated habitats such as estuaries and lagoons, would show low to no connectivity. This connectivity, in turn, can be modulated by the durations and behaviors of the early life stages of different species.In order to determine if connectivity is mediated through a continuous stretch of suitable habitats along the coastline, genetic analyses need to be combined with habitat mapping. Species with supposed low dispersal abilities, such as G. divaricata, and their ecological interactions, will shed light on the actual dispersal ability of the species and enlighten ongoing phylogeographic studies.Overall, our findings indicate weak differentiation among the studied populations belonging to the western and the eastern coasts of the central-south Adriatic. Although significant, the extent of differentiation and the strength of barriers were low, and some level of migration was evident, which taken together suggest very recent isolation or a weak restriction to gene flow. Multiple physical and biological factors may be influencing this population structure. Therefore, much research is still needed to determine the extent to which these factors affect connectivity in this species, not only in the Adriatic region but throughout its distribution. In addition, knowledge of the life cycle phases of Connectivity is increasingly recognized as a key conservation objective because of its importance for species replenishment . ChangesVL-M conceived the research, conducted field work and laboratory and data analyses, and wrote the manuscript. JT and AM conceived the research, were responsible for resource acquisition, conducted field work and wrote and provided comments on the manuscript. DB collaborated in data analyses and reviewed the manuscript. LZ conducted field work, collaborated in data analyses, and provided comments on the manuscript. IM, EB, and DM conducted field work and reviewed the manuscript. This research is part of VL-M Ph.D. thesis, co-directed by JT and AM.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aphis gossypii Glover (Hemiptera: Aphididae) is a serious polyphagous agricultural pest worldwide. In the present study, we used eight microsatellite markers to investigate the genetic structure and diversity of A. gossypii populations in Korea. Samples were collected from 37 locations in Korea  from pepper plants. A. gossypii had low to moderate genetic diversity, and expected heterozygosity (HE) ranged from 0.354 to 0.719. A Mantel test of isolation by distance indicated no relationship between genetic structure and geographic distance among all populations , suggesting high gene flow among populations in Korea. Populations of A. gossypii in Korea were divided into two distinct genetic clusters (ΔK = 2). In 2016 and 2017, the genetic clusters changed into opposite genetic structures within one year mostly in northwest and southeast parts of Korea. Possible relevance of study results was discussed. Chemical control, cyclical parthenogenesis, and immigrants from the exterior might have resulted in this low genetic diversity and opposite genetic clusters. Aphis gossypii Glover (Hemiptera: Aphididae), is a worldwide polyphagous insect species, with a large ecological and host range ) using the FreeNA program ) . Pairwislations) . Pairwislations) . Pairwislations) . FST adjA. gossypii expanded spatially without severe bottleneck in most regions of Korea. Population YC_16, JiJ_16, and HS_18 showed a significant bottleneck effect at p = 0.05 and a shifted shape (S) allele frequency distribution, providing evidence for recent population reduction in these populations  and three of 37 populations using the stepwise mutation model (SMM). Accordingly, there was potential evidence for recent population reductions in a few populations. Most populations had a normal L-shaped distribution , indicating that ulations .A. gossypii populations revealed a high variance component within individuals (85%), followed by among populations (15%) (FST). Based on the Mantel test for IBD, no significant correlation was found between genetic and geographical distances among populations , suggesting high gene flow among populations in Korea  for ns (15%) . The comin Korea .K (Evanno method) occurred at K = 2 with a maximum value of 1251.3  showed that the pattern of genetic structure was similar to the genetic cluster analysis result (two distinct groups) . Total vA. gossypii in Korea. In the present study, we analyzed genetic diversity within and between A. gossypii populations collected from pepper plants (mostly in greenhouses). Genetic variability found in A. gossypii through the use of molecular markers revealed that its clonal diversity is structured by its host plants [A. gossypii had low to moderate genetic diversity based on microsatellite data (HE varied from 0.354 (GwJ_16) to 0.719 (BS_16). Most HE values were lower than the average (average = 0.524). A similar level of genetic variation was shown in other aphid species (Sitobion avenae), for which HE ranged from 0.409 to 0.873 on the basis of eight microsatellite loci [A. gossypii, clonal diversity declined significantly as spring/summer season progressed [S. avenae, genetic variation significantly decreased from spring to summer [A. gossypii populations, collected from cotton crops, vegetable crops, and weeds in northern Cameroon, show very low genetic diversity (only 11 multilocus genotypes identified). The final predominance of the clone may occur through a combination of genetic drift related to population foundation, demographic explosion, and/or clonal competition. The final fittest founder genotype will become dominant during the period of rapid population growth, while recessive genotypes will decrease in frequency or may extinct completely. Moreover, Eriosoma lanigerum and Myzus persicae show low levels of genetic diversity due to adaptation of aphids to heavy selection pressures, including distribution of host plants and the use of insecticides [A. gossypii samples were collected from greenhouse peppers in the summer, during which most A. gossypii were under cyclical parthenogenesis. Chemical control was common in all greenhouses. This apparently led to heavy selective pressure for A. gossypii. These points may explain such low levels of genetic variability of A. gossypii observed in Korea. Moreover, A. gossypii developed resistance to insecticides such as carbamates and organophosphates. Its exponential growth due to parthenogenesis favors rapid selection for insecticide resistance traits [A. gossypii populations, to estimate insecticide resistance over temporal and spatial scales and investigate distribution of resistance genes among A. gossypii populations because resistance genes are related to genetic factors.This study is the first attempt to understand the pattern of genetic variability in t plants . In the ite data , in whic.354 GwJ_ to 0.719o summer . Brévaulo summer  revealedcticides ,44. Cyclcticides . In our e traits ,47. TherA. gossypii population structure in Korea  showed a significant p-value in the TPM model. Moreover, a change in genetic cluster in a period of one year might be caused by different fitness between two genetic clusters of A. gossypii in Korea. Two genetic clusters of A. gossypii might have coexisted in the same regions, and one genetic cluster could be a dominant genotype if there is a fitness difference between them.The moderate flight and dispersal ability of aphids might allow short-distance movement. Usually, the IBD test is widely used to examine spatial patterns of gene flow and genetic relatedness between populations . Resultsin Korea . In the in Korea . InteresA. gossypii populations in Korea based on eight microsatellite loci. A. gossypii populations in Korea appeared to be classified into two genetic clusters. However, its genetic structure rapidly changed into opposite clusters in several regions, although samples were collected from the same locations. Based on the results of bottleneck analysis , most A. gossypii populations experienced a recent spatial expansion without any severe bottleneck in most regions of Korea. However, several locations  were shown to be significant in TPM, which may affect the rapid turnover in genetic structure. These results provide important information for understanding its feasible local adaptation and dispersal patterns. A. gossypii populations in pepper-growing (field and greenhouse) areas in Korea showed low genetic diversity and high gene flow due to cyclical parthenogenesis and heavy insecticidal selection pressure. Thus, future work should elucidate any phenotypic fitness difference between the two genetic clusters of A. gossypii in Korea. Also, further studies should focus on the relationship between the genetic structure of A. gossypii populations in various crop-producing areas and the extent of insecticide selection pressure.Genetic diversity was revealed among


