Prunus armeniaca L.), which was probably primarily domesticated in China. By comparing genetic diversity among regional apricot gene pools in several Mediterranean areas, we investigated the loss of genetic diversity associated with apricot selection and diffusion into the Mediterranean Basin.Domestication generally implies a loss of diversity in crop species relative to their wild ancestors because of genetic drift through bottleneck effects. Compared to native Mediterranean fruit species like olive and grape, the loss of genetic diversity is expected to be more substantial for fruit species introduced into Mediterranean areas such as apricot  was structured into three main gene pools: ‘Irano-Caucasian’, ‘North Mediterranean Basin’ and ‘South Mediterranean Basin’. Among the 25 microsatellite markers used, only one displayed deviations from the frequencies expected under neutrality. Similar genetic diversity parameters were obtained within each of the three main clusters using both all SSR loci and only 24 SSR loci based on the assumption of neutrality. A significant loss of genetic diversity, as assessed by the allelic richness and private allelic richness, was revealed from the ‘Irano-Caucasian’ gene pool, considered as a secondary centre of diversification, to the northern and southwestern Mediterranean Basin. A substantial proportion of shared alleles was specifically detected when comparing gene pools from the ‘North Mediterranean Basin’ and ‘South Mediterranean Basin’ to the secondary centre of diversification.A marked domestication bottleneck was detected with microsatellite markers in the Mediterranean apricot material, depicting a global image of two diffusion routes from the ‘Irano-Caucasian’ gene pool: North Mediterranean and Southwest Mediterranean. This study generated genetic insight that will be useful for management of Mediterranean apricot germplasm as well as genetic selection programs related to adaptive traits. Domestication of plants is a complex evolutionary process in which human selection favours phenotypic transitions making them more useful for humans and better adapted to landscape management. It is a crucial step in the evolution of crop species since humans have an important impact on their origins and changes. Moreover, selection pressure and local diversification lead to an ongoing process . Two majThe second major consequence of domestication is the reduction of genetic diversity in crops relative to their wild progenitors due to human selection and genetic drift through bottleneck effects . ContrarSpondias purpurea, within the Mesoamerican centre of domestication. About 29% of the total diversity was not recovered in wild populations, suggesting that either new alleles have arisen during cultivation or, alternatively, contemporary extinction of tropical dry forests has occurred in Mesoamerican areas leading to genetic erosion of the wild gene pool. In Mediterranean zones, only a weak bottleneck effect on diversity in olive and grapevine was observed when comparing the wild and cultivated forms  by STRUCTURE analysis . However, estimation of genetic diversity reduction due to bottleneck domestication could be biased by human selection [Prunus genome and not linked to the self-incompatibility locus [For most crop species, domestication processes cause a loss of genetic diversity due to the bottleneck effect and genetic drift -12. In oelection  and the election . Therefoty locus .vs. sexual reproduction in South Mediterranean, especially in the oasis agroecosystems in the Maghreb area [The results obtained using the two sets of markers  were congruent for all diversity estimators. A substantial decrease in genetic diversity was observed from the eastern (cluster 1 = ‘Iran-Caucasian’) to the western Mediterranean Basin (cluster 3 = ‘North Mediterranean Basin’ and cluster 4 = ‘South Mediterranean Basin’). Such a loss of genetic diversity was significant when comparing clusters 1 (‘Iran-Caucasian’) and 3 (‘North Mediterranean Basin’) and clusters 1 and 4 (‘South Mediterranean Basin’). Nevertheless, it was not significant when cluster 3 was compared to cluster 4, despite the different modes of apricot propagation: vegetative in North Mediterranean reb area ,34.We thus noted a substantial loss of genetic diversity that was independent of the selection impact due to the domestication bottleneck. Such a loss of genetic diversity is closely in line with the apricot diffusion routes, and contrasting with patterns in other