Arabidopsis thaliana derived from 30 natural populations not belonging to the 1001 genomes, collected at increasing altitudes, between 200 and 1800 m in the Pyrenees. Class III peroxidases and ribosomal RNA sequences were used as markers to determine the putative genetic relationships among these populations along their altitudinal gradient. Using Bayesian-based statistics and phylogenetic analyses, these Pyrenean populations appear with significant divergence from the other regional accessions from 1001 genome . Individuals of these populations exhibited varying phenotypic changes, when grown at sub-optimal temperature (22 vs. 15°C). These phenotypic variations under controlled conditions reflected intraspecific morphological variations. This study could bring new information regarding the west European population structure of A. thaliana and its phenotypic variations at different temperatures. The integrative analysis combining genetic, phenotypic variation and environmental datasets is used to analyze the acclimation of population in response to temperature changes. Regarding their geographical proximity and environmental diversity, these populations represent a tool of choice for studying plant response to temperature variation.Natural variations help in identifying genetic mechanisms of morphologically and developmentally complex traits. Mountainous habitats provide an altitudinal gradient where one species encounters different abiotic conditions. We report the study of 341 individuals of -A. thaliana in the Pyrenees mountains helps to understand European population structure and to evaluate the phenotypic trait variation in response to climate change.Studying the natural diversity of  Plant diversity represents a huge reservoir of variations maintained by different evolutionary processes, such as natural or artificial selection. The intraspecific natural variation, i.e., the within-species genetic and phenotypic variations, may reflect species local adaptations to different natural environments . AnalysiFor instance, with altitudinal gradients, plants must cope with multiple environmental variations including the decrease of temperatures and air humidity, the increase of UV radiation and a diminution of atmospheric pressure with rising altitudes . In respArabidopsis thaliana is an annual plant species distributed worldwide. It is an interesting model for plant molecular sciences, as well as for ecological and evolutionary studies. Several studies about natural genetic variation of A. thaliana have been published at European distribution scale. A large genetic diversity of A. thaliana with a strong geographic structure was observed and brought the hypothesis of multiple Iberian glacial refuges that contributed to the post-glacial recolonization of Europe by plants  and 15°C . Contrasting temperature regimes using 15°C as cool condition have been recently tested and have demonstrated the leaves plasticity of two A. thaliana ecotypes  have only been detected in green plants, they constitute a large multigenic family in land plants and due to their cell wall location, they could have important role during cell wall dynamic . In addiA. thaliana contains hundreds of 45S rDNA units, each of them encoding the 18S, 5.8S and the 25S rRNA genes separated by external (5′ETS and 3′ETS) and internal transcribed spacer (ITS1 and ITS2). The characterization of the 45S rDNA 3′ETS sequence from various A. thaliana populations originating from different geographic regions has revealed the existence of one to four 45S rDNA variants  genes or rDNA. In plants, two major classes of nuclear rDNA can be distinguished: 45S rDNA and 5S rDNA . The genvariants . Besidesvariants .A. thaliana populations from the French part of Pyrenees mountains have been tested using 1001 genome accessions as reference, (ii) the phenotypic variability in these populations at 22 and 15°C has been analyzed and (iii) the ability of a given population to acclimate or adapt to different growth temperatures has been evaluated via integrative approaches. This study highlights strong variations in the phenotypic response to environmental constraints, notably temperature variation, which seems to be decoupled from population structure of A. thaliana in the Pyrenees mountains.In the present study, (i) the genetic diversity and structure of A. thaliana in Pyrenees mountains allowed to identify 30 independent locations at different altitudinal levels all along the Pyrenees mountains . Population names correspond to the first four letters of the closest village or location where the plants were found . After 48 h of stratification at 4°C in darkness, plants were grown at 90 μmol photons m-2 s-1 light intensity, 70% humidity and 16 h light/8 h dark photoperiod at two different temperatures: 22°C  or 15°C . For rosette phenotyping and biochemical analyses, four- and six week-old rosettes were collected, respectively, at 22 and 15°C, corresponding to emergence of the first flower buds for Col and Sha . They correspond to the loci of 5 CIII Prx . PCR were performed using high fidelity recombinant Pfu DNA polymerase  according to the manufacturer’s instructions. PCR products were sequenced using the same primers and on both strands to ensure reading quality. Paired end sequencing and manual editing using BioEdit , 10 independent runs were performed, and likelihood values obtained from these 10 runs were averaged. To identify the appropriate K value, Evanno’s method based on data likelihood variation over successive K was used  and 22 accessions available from the 1001 Genomes Project (11 in South France and 11 in North Spain the most closely to the Pyrenees mountains) were analyzed , S2. Tajwas used . The opt K value .The analysis involved 375 nucleotide sequences . Phylogenies can be inferred separately from each gene or directly from concatenated supergene with various advantages . The con-2 s-1 continuous white light. The Petri dishes were observed using a Zeiss Axiozoom V16 stereomicroscope. The seed pictures were analyzed using ImageJ. Testa and endosperm ruptures (TR and ER) were quantified, using the testa integrity and the radicle tip protrusion as markers, respectively (Seeds were sterilized  and sown in Petri dishes containing half-concentrated MS medium  supplemeectively . The perRosettes grown at 22 and 15°C were analyzed at 4- and 6-weeks, respectively. These durations correspond to the necessary times for the two Col and Sha accessions to reach the bolting stage at these temperatures . RosetteTo identify the maximum growth speed of the floral stems, a logistic function was used . The datg. The absorbance of chlorophyll α and β was measured in the supernatant, respectively at 663 and 647 nm. Chlorophyll concentration was calculated using the following formula: (A663 × 7.15 + A647 × 18.71)/mg of fresh material = μg chlorophyll/mg fresh material (530 – 0.25 × A657)/mg of fresh material)  was vigorously vortexed with 1 mL of 80% acetone solution for 5 min or with 1 mL of 95% ethanol/1% HCl and stored at 5°C for 24 h in darkness for chlorophyll or anthocyanin contents, respectively. The samples were centrifuged for 10 min at 1000 material . The antaterial) .4 to allow a better visualization of the different sets when they correspond to averages. Kruskal–Wallis and Wilcoxon non-parametric tests were carried out to compare groups. To investigate the underlying variation between population data (phenotypic), Principal Component Analysis (PCA) with multilevel function was used with the mixOmics package . Student’s statistical tests for two samples were carried out in order to determine the temperature effects on the populations. In addition, Duncan’s multiple range tests were performed with the package agricolae package  availablA. thaliana as well as Lant, Sha and Col, were described using the altitude (m) and the following 5 climatic variables (2).The 30 Pyrenean populations of ariables : annual The variables describing temperature spectrum  are highly positively correlated but negatively correlated with altitude, annual rain accumulation and solar radiation . To analCIII Prx loci  was analyzed. Those loci played major roles in Col, during most of development processes and in response to many biotic and abiotic stresses , meaning that null hypothesis of neutral mutation drift equilibrium for these sequences could not be rejected. Consequently, these markers could then be considered as neutral and used for genetic analysis of population structure. The polymorphism within the populations was evaluated using different parameters: the haplotype diversity and frequency distribution, the number of SNPs and the percentage of identity of haplotypes contents in each population  and those of 22 accessions available from the 1001 Genomes Project  and locapulation . This anpulation .A. thaliana populations, two different approaches were used: a Bayesian clustering analysis and a phylogenetic analysis. These two analyses complement to evaluate the different polymorphism parameters in the genetic analysis.To analyze the genetic structure and polymorphism of the Pyrenean K value), each characterized by a set of haplotype frequencies. These STRUCTURE analyses allowed to identify two genetic clusters for the Pyrenean populations  method using the concatenated sequences of the 5 CIII Prx markers. The tree segregated the populations into three major clusters  and the 3 out-groups Lant, Col and Sha . Color represents the population allocations into their estimate membership proportions in each genetics cluster determined by STRUCTURE and illustrated in the Molecular phylogenetic analysis (maximum likelihood method) based on five independent peroxidase gene markers of 341 individuals from the 30 The germination rate was constant for all the Pyrenean populations tested with a rate higher than 90% (data not shown). It demonstrated a high seeds viability in all the populations independently of the temperatures used for the germination assays. However, the germination speed differed between the populations.A. thaliana, successive early steps of germination comprise testa rupture (TR) and endosperm rupture (ER) (In ure (ER) . The perure (ER) . The gerure (ER) . Interesure (ER) . This coure (ER) .A. thaliana grown at 22°C  were compared to those of plants grown at 15°C . Contrasted rosettes phenotypes were observed between the two temperatures , and Biel (shorter stems at 22°C than at 15°C). The stems growth speed was also significantly faster at 15°C. For example, Eaux, Fos and Chau stems grew slower at 22°C than at 15°C .A. thaliana Pyrenean plants grown at different temperatures, anthocyanin content was quantified in the leaves of individuals cultivated at 15 and 22°C. Most of the populations  showed an increase of anthocyanin content of rosettes grown at 15°C as compared to 22°C  and those with more chlorophyll at 22°C than at 15°C . It should be noted that regardless of the variation between the two growth temperatures, all the populations revealed about the same chlorophyll content at 15°C .Arabidopsis to the different growth temperatures. In fact, the 10 traits measured  varied not only between the growth temperatures but also between the populations/accessions; hence, PCA was performed to get more integrative results. Multilevel methods of PCA can be used if repeated measurements from different treatments are applied on the same individual. This multilevel approach was developed to highlight treatment effects within subject separately from the biological variation between subjects . This separation was mainly due to the chlorophyll overall content which was more abundant in rosettes from plants cultivated at 22°C as compared to 15°C, and to the other phenotypic parameters generally higher at 15°C than at 22°C in most of the populations. Indeed, highland population such as Prad and Hosp appeared to contain more chlorophyll at 22°C than at 15°C unlike to the lowland populations Chau and Biel. The second axis differentiated Pyrenean populations according to their speed to reach the bolting stage, which was negatively correlated with the floral stem speed regardless of the temperature. For example, Bran, Fos and Eaux reach the bolting stage earlier when grown at 22 vs. 15°C and have a faster stem growth speed than the other Pyrenean populations when cultivated at 22°C. Therefore, the time to reach bolting stage and stem growth speed appear to be independent and not correlated with the developmental stages, and hence useful to compare different genotypes.The effect of genetic clusters or environmental data on phenotypic trait variation was evaluated by Kruskal–Wallis non-parametric tests A. thaliana in the Pyrenees is to have bigger rosette at low temperature growth conditions.In the populations assigned to the three distinct genetic clusters by STRUCTURE analysis, rosettes and floral stems phenotypes were impacted by growth temperature. Indeed, 9 of the Pyrenean populations considered as homogeneous (cluster 1 and 2) have systematically wider stems than the population of mixed ancestry (cluster 3) at both growth temperature conditions . MoreoveFinally, climate PC1 value obtained from normed PCA of six environmental datasets, allows classifying populations as a function of their environmental conditions considering altitude levels. The populations were distributed as plain , medium  and high altitudes  . Some coAll the climatic variables are usually well correlated with altitude, although the field topology may play an important role. Precipitation and seasonality exert the largest influence on the regional climate variation . PyreneeSequence polymorphism is currently used to analyze genetic variability and structure of natural populations  and to sA. thaliana populations collected in the Pyrenees mountains are genetically separated from the 22 accessions originated from the south of France and the north of Spain and described in the 1001 Genomes Project. The specific structure of these populations in this geographic area makes them very interesting to understand regional population structure in A. thaliana. However, the genetic diversity in the Pyrenees mountains is not just defined by a single genetic lineage; rather, fine-scale patterns of population structures were depicted by the phylogenetic tree. These inter- and intra-population variations may be associated with the variability of the natural environment and may demonstrate a specific adaptation of A. thaliana within this topographic complex area that may have acted as geographic barriers. Nevertheless, they could also have resulted from a prehistoric introduction or from a glacial refuge. It is interesting to note the complementarity of the results obtained with the different approaches . Indeed, populations that are homogeneous according to the Bayesian clustering approach (STRUCTURE) are also those with a high percentage of sequence identity and were found on one branch of the tree . Conversely, the populations considered as heterogeneous were distributed into two main branches of the tree .According to the 5 CIII Prx markers used, most (70%) of the Haplotype diversity and frequency , BayesiaAethionma arabicum  a rapid germination which allows the plant to grow faster in the frame of favorable climatic conditions and (ii) a slow germination speed which allows the seed to optimize the timing to germinate in different environmental constraints. This later strategy could be correlated to seed dimorphism as observed in arabicum . In addiarabicum  and, conAnthocyanin accumulation in leaves is considered as a general stress marker in plants  but in sin natura : 9 Pyrenean populations appear genetically close and homogeneous with regard to other accessions known in the region; 12 Pyrenean populations appear genetically close, homogeneous and specific of these mountains and 9 Pyrenean populations appear as populations of mixed ancestry.In summary, the present characterization of 30 populations of A. thaliana across abiotic gradient modeled here with the temperature change. Some of them are correlated with identified genetic clusters or with environmental data. These analyses contribute to enrich knowledge on abiotic stress acclimation in natural plant populations. Preliminary analyses suggest that the phenotypic acclimation may be due to the cell wall modification affected by low temperature (This study also revealed phenotypic variations in acclimation of perature . TranscrA. thaliana populations to heterogeneous abiotic conditions found in mountain environments.Adaptation of plants to changing environments is a complex process in the context of rapid and transitory environmental changes. It is not possible to conclude that the most adapted phenotype to the temperature elevation is always the most plastic phenotype, but phenotypic trait variation could drive the acclimation of HD, PR, and CD planned, designed the research and performed the bioinformatics analysis. HSC and MaB performed genetic analyses. HD and DLMM performed the phenotyping analyses. AE and SV performed germination assays. NE and MB collected of natural population and supervised the genetic analyses. PR performed the DNA extraction and the sequencing data. VD-H, JS-V, and J-PR supervised the rRNA genetic analyses and analysed the data. HD and SD designed the statistical analyses and analysed the data. HD and CD wrote the article. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oncorhynchus mykiss) was utilized to detect polymorphisms in seven salmonid species, including Hucho taimen, Oncorhynchus masou, Salvelinus fontinalis, Brachymystax lenok, Salvelinus leucomaenis, O. kisutch, and O. mykiss. The number of polymorphic markers per population ranged from 3,844 (O. kisutch) to 53,734 (O. mykiss), indicating that the rainbow trout SNP array was applicable as a universal genotyping tool for other salmonid species. Among the six other salmonid populations from four genera, 28,882 SNPs were shared, whereas 525 SNPs were polymorphic in all four genera. The genetic diversity and population relationships of the seven salmonid species were studied by principal component analysis (PCA). The phylogenetic relationships among populations were analyzed using the maximum likelihood method, which indicated that the shared SNP markers provide reliable genomic information for population genetic analyses in common aquaculture salmonid fishes. Furthermore, this obtained genomic information may be applicable for population genetic evaluation, marker-assisted breeding, and propagative parent selection in fry production.Various salmonid species are cultivated in cold water aquaculture. However, due to limited genomic data resources, specific high-throughput genotyping tools are not available to many of the salmonid species. In this study, a 57K single nucleotide polymorphism (SNP) array for rainbow trout ( Salmonid fishes are naturally distributed in freshwater and marine habitats around the world. Salmonid fishes are of high economic, recreational, and ecological value. However, the genetic variation of salmonid species has decreased to different degrees because of increased inbreeding due to human activities. For aquaculture species, supportive or assistant breeding causes inbreeding depression or loss of genetic variability ,2. InbreOncorhynchus, Salvelinus, Brachymystax, and Hucho are major cold water cultural fishes in China. Among these salmonid fishes, rainbow trout, O. mykiss, is the most widely cultivated species. It is also a worldwide aquaculture species with relatively well investigated resources of genomic data [The family Salmonidae consists of three subfamilies and 11 genera, including more than 70 species . Speciesmic data –12. Basemic data . Abundanmic data , RNA-seqmic data ,16, exprmic data , restricmic data , and genmic data . Out of mic data . This armic data , genome-mic data , and genmic data .Salmo salar), two salmonid fishes with whole genome sequencing projects, genomic resources for other salmonid fishes are limited. Mitochondrial DNA and microsatellites have thus been used as resources for genetic analysis of other salmonid species [Cyprinus carpio) we observed that a considerable number of polymorphic SNPs are shared among phylogenetically related species. Thus, the common carp SNP array may be utilized in eight other species of family Cyprinidae [However, except for rainbow trout and Atlantic salmon , Oncorhynchus masou , Salvelinus fontinalis , Brachymystax lenok , Salvelinus leucomaenis , Oncorhynchus kisutch , and Oncorhynchus mykiss rainbow trout . All sampling procedures complied with the guidelines of Animal Care and Use Committee (ACUC) of the Centre for Applied Aquatic Genomics, Chinese Academy of Fishery Sciences. All sampling procedures and experimental manipulations were approved as part of obtaining the field permit. Approximately 1 cm2 tail fin tissue was cut from each individual, and then dried at 56 oC before short storage at room temperature. The sampled individuals were released to the ponds for natural fin regeneration. Genomic DNA was extracted from fin tissues using a Marine Animal Genomic DNA Extraction Kit , following the manufacturer’s recommendations. The integrity of DNA was analyzed on 1% agarose gels. DNA was quantified using a NanoDrop 8000 device .In this study, 96 cultured samples representing seven salmonid species were collected for genotyping and further analyses. Fish samples were collected at three sites: Mudanjiang in Heilongjiang, Dujiangyan in Sichuan, and Huairou in Beijing, China. Unrelated individuals from seven salmonid species were sampled: http://media.affymetrix.com/support/downloads/manuals/ axiom_best_practice_supplement_user_guide.pdf). SNP validation and statistics was processed using PLINK v1.09 (https://www.cog-genomics.org/plink2) [More than 5,000 ng of genomic DNA from each sample were sent to a commercial service provider  for genotyping, which was performed using the 57K rainbow trout SNP array, according to Axiom genotyping procedures described by Affymetrix. Affymetrix Power Tools (APT) and SNPOLISHER software packages were used for genotyping and quality control, following the manufacturer’s recommendations (/plink2) ,29. A mo2 < 0.2 and MAF > 0.02, respectively. The R package (www.r-project.org/) was used to visualize the PCA results. Customized high-resolution Venn diagrams were constructed to visualize the shared and unique SNPs among the four salmonid genera using the VennDiagram suite in the R package [Hucho, Oncorhynchus, Salvelinus, and Brachymystax genera were evaluated. One species from each genus was selected as representative.Principal component analysis (PCA) was performed to assess the genetic relationships among seven salmonid species using PLINK v1.09 and GCTA  software , respect package . The disPhylogenetic analysis of the salmonid populations was performed with CLC Genomics Workbench 9.5.4, and a maximum likelihood phylogenetic tree was generated. Bootstrapping was performed using the group population option with 1,000 replicates over loci. Phylogenetic analysis was conducted using the genotyping data of 11,643 SNPs  shared by all populations. A phylogenetic tree was constructed using EvolView v2 ,33.The 96 salmonid individuals used in this study represented seven different populations based on species and strains, namely, HT, OM, SF, BL, SL, OK, and RT. The call rate distribution of each sample is shown in Oncorhynchus, the OK and OM populations showed low polymorphic rates . The number of SNPs and polymorphic SNPs of each population are summarized in While a moderate call rate threshold was set (>85%), all the non-rainbow trout samples passed quality control. The number of informative SNPs in the seven populations varied from 47,347 to 55,955. The number of polymorphic SNPs in each group ranged from 3,844 in the OK population to 53,734 in the RT population. The RT population exhibited a high SNP polymorphic rate (96%). Although belonging to the same genus Hucho, Oncorhynchus, Salvelinus, and Brachymystax genera were 1,151, 1,386, 612, and 1,003, respectively. In contrast, in terms of polymorphic SNP sets . The number of informative and polymorphic SNPs in the overlapping area of the Venn diagram among the four genera was 28,882 and 525, respectively. Hucho and Brachymystax shared the highest number of SNPs  between two genera, whereas Salvelinus and Brachymystax shared the highest number of polymorphic SNPs  between two genera.The shared SNP statistics of the four genera are presented using Venn diagrams . In termSNP sets , by contThe genetic relationships among salmonid individuals and populations were analyzed by PCA. Different SNP datasets were obtained by different filter thresholds: 1) 57,501 SNPs with no filter; 2) 53,436 SNPs with a call rate >85% for all 96 individuals; 3) 28,882 SNPs with call rate >85% in each population; and 4) 2,393 SNPs with a call rate >85% in each population and MAF >5% in each population. PCA of each dataset was performed using PLINK v1.09 and GCTA. With varied principal component (PC) values, the clustering distributions of individuals were highly similar among the PCA results while using 1) - 3) datasets and both software. The 96 individuals were clustered into four groups: 48 rainbow trout individuals were clustered as a loose group, and the other six species were classified into three separate groups. While the number of SNPs was reduced to 2,393, all the non-rainbow trout individuals belonged to a single group.Oncorhynchus mykiss from the other six populations. The HT and BL populations were clustered together, whereas the OM and OK populations and the SF and SL populations were respectively clustered into one group.The GCTA results of 28,882 SNPs was adopted in this study . In the Oncorhynchus, were clustered with the O. mykiss clade at one branch. A parallel branch consisting of the SL clade and clades were classified under genus Salvelinus.Phylogenetic