native Mediterranean fruit species such as olive and grape for which a weak loss of genetic diversity between varieties and wild relatives has been observed ,18. The Prunus species [The Irano-Caucasian area is considered as a secondary diversification zone  for at l species . Second, species . Similar species . Althoug species ,23, our  species , or two  species  and Mehl species ?th century [The distinction of ‘South-Mediterranean’ apricots from the ‘Irano-Caucasian’ cluster confirmed the findings of a previous study  based on century ,26.vs. 3 and 1 vs. 4, we observed a significant loss of genetic diversity. Conversely, we noted no significant loss of genetic diversity between clusters 3 vs. 4, while they were more differentiated than cluster pairs 1 vs. 3 and 1 vs. 4  and the southern route (clusters 1 vs. 4 geographic group A vs. C). These results strongly indicate that apricot was diffused through two main routes: the first one through countries north of the Mediterranean Sea (cluster 3/region B) and the second one probably brought by the Arabs through North African countries (cluster 4/region C), as previously proposed [By comparing cluster pairs 1 proposed ,52. Our proposed ,26, but proposed , who repi) improve management and conservation strategies for Mediterranean apricot germplasm, and (ii) propose a genetic basis for apricot breeding programs.Based on the three main identified gene pools, we observed a significant and substantial loss of apricot genetic diversity, ranging from about 37 to 49% from the secondary apricot diversification zone (‘Irano-Caucasian’) to the southwestern Mediterranean Basin, depicting a genetic signature of apricot domestication and diffusion into the Mediterranean Basin. Unlike Kostina’s assumptions , we propInstitut National de la Recherche Agronomique , the Italian collection of the Department of Fruit Science and Plant Protection of Woody Species , the Spanish collection of CEBAS-CSIC , and the Turkish collection of the Inonu University , or from different collection surveys as was the case for the Algerian, Moroccan and Tunisian accessions. The French material studied contained native cultivars as well as a few introduced accessions initially acquired from other collections around the world , which were also considered in the sample set in order to span a broad eco-geographical apricot distribution range , France (49), Italy 19), Morocco (34), Spain (8), Tunisia (42) and Turkey . For these latter, total genomic DNA extraction was conducted from 150 mg of fresh young leaves, using the DNeasy Plant Mini Kit  according to the manufacturer's instructions, with minor modifications: addition of 1% w/v of PVP-40 to the AP1 buffer solution. For the Turkish accessions, total genomic DNA was extracted at the Apricot Research Center of the Inonu University of Malatya according to the same protocol. Then DNA aliquots were purified in the INRA Montpellier laboratory according to the protocol described above.Prunus reference genetic map: ‘Texas’ x ‘Earlygold’ [Prunus genome . The forward primer was 5’-labeled with one of the three fluorochromes . The PCR conditions were as follows: 35 cycles at 94°C for 30 s, T° annealing (depending on the locus) for 60 s, and 72°C for 60 s, with a final extension step at 72°C for 10 min. Amplified products were resolved using an ABI prism 3130 XL automatic DNA sequencer . Allele sizes were determined with GeneMapper 3.7 software .Twenty-five microsatellite markers were selected on the basis of the ease of amplification in apricot and their location on the rlygold’ ,54 as thme Table . These SK value (number of genetic clusters), for K ranging from 1 to 6. Each run involved a burning period of 100000 iterations, and a post-burning simulation length of 1000000. The run with the maximum likelihood was used to assign the most probable number of clusters, which was validated with an ad hoc statistic based on the second order rate of change in the log probability of data between successive K values [H’) of runs. The output obtained was used directly as input by the cluster DISTRUCT version 1.1 visualization program [The model-based clustering approach, as implemented in the STRUCTURE 2.2 software program , was useK values . To findK values  was used program .NA), number of alleles with frequency higher than 5% , and observed (Ho) and expected (He) heterozygosities. PowerMarker 3.25 [FIS) and the genetic differentiation (FST) were computed according to the formula of Weir and Cockerham [FST