reconstruction of the 96 individuals representing seven salmonid populations was performed using a dataset of high-genotyping quality 11,643 SNPs, which were obtained using a call rate threshold of >99%. The seven species were clearly differentiated in the maximum likelihood tree , with hiComparative genomic studies have indicated that salmonid fishes emerged after a whole genome duplication event involving an autotetraploid ancestor, which was estimated to have occurred 88–103 million years ago ,34–37. TB. lenok, a native salmonid species in China, exhibits high genetic diversity. However, some aquaculture species that were introduced from foreign countries, which include O. kisutch, showed limited polymorphisms, suggesting that their genetic diversity is low, and introduction of new broodstock is necessary.The shared SNPs varied among non-rainbow trout populations, indicating that these markers may be utilized for genetic analyses. With eight individuals sampled in each population, polymorphic rates of 7.8% (OK) to 38.3% (BL) were observed. These observations suggest that the aquaculture population of O. clarkii (N = 5), O. tshawytscha (N = 3), and O. kisutch (N = 4) [The 57K SNP array has been utilized in three non-rainbow trout species namely  (N = 4) . A relatPCA is a dimension-reducing technique that represents all datasets with a handful of SNPs corresponding to the most significant PCs and can be used to visualize population relationships. As shown in our results, a large dataset  was necessary to extensively illustrate the genetic diversity and population relationships among representative salmonid populations in China . The nonO. mykiss was significantly longer than other species, suggesting that over centuries of domestication, this species underwent strong selection, thus its mutation rate was higher than that of other species. Genetic relationships among individuals could also be visualized in the phylogenetic tree, which could help identify subgroups within the investigated populations, as well as select proper propagative parents with enough genetic distances.The phylogenetic tree was constructed based on both polymorphic and monomorphic SNPs, with the latter because providing essential information on conserved sites. The positions of different species in the phylogenetic tree coincide with their evolutionary relationships, indicating that the genotyping results are highly reliable. The tree branch of In most hatcheries in China, salmonid fishes are cultured in nearby areas because these have similar environmental conditions. As a universal genetic tool, the 57K SNP array may facilitate genetic analysis these poly-cultured salmonid species. For different purposes and species, suitable sets of SNP markers from the 57,501 candidate SNPs could be selected for genotyping. In this work, we presented the genotyping results of 96 individuals from seven populations, which belonged to four genera and seven species. O. kisutch, showed low genetic diversity. Genetic distances among individuals and subgroups within the populations were discovered. The results of the present study may be potentially used in the genetic evaluation of germplasm resources, as well as genomic selection in salmonid breeding.In this study, a 57K rainbow trout SNP array was utilized to explore the genetic diversity and population relationships in a variety of salmonid populations in China. High genotyping call rates (>90%) were obtained, whereas the polymorphic rates within populations ranged from 7.8% to 96%. A number of shared SNPs across species was developed and used for population genetic analyses, including PCA and phylogenetic reconstruction. One of the study populations, S1 Table(CSV)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


Aedes aegypti transmits viral diseases that have plagued humans for centuries. Its ancestral home are forests of Africa and ~400–600 years ago it invaded the New World and later Europe and Asia, causing some of the largest epidemics in human history. The species was rarely detected in countries surrounding the Mediterranean Sea after the 1950s, but during the last 16 years it re-appeared in Madeira, Russia and in the eastern coast of the Black Sea. We genotyped Ae. aegypti populations from the Black Sea region to investigate whether this is a recent invasion  or a remnant of pre-eradication populations that extended across the Mediterranean. We also use the Black Sea populations together with a world reference panel of populations to shed more light into the phylogeographical history of this species.The yellow fever mosquito Microsatellites and ~19,000 genome-wide single nucleotide polymorphisms (SNPs) support the monophyletic origin of all populations outside Africa, with the New World as the site of first colonization. Considering the phylogenetic relationships, the Black Sea populations are basal to all Asian populations sampled. Bayesian analyses combined with multivariate analyses on both types of markers suggest that the Black Sea population is a remnant of an older population. Approximate Bayesian Computation Analysis indicates with equal probability, that the origin of Black Sea populations was Asia or New World and assignment tests favor the New World.Ae. aegypti left Africa and arrived in New World ~500 years ago. The lineage that returned to the Old World and gave rise to present day Asia and the Black Sea populations split from the New World approximately 100–150 years ago. Globally, the Black Sea population is genetically closer to Asia, but still highly differentiated from both New World and Asian populations. This evidence, combined with bottleneck signatures and divergence time estimates, support the hypothesis of present day Black Sea populations being remnants of older populations, likely the now extinct Mediterranean populations that, consistent with the historic epidemiological record, likely represent the original return of Ae. aegypti to the Old World.Our results confirmed that The online version of this article (10.1186/s13071-018-2933-2) contains supplementary material, which is available to authorized users. Aedes aegypti bears the official common name of “the yellow fever mosquito”, although today it is more feared as the major vector of viruses causing dengue, chikungunya and Zika fevers. There is little doubt that the origin of Ae. aegypti is Africa. This is consistent with ecology, biogeography, genetics and historical records  for the ancestral forest form and Ae. aegypti aegypti (Aaa) for the domestic form.view see  and refeod meals , 3. At sod meals , 4. ThesAe. aegypti outside Africa has been dynamic, as expected of an invasive human commensal  , 15; andThe Black Sea region is represented by samples from Turkey and Georgia. Two locations were sampled from Turkey (~50 km apart) and three locations from Georgia . Since the sample size of the two inland Georgia localities was small , the preliminary analysis indicated that they form one homogenous group with low differentiation among them, and due to their geographical proximity (within ~10 km), these samples were pooled and treated as a single population sample of 15 individuals labeled as Georgia-inland.Ae. aegypti SNP-Chip  it should have close genetic affinities with some other population(s) in the reference panel, as observed in several other cases of recent re-invasions around world –67. As nAe. aegypti from the New World arrived in Asia ~150 years ago, coinciding with previous studies and the historic record [Ae. aegypti populations studied, however, we note that given the limitations presented above, we cannot reject that the actual age of the BS population could exceed the ~100–150 years, which would allow the estimate to be consistent with historical epidemiological data.The most likely phylogeographical scenario, supported by two independent analyses (ABC and RASP) is that c record . HoweverAe. aegypti). Urban dengue and chikungunya first appeared in Asia in 1879–1890. The opening of the Suez Canal in 1869 has been proposed as the path that facilitated this dispersal [Ae. aegypti arrival to Asia ~150 years ago reinforce this hypothesis. Although we included a similar scenario in our ABC analysis  and indicative worldwide sampling localities where Ae. aegypti is established as downloaded from www.worldclim.com. Table S4. Genetic diversity. Summary of the population genetic diversity statistics for the 56 populations of Ae. aegypti consisting the microsatellite dataset. Figure S1. STRUCTURE bar plots based on the microsatellite dataset, including equal number of populations from each region. Population names are reported on their X axes. For each STRUCTURE run only the number of genetic clusters supported by the Evanno method is presented. For details see legend of Fig. Figure S2. STRUCTURE bar plots based on the microsatellite dataset, including equal number of Ae. ae. aegypti populations from each region. For each STRUCTURE run only the number of genetic clusters supported by the Evanno method is presented. For details see legend of Fig. Figure S3. Principal Components Analysis (PCA) on the global (a) and the Ae. ae. aegypti (b) microsatellite dataset as implemented and plotted using the ade4 package in R. Figure S4. Discriminant Analysis of Principal Components (DAPC) for the Ae. ae. aegypti populations based on the microsatellite dataset. Figure S5. Discriminant Analysis of Principal Components (DAPC) for the Ae. aegypti populations collected from Turkey and Georgia based on the microsatellite dataset. Figure S6. Assignment test as implemented in Geneclass2 for the Black Sea populations and using the remaining worldwide populations as reference panel. (PDF 8863 kb)


Brassica napus L.) is one of the most economically important oilseed crops worldwide. In Japan, it has been cultivated for more than a century and has formed many feral populations. The aim of this study was to elucidate the genetic diversity of feral rapeseeds by genotyping 537 individuals  sampled from various regions in Japan. Analysis of 30 microsatellite markers amplified 334 alleles and indicated moderate genetic diversity and high inbreeding  within the feral populations. The Mantel test showed only an insignificant weak positive correlation between geographic distance and genetic distance. Analysis of molecular variance showed a greater genetic diversity among individuals than between populations. These results are in accordance with population structure assessed by using principal coordinate analysis and the program STRUCTURE, which showed that the 537 individuals could be assigned to 8 genetic clusters with very large genetic differences among individuals within the same geographic population, and that among feral individuals, many are closely related to rapeseed accessions in the NARO Genebank but some have unknown origins. These unique feral rapeseeds are likely to be affected by strong selection pressure. The results for genetically modified individuals also suggest that they have two different sources and have a considerable degree of diversity, which might be explained by hybridization with nearby individuals and separation of hybrid cultivars. The information obtained in this study could help improve the management of feral rapeseed plants in Japan.Rapeseed ( Brassica napus L.; genome AACC, 2n = 38) is one of the most economically important oilseed crops worldwide with 75 million tonnes produced per year [Rapeseed , has produced genetically modified (GM) rapeseed since the late 1990s; in recent years, the percentage of GM rapeseed in Canada has reached nearly 100% ,14. In aIn a previous study, we performed multiplex PCR using single-locus simple sequence repeat (SSR) primers to analyze the genetic diversity of Japanese and overseas rapeseed accessions preserved in National Agriculture and Food Research Organization (NARO) Genebank . By idenThis study had three objectives: (1) Use SSR markers to characterize the genetic diversity of feral rapeseed populations collected from various locations in Japan. (2) Compare the SSR marker-based genotype data of feral rapeseed and NARO Genebank resources to explore the possible sources of feral rapeseed. (3) Ultimately, explore the possible origin of GM feral rapeseed.B. napus  were obtained from the National Institute for Environmental Studies (NIES) of Japan (143 accessions) and the Ministry of Agriculture, Forestry and Fisheries (MAFF) of Japan (394 accessions). The seeds of the former materials were collected in 2005 [Samples of feral  in 2005 , and the in 2005 . If at lSeeds were germinated on moistened filter paper in 9-cm Petri dishes and then transplanted into 72-cell trays filled with Nippi-Engei-Baido-1gou soil . Seedlings were grown in the farm of Tsukuba Plant Innovation Research Center, University of Tsukuba, until the first true leaves had fully expanded. Genomic DNA was extracted from the first true leaf of each seedling and the collected leaves by using the DNeasy Plant Mini Kit .2, 0.1 U KAPA 2G Fast DNA polymerase, 2 pmol reverse primer, and 0.5 pmol forward primer. The forward primers were 5′-labeled with the fluorescent dyes 6-FAM, VIC, NED, or PET [Thirty single-locus SSR markers screened in our previous study  were use, or PET . PCR wasF statistics indices, and Nei’s genetic distance [Ho) , expected heterozygosity (He), and Shannon’s information index (I) were calculated in GenAlEx v. 6.502 software [The number of alleles, major allele frequency, polymorphism information content (PIC), distance  were caldistance . Observesoftware .https://www.google.com/earth/) from the sampling location information.Hierarchical analysis of molecular variance (AMOVA) and the Mantel test of the association between genetic distance and geographic distance were calculated in Arlequin v. 3.5.2.2 software . The geoK value) and to assign rapeseed individuals to different clusters. The K value was determined by running an admixture and related frequency model with K = 1 to 15 ; the burn-in period of each run and the Monte Carlo Markov Chain lengths were both set to 100,000. The website program STRUCTURE HARVESTER was used to estimate the optimal K value [K method of Evanno et al. [Genotype data for the SSR markers were analyzed in the model-based STRUCTURE v. 2.3.4 software  to deter K value ; this pro et al. .To reveal the population structure, we combined the genotype data from the current study with the NARO Genebank data for 582 accessions from our previous study that used the same markers . Then prHe (indicating moderate to high levels of polymorphism), 0.16 for Ho , and 0.61 for PIC (indicating that these markers were informative). The FIS, which is the deviation of the actual frequency of the genotype from the theoretical expected frequency in the population, was 0.68 , indicating a lack of heterozygosity across all markers in the feral populations, and implying that the genotype frequencies at all markers may deviate from Hardy–Weinberg equilibrium in the feral populations.The 30 SSR markers amplified a total of 334 alleles, ranging from 2 (BoEMS0049) to 39 (BrGMS0070) alleles per marker  to 0.27 (Shibushi). In all populations, Ho was lower than He, and the inbreeding coefficient within individuals FIS in each population was substantially greater than 0 , implying that each population may deviate from Hardy–Weinberg equilibrium.The genetic diversity indices for geographic populations are summarized in ST) ranged from 0.038 (Fukuoka and Yokkaichi) to 0.566 (Kitakyushu and Tomakomai) (ST > 0.4). A similar tendency was seen for the Nei’s genetic distance: the Kitakyushu population showed a relatively high distance from the other populations  between geographic distance and genetic distance (The pairwise genetic differentiation coefficient (Fmakomai) . Among tdistance .AMOVA of the geographic populations  showed tK indicated K = 8 as the most likely model , and PIC  indicate that the SSR markers are suitable for diversity analysis of feral rapeseed. Feral rapeseed populations are found in countries other than Japan [He of the feral populations in Japan is higher than that of populations in Austria (0.62 versus 0.32) [In this study we explored the genetic diversity and population structure of feral rapeseed populations in Japan by using the SSR markers which we used in our previous study . By genoan Japan , but theus 0.32) . Japan mIS), which ranges from −1 to +1, indicates the degree of inbreeding. Extremely positive significant values mean that outcrossing predominates in the population, whereas extremely negative significant values mean that inbreeding predominates [IS value of the feral populations was 0.68 , which ranges from 0 to 1, is an important indicator of genetic differentiation between populations; values over 0.15 are considered to indicate substantial genetic differentiation [P < 0.05; ST values around 0.2, indicating that the differentiation between them and other populations was moderate; the Kitakyushu population had pairwise FST values of >0.4, indicating strong differentiation. These results show that individuals within these populations were different from those in other populations, indicating that all of the feral populations except Kitakyushu may have originated from several founding events including but not limited to escapes of cultivars with different genetic backgrounds, or from seeds dropped during transportation. Such origin of these