values. The generalized rarefaction approach ADZE [Ar) and the private allelic richness (Apr).GENETIX 4.05  was usedker 3.25  was usedker 3.25 . The inbockerham  using GEockerham  and Fishockerham  was applach ADZE  with stai) a group level identifying the eleven apricot geographic groups; and (ii) a region level distinguishing the three geographic regions A (Iran and Turkey), B , and C .Pairwise standard genetic distances of Nei  were calFST levels by plotting FST against heterozygosity on the set of markers. The Fdist2 analysis was conducted on three comparisons of the three main genetic clusters. We compared (i) clusters 1 (‘Irano-Caucasian’) and 3 (‘North Mediterranean Basin’), (ii) clusters 1 and 4 (‘South Mediterranean Basin’), and (iii) clusters 3 and 4. For each comparison, 50000 simulations were run using the infinite allele model for markers. A first analysis revealed a first set of outliers. Then they were removed and a new FST was calculated, which was used to make a new analysis and reveal a new set of outliers. Analyses were iterated until no further locus fell outside of the expected distribution. The last FST value was used as the neutral value to detect outliers on the whole set of data.The observed variation of genetic diversity among clusters might be due to two evolutionary factors: selection and bottleneck effects. Selection force could affect the estimation of bottleneck impact on the apricot diffusion history. A sub-sample of neutral markers was thus defined and used for the genetic diversity analysis. For this purpose, the method developed by Beaumont and Nichols  was usedDIV1/DIV2), where DIV1 is the estimator of diversity in the supposed derivating genetic pool and DIV2 is the estimator of diversity in the supposed originating genetic pool. The relative reduction of diversity was estimated using all SSR markers for the comparison among regional groups, and based on all 25 SSR markers and also only on neutral SSR markers when comparing the three main genetic clusters.The relative reduction of diversity was estimated with five diversity estimators: the total number of alleles, allelic richness, private allelic richness, observed heterozygosity and the expected heterozygosity, as described by Vigouroux et al. . The proBK designed and coordinated the study, performed the statistical analyses and drafted the manuscript. HB participated in the design of the study, conducted the molecular analysis, participated in the statistical analyses, and drafted the manuscript. JMA supervised the research project, prepared the plant sampling, helped in designing the study and drafting the manuscript. LK, AM, ST, CD and BMA contributed to plant sampling and descriptions of accessions. NTF helped in designing the study and drafting the manuscript. SS supervised the molecular analysis. All authors participated in the finalising the text and approved the final manuscript.Laboratoire d’Arboriculture at the University of Blida. CD is an Assistant Professor at the University of Pisa, Head of the Laboratory of Molecular Biology of Fruit Science and in charge of local fruit resource genotyping. BMA is a scientist in the Department of Biology at Inonu University. SS is an engineer, molecular biologist, and is in charge of the development and use of molecular markers. BK is a geneticist in the conservation biology field, focusing on diversification of Mediterranean fruit species and on the management of agrobiodiversity and agroecosystems.HB is a PhD student who will defend her thesis entitled “Apricot genetic structure in the Mediterranean Basin: domestication and diffusion process” in 2012. JMA is a scientist at INRA, the French national institute for agricultural research, and is in charge of INRA’s fruit research group and in particular apricot research program including genetic resource management and breeding programs. LK is an Assistant Professor at the University of Sciences of Gafsa and a scientist in the Laboratory of Molecular Genetics, Immunology and Biotechnology at the University of Sciences of Tunis and is in charge of the apricot genetic resource management. NTF is a Professor at the University of Sciences of Tunis and Director of the Laboratory of Molecular Genetics, Immunology and Biotechnology at the University of Sciences of Tunis and is in charge of fruit genetic resource management. AM is a scientist at the Moroccan national institute for agricultural research and is in charge of apricot genetic resource management and breeding. ST is a scientist at the Table S1. Genetic diversity at each of the 25 SSR loci used in the three geographic regions. FIS fixation index values; bold values represented exact test significant at P < 0.01. Click here for fileTable S2. Apricot germplasm accessions, code, origin as well as geographic group and genetic cluster to which each accession was assigned.