populations can also explain why the Mantel test revealed no significant correlation between genetic distance and geographic distance among the different populations. The result of AMOVA hierarchical analysis showed genetic variation mainly among individuals and not between populations, providing further evidence that most feral populations were composed of individuals that differed from each other. The Kitakyushu population showed strong identity in the PCoA and STRUCTURE analysis, and a different genetic background from other populations, perhaps because it had a single source or because only nine samples were collected from it. In a future study, more samples should be collected from the Kitakyushu population to confirm the results.The population genetic differentiation coefficient Click here for additional data file.S2 Fig(EPS)Click here for additional data file.S3 FigK = 4, all individuals were classified into four groups.At (EPS)Click here for additional data file.S4 FigThis figure is an enlargement of part of (EPS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Myzus persicae (Sulzer), is a worldwide pest of many crops, and the most important aphid pest of peach and potato crops in Tunisia, mainly due to virus transmission, for which insecticides are frequently applied. We studied the genetic structure of M. persicae populations in Tunisia, in order to further our understanding of the biotic and abiotic factors shaping populations and to predict their evolutionary responses to the present management practices. We monitored peach orchards and seed potato crops in different seasons and regions from 2011–2013 and in 2016 (19 populations), assessing the genetic diversity of M. persicae at six microsatellite loci. Temporal and spatial changes in the frequency and distribution of 397 genotypes in 548 sampled aphids were studied. Only 37 genotypes were found more than once , as most genotypes were found only once . A similarly high genetic diversity was observed in aphids sampled from peach  and potato . Only a weak genetic differentiation among populations was found, mainly between geographic locations. Clustering analysis revealed genotypes to be grouped mainly according to host plant. The availability of the primary host, high proportion of unique genotypes, high genetic diversity and lack of structuring suggest that the aphid reproduces mainly through cyclical parthenogenesis in Tunisia. On the other hand, we provide a farm-scale study that shows how easily M. persicae can colonize different areas and hosts, which may have important implications in relation to plant virus vectoring.The peach potato aphid,  In agroecosystems, insect pest populations can rapidly evolve to the latest pest management practices and global changes . UnderstAphids (Hemiptera: Aphididae) can pose a major threat to food crop safety due to direct physical damage and vectoring viral plant diseases . Aphids Myzus persicae (Sulzer), is one of the most exceptional aphid pest species globally. Originating in Asia, this aphid has a cosmopolitan distribution, being found anywhere crops are cultivated  and 2.8 migrants per generation after correcting for sampling size. Migration between geographic zones  was even higher (p(1) = 0.005; 17.6 migrants).M. persicae populations worldwide. For instance, a recent study on Italian populations of the peach potato aphid showed a total of 90 alleles at 8 microsatellite loci  [M. persicae aphids revealed 49 alleles  at five microsatellite loci [All of the six microsatellite loci studied were polymorphic and produced a total of 92 alleles , results which in terms of the level of polymorphism are comparable to those found for other r locus) . Similarr locus) , while aite loci .M. persicae from Tunisia [P. persica), the primary host where M. persicae reproduce sexually, and (ii) the harshness of the winter climate, and the management practices involved . Parthenogenetic reproduction can quickly produce so-called ‘genetic inflation’, a population genetic phenomenon where a few asexual genotypes are overrepresented, while unique genotypes are rarely found [M. persicae aphids collected on secondary hosts and weeds, on which M. persicae females multiply parthenogenetically during spring and summer. This has been reported for Scottish populations (G/N = 0.042), where most overrepresented genotypes were also resistant to insecticides [In terms of the genetic diversity, our study (2011–2013 and 2016) shows an overall clonal diversity (G/N = 0.72) similarly high to that previously reported  for  Tunisia . Further Tunisia  When samcticides  and confcticides . Similarcticides . In contcticides . Also, hcticides .M. persicae populations from areas with warmer winters tend to show lower clonal diversities than in areas with colder winters, as reported for countries from the northern and southern hemispheres. For example, in the southeast of Spain, where agroecosystems are quite heterogeneous comprising peach orchards surrounded by cultivated and wild herbaceous hosts, the clonal diversity is substantially lower (G/N = 0.46) than in Tunisia or France [Regarding the impact of climate conditions on clonal diversity, r France . Similarr France , New Zear France  and Chilr France , clonal r France .M. persicae in Tunisia mainly reproduce through CP. Nevertheless, two MLGs (G6 and G331) were overrepresented and time-persistent. Genotype G6 was the most abundant genotype over the whole sample , while genotype G331, the only one found through the all sampled years (from 2011–2013 and 2016), was restricted to potato crops regardless of the season and the sampling sites . Whether these genotypes constitute ‘superclones’ [In our study, most aphids were sampled during their asexual phase (spring/summer), so clonal diversity is affected by genetic inflation . HoweverFST = 0.038; one copy per genotype), as well as significant pairwise FST comparisons among the 19 populations of M. persicae, ranging from 0.013–0.083, with most (99%) variation found within each sampling localities. Our results are similar to those previously reported by Charaabi et al. [FST = 0.024) with most variation (94%) present within localities. These findings suggest that the predominance of sexual reproduction explains the genetic homogeneity among populations, as aphids migrate from different hosts to peach trees where they mate, involving recombination of the available population [The global AMOVA analysis revealed a weak but significant genetic differentiation  [FST = 0.012) [FST = 0.137) [That the peach potato aphid has become a cosmopolitan pest in part due to its ability to move over large geographic distances. Thus, it has colonized all continents through clonal propagation, for which human transportation and global commerce have aided in expanding its range both at global and regional scales ,60. Althom China , individom China ,61, as t= 0.021) , Chile (= 0.012)  and Ital= 0.137) . Our stu= 0.137) , as sugg= 0.137) , althougM. persicae can be governed by positive selection exerted by seasonal changes related to variable biotic and abiotic selective regimes, such as host plant availability, abundance of natural enemies, weather conditions, and management practices [M. persicae are positively correlated with the maximum temperatures and with the presence of natural enemies  and negatively correlated with the relative humidity during the evening [M. persicae, insecticide resistance has accelerated the widespread distribution of this aphid pest, even favoring some genotypes being shared in different countries [M. persicae is variable according to regions, hosts and season, as previously reported [M. persicae in Tunisian populations and their time-persistence among seasons and years could be attributed to selection by insecticides. Further research is needed to confirm this and understand how insecticide resistance is evolving in Tunisian populations.Temporal dynamics of the genetic diversity in populations of ractices . Our stu evening . On the  evening . In the ountries ,52. The reported . Thus, tM. persicae is important for transmission efficiency of phytopathogenic viruses. This is because the affinity for some viruses is variable among different M. persicae genotypes; some M. persicae genotypes transmit the virus in a non-persistent way while others are persistent, as described for PVY and PLRV, respectively [M. persicae genotypes may increase virus transmission [M. persicae is comparatively less important in the spread of PPV, due to the fact that during the period when peach orchards are more susceptible the abundance of M. persicae is lower, it has been shown that M. persicae is highly efficient in PPV transmission [M. persicae for adopting pre-emptive rational control measures.The population dynamics of ectively . Howeversmission . Severalsmission . Althougsmission . This emM. persicae [M. persicae populations in Tunisia, in order to increase our understanding of the factors shaping populations in order to predict their evolutionary responses to the present management practices. As the peach potato aphid is under strong selection pressures , this will certainly result in the selection of resistant genotypes. Thus, the evolution of genetic diversity and population structure in time and space could be faster, making pest control harder and more expensive. Our results should certainly be considered, we believe, when considering novel management strategies that better fit with the biology of the pest, given that aphids take advantage of the oversimplified design of current agroecosystems and succeed in a range of environments. Following this survey, we are studying the temporal and spatial dynamics of insecticide resistance in the peach potato aphid, as key knowledge for improving its control in Tunisia.In Tunisia, about 20,000 tons of seed potatoes are imported annually from France and The Netherlands , from whpersicae . By usin


Plagiorhegma dubium, a rare myrmecochorous herb, populations sampled across northeast Asia and Siberia. We found low within‐population genetic variation associated with historical bottlenecks. Although pairwise FST values were not much higher than the ones found in similar life form species, STRUCTURE and PCoA revealed a clear broadscale spatial pattern of genetic structure. Bayesian clustering (best K = 6) and PCoA identified three populations that are distinctive from neighboring populations in the Korean peninsula, which suggests potential units for conservation and management plans in Korea. MIGRATE‐N and BAYESASS showed that both contemporary (0.003–0.045) and historical migration rates (2 × e−5−4.6 × e−4) were low. Our findings provide a good example, where genetic considerations should be integrated for conservation and management plans of rare and threatened species.Distribution of genetic variation over time and space is relevant to demographic histories and tightly linked to ecological disturbances as well as evolutionary potential of an organism. Therefore, understanding the pattern of genetic diversity is a primary step in conservation and management projects for rare and threatened plant species. We used eight microsatellite markers to examine the level of genetic diversity, spatial structure, and demographic history of  For example, a molecular marker analysis revealed significantly lower heterozygosity and higher inbreeding in Pacific jumping mice  how the species is genetically structured over heterogeneous landscapes; (c) the historical and contemporary gene flow; and (d) whether there have been recent and/or historical population bottlenecks. Using microsatellite markers, we investigated genetic diversity patterns of P. dubium throughout the global distribution (northeast Asia and Siberia). Due to rarity of the species, lower within‐population genetic diversity is expected. We hypothesize that populations of P. dubium may be largely structured over varying landscapes with limited migration rates through myrmecochory. Given that the species has not been commonly found until recently  the level of genetic diversity in 22.1Plagiorhegma dubium Maxim.  Perss; Bentham & Hooker, Plagiorhegma dubium following the plant list (http://www.theplantlist.org/). Plants normally flower in the early spring around March to April and produce capsule fruits once a year . We collected samples throughout the global distribution  except for North Korea due to the inaccessibility. For the seven Korean domestic populations, we randomly chose collection sites: the distances between domestic populations in South Korea vary from 30 km for the closest population pairs to 150 km for the most distant population pairs Figure . To avoi2.3Plagiorhegma dubium , fluorescently labeled  forward and reverse primers, 0.5 μl (10 pmol each). PCR cycling conditions were as follows: 5 min predenaturation at 95°C followed by 30 cycles of 1 min at 95°C, 1 min at 50°C, and 1 min at 72°C, followed by a final 10 min extension step at 72°C. The fluorescently labeled PCR products were pooled with a size standard GS500LIZ . ABI 3730XL automated sequencer (Applied Biosystems) was used to separate out the amplified fragments. Using Gene Marker program , we examined microsatellite profiles with automated scoring. We then manually checked the result to avoid scoring errors. We tested for presence of null alleles using FreeNA for each of 14 loci within each population  values for HWE and LD. Six of 14 markers were with moderate to high frequency of null alleles in more than two populations. The six markers were either significantly departure from HWE or not independent from one another  in over two or more populations. Some of statistical analyses such as STRUCTURE depend largely on two strong assumptions, that is, Hardy–Weinberg equilibrium (HWE) and marker independence within populations  was calculated in each population (cut off for PgenFIS < 0.05).We tested whether there is a significant deviation from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) within each population for each of the 14 microsatellite markers using Fisher's exact test  using Arlequin v. 3.5 and GENALEX v. 6.5 , number of alleles (Na) allelic richness (Ne), and inbreeding coefficient ( Slatkin  suggesteK from 1 to 12. To infer the number of clusters (K) best explaining the data, ΔK was calculated ) and log‐transformed Euclidean distance for all population pairs  between the pairwise FST and the Euclidean distance was significantly different from zero.We performed a Mantel test to assess isolation by distance (IBD) based on pairwise genetic distance  and asymmetric migration rate, M  between populations over a long time scale . To reduce the computational challenges, we reformed groups based on the genetic clusters identified by STRUCTURE . We also estimated relatively recent reduction in population size within the past few generations using BOTTLENECK v. 1.2.02  implemented in Arlequin v. 3.5 . We found significant excess of heterozygotes in those 6 loci (observed heterozygosity > 0.7). Two of the six loci for three populations were with moderate (0.13) to high frequency (0.30) of null alleles. There were a few identical MLGs found in two populations (DG and GJ). However high values of PgenFIS (average = 0.71) indicate that those identical MLGs were not true clones. Therefore, we treated all individual samples were not clones for the subsequent analysis. The mean expected heterozygosity per population ranged from 0.14 in BS . The mean number of alleles for each population and allelic richness were the highest in VLT , whereas the lowest allelic richness was shown by populations BS, DG, DOM, and SDJ  than the rest of the population pairs  in more than two populations. After filtering the six microsatellite markers with null alleles, all 8 markers did not deviate from HWE nor were in LD after Bonferroni correction  that best explain the data was 6  than within groups  and geographic distance  of MIGRATE‐N ranged from 0.184 (US to CL1) to 4.65  with a unique pattern of genetic diversity, which might be an important unit for conservation in Korea.Most rare and threatened plants may severely suffer from erosion of genetic diversity, inbreeding depression and limited gene flow derived by reduced population size and/or fragmented habitats  relative to the one with 8 loci that didn't show excess of heterozygote and/or presence of null alleles . Those six loci screened out were nearly fixed for heterozygotes in most of the 10 populations, which makes the two alternate alleles equally frequent resulting in high expected heterozygosity . Given that most of the loci only showed 1–3 alleles per population, the six loci with heterozygote excess also were contributing greatly to variation in allele numbers.We found inflation of genetic variation when using all 14 loci , M ratios (G‐W index) appeared to be lower than the threshold (M = 0.73) estimate from Critical_M in all 10 populations. It suggests that there were drastic changes in population sizes over a long period of time. Although P. dubium is autogamous allowing selfing  was comparable but not significantly higher than the ones found in similar life form plant species  assume rather unrealistic island model with no mutation, no selection and particularly equal population sizes, FST does not directly reflect migration and gene flow .Microsatellite data are available in the DRYAD Digital Repository ( Click here for additional data file.