*in situ sampling. a The clusters were assigned based on STRUCTURE analysis with cluster 1 = ‘Irano-Caucasian’ (blue), cluster 2 = ‘Adaptive Diversity’ (green), cluster 3 = ‘North Mediterranean Basin’ (yellow) and cluster 4 = ‘South Mediterranean Basin’ (red). Some accessions were assigned as mixed clusters because they were assumed to have admixed ancestry ; Global FST = 0.111 *P < 10-4; **P < 10-6. s. Nei’s  genetic Click here for fileFigure S1. Description of the four steps for the graphical method allowing determination of optimal K according to Evanno’s parameters. Click here for fileTable S4. Information on the number of assigned accessions per geographic group and region.Click here for fileTable S5. Pairwise FST among the four genetic clusters defined by the STRUCTURE analysis. All FST values were significant at P < 10-6; Global FST = 0.122; Global FST among the 3 main clusters = 0.109 significant at P < 10-4. Click here for fileFigure S2. Histogram illustrating the frequency distributions of alleles for each microsatellite among geographic regions. Arrows indicate specific alleles detected in each geographic region pairwise with A vs. B, A vs. C and B vs. C. Click here for fileTable S6. Specific alleles at each microsatellite locus within genetic cluster pairs.1 total allele observed at each locus. 2 alleles shared by at least two of the three clusters. a alleles observed in each cluster pairwise . b specific alleles observed in each cluster pairwise . c frequency of alleles based on the total number of alleles observed at the 25 SSR loci in the 207 apricot accessions studied. d frequency of specific alleles based on the total number of alleles detected at the 25 loci.Click here for file


The emotional impact of genetic testing has been extensively studied in individuals at high risk for Hereditary Breast and Ovarian Cancer Syndrome (HBOS). Studies on the impact of genetic testing for Lynch Syndrome (LS) are not as prevalent and few studies have compared the two cohorts.To compare the psychological impact of genetic testing in participants at high risk for LS and HBOS and identify variables associated with genetic testing distress, anxiety and depression at one week and one year post disclosure of genetic test results.140 individuals undergoing genetic testing for LS and 133 undergoing genetic testing for HBOS at the Dana-Farber Cancer Institute were enrolled in a longitudinal questionnaire study. We assessed levels of genetic testing distress, anxiety and depression using The Multidimensional Impact of Cancer Risk Assessment (MICRA) and the Hospital Anxiety and Depression Scale (HADS). Multivariable regression analysis was used to identify factors associated with psychological distress in the LS and HBOS cohorts. One year follow-up data is based on 96 LS and 94 HBOS tested individuals that completed the one year questionnaire.For both cohorts, individuals who had the expectation for carrying a deleterious gene pre-test and/or those who received a positive or variant of uncertain significance (VUCS) test result had significantly higher genetic testing distress at one week post-disclosure (p ≤ 0.05). Additional independent predictors of higher genetic testing distress in the LS cohort included not being married and having a personal history of cancer. In the HBOS cohort, predictors included having a higher HADS anxiety score at baseline and having an annual household income of ≤ $50,000.At one year post-disclosure, individuals that tested positive or VUCS and/or had the expectation of carrying a deleterious gene pre-test continued to have significantly higher genetic testing distress (p ≤ 0.01). While there were no differences between the cohorts in baseline or post-disclosure HADS anxiety or depression scores, significantly more individuals in the LS cohort had HADS depression scores ≥ 8 one year after testing .Individuals at risk for LS and HBOS whose genetic tests reveal a pathogenic mutation or VUCS can continue to demonstrate genetic testing distress one year after receiving genetic test results. These individuals may benefit from counseling beyond the immediate disclosure period.


Salmo salar) strains and their wild progenitor populations in an effort to detect potential signals of domestication selection by typing of 261 SNPs and 70 microsatellite loci. By combining information from four different neutrality tests, in total ten genomic regions showed signs of directional selection based on multiple sources of evidence. Most of the identified candidate regions were rather small ranging from zero to a few centimorgans (cM) in the female Atlantic salmon linkage map. We also evaluated how adaptation from standing variation affects adjacent SNP and microsatellite variation along the chromosomes and, by using forward simulations with strong selection, we were able to generate genetic differentiation patterns comparable to the observed data. This study highlights the significance of standing genetic variation during the early stages of adaptation and represents a useful step towards identifying functional variants involved in domestication of Atlantic salmon.Domesticated animals provide a unique opportunity to identify genomic targets of artificial selection to the captive environment. Here, we screened three independent domesticated/captive Atlantic salmon ( Over the last decade, advances in genomic technologies together with developments in methods of statistical analyses have resulted in considerable progress in the systematic detection of genomic regions influenced by artificial selection –5. TheseSalmo salar L.) represents a useful model for studying the genetic basis of recent, ongoing domestication since reared salmon strains have experienced intense artificial selection for only a short period of time (5–12 generations), and there are still many wild populations available for comparative purposes, including progenitor populations. The traits that have been targets of selective breeding in reared Atlantic salmon strains include higher growth rate, delayed sexual maturity, bacterial resistance, flesh colour, and fat content. The selection response of many of these traits has been as high as 10% per generation [Atlantic salmon , Ume/Vindelälven Rivers , and the Saint John River , in an attempt to identify genomic regions affected by domestication/captive breeding. A short description of the history of each reared strain and sampling details are provided below.Artificial rearing of juvenile salmon for ranching from the Burrishoole river began in 1960 and recruitment from the wild population continued until 1964. Apart from the addition of a small number of wild fish in the reared broodstock between 1970 and 1975, the breeding strain has been effectively closed since that time; the hatchery born fish were distinguished from their wild counterparts by the absence of a previously-clipped adipose fin. Assuming a generation time of 5 years for the wild fish, wild and reared (ranched) Burrishoole salmon have experienced different selection pressures for approximately 8 generations during the early freshwater phase (from fertilization to smoltification). Today, the Burrishoole-ranched strain is composed of three almost distinct coexisting cohorts with little overlap between year classes . BurrishThe wild River Umeälven salmon were extirpated by power plant dam construction in the early 1950s but River Vindelälven (a tributary of Umeälven) continued to support wild salmon. The present hatchery stock of River Umeälven originates from the wild Vindelälven population and since 1971, all hatchery (ranched) smolts have had their adipose fin removed. Since 1974 all salmon without adipose fins (hatchery fish) or with intact adipose fins  have been recorded at the fish trap below the Norrfors hydroelectric dam and only salmon of wild origin are released above the dam to continue their upstream spawning migration to the Vindelälven. As a result, wild and captive (ranched) Ume/Vindelälven salmon have experienced different selection pressures for approximately 5-6 generations during the early freshwater phase (from fertilization until smoltification) assuming a generation time of 5-6 years. River Ume/Vindelälven wild samples consisted of 16 returning individuals in 2006, while the samples from the reared (ranched) strain were obtained in 2009 (16 1+ old juveniles).The Saint John reared strain used for farming was founded in 1990 from wild Saint John River salmon, and in contrast to IRL and SWE populations, by 2006 it has experienced four generations of selection for higher growth and reduced early maturation . As a reIn total, 320 SNP developed by Moen et al.  were genμL volumes including 10–100 ng of genomic DNA and 0.033–0.3 μM of locus specific primers, the forward primer being labelled with one of four fluorescent dyes , and 1 × QIAGEN multiplex PCR master mix. The thermal cycling conditions used were as follows: 15 min initial activation step at 95°C followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55–60°C for 90 s and extension at 72°C for 60 s and ending with a final extension at 60°C for 10–30 min. Amplifications were performed using an Applied Biosystems 2720, a PTC-100, or a PTC-200 (Bio-Rad) thermal cycler. The PCR products from separate multiplex reactions were pooled (1–2.5 μL) and made up to the final volume of 120 μL of distilled water, mixed with GS600LIZ size standard (Applied Biosystems), and formamide for electrophoresis on an ABI 3130 × l automated sequencer. Detailed multiplex amplification information, including specific primer concentrations, annealing temperatures, and post-PCR pooling procedures, are available on request.A total of 70 variable number tandem repeat markers were also included in this study. They included two MHC linked mini- and microsatellite loci, 21 EST-derived microsatellite loci , 36, andFST estimates [FST estimates between pairwise comparisons was tested by permutation of individuals between samples using FSTAT 2.9.3 [Diversity indices were estimated using the program FSTAT 2.9.3 . Deviatistimates . The sigAT 2.9.3 .FST for each locus in the sample, and coalescent simulations based on a symmetrical island model are used to generate data sets with the mean FST similar to the empirical distribution. To calculate approximate P values for each locus, 100,000 independent loci were generated and the simulated distribution of FST between two populations was then compared to the observed FST values, conditional on heterozygosity, to identify potential outliers as implemented in the software FDIST2. Sample sizes used to calculate FST from coalescent simulations were set to 16 individuals, corresponding to the empirical data. Two different tests aimed at detection of loci subject to directional selection during domestication/captive breeding were used. Both approaches rely on the rationale that directional selection increases genetic differentiation between populations and reduces variability at linked loci. However, because both of the tests are based on different assumptions, identification of outlier loci simultaneously with both approaches provides additional support for the candidate status of a particular locus . The firFST is modelled using a logistic regression model implementing a locus effect and a population effect, relaxing the assumption of symmetrical island model by allowing for population structure asymmetries. The estimation of the model parameters was automatically adjusted on the basis of short pilot runs (10 pilot runs with 5000 iterations each). We used a burn-in of 50,000 iterations and the total chain length of 500,000 iterations  to identify loci under selection. Altogether, three independent runs were performed for each wild and domesticated/captive population pair. Bayescan estimates the posterior probability that a locus is under selection by calculating a Bayes factor, which is given by the ratio of posterior model probabilities of two models , given the data. For example, a Bayes factor of two indicates that the data favor one model over the other at odds of two to one. According to Jeffreys' interpretation, Bayes factor between 3 and 10 (log10 = 0.5–1) is considered “substantial evidence” to favour one model over the other and corresponds to a posterior probability between 0.76–0.91. A Bayes factor between 10 and 32 (log10 = 1–1.5) is considered “strong evidence” of different statistical support for the two models and corresponds to a posterior probability between 0.91–0.97. However, this probability cannot be compared directly to the P value from FDIST2, since Bayescan explicitly takes all loci into account in the analyses. The second method to detect footprints of selection also identifies loci that exhibit extreme differentiation compared to the rest of the genome using the Bayesian likelihood method implemented via reversible jump Markov Chain Monte Carlo . Using tFST) or large differences in genetic diversity (lnRH) [FST and mean absolute lnRH for pairs of markers along specific chromosomes and by using permutations evaluated whether the empirical pattern differs from random expectations . We subsequently identified locus pairs that exhibited elevated differentiation (FST) or large differences in genetic diversity (lnRH), against the permutation-derived 95% significance threshold. Absolute lnRH was used as a parameter for quantifying the difference in genetic diversity (lnRH) between populations, as very short-term directional selection can either reduce or increase the genetic variation of a particular locus depending on the frequency of the allele linked to an advantageous mutation. For example, short-term directional selection initially increases the diversity when the positively selected (or linked) allele is at low frequency, while it reduces the variation when the positively selected (or linked) allele is at high frequency in the population. Loss of diversity measured as lnRH has been commonly used to identify outliers in microsatellite datasets but this statistic can also be applied to SNPs markers [As a complementary approach to the outlier tests described above, we evaluated whether there is a tendency for adjacent loci to exhibit high differentiation (y (lnRH)  between  markers  as lnRH  markers .q value to evaluate the proportion of putative outliers that are likely to be false positives ; FDRs  [P values derived from (i) coalescent simulations (FDIST2 test), (ii) from expected normal distributions (lnRH test), and (iii) from permutation tests aimed at detecting clustering of loci with extreme FST and lnRH values. A bootstrap method was used for estimating the overall proportion of true null hypotheses as a more conservative approach compared to the smoother method as suggested by Storey and Tibshirani [To control for the multiple testing problem that arises when large numbers of markers are tested against the neutral null hypothesis, we employed the false discovery rate (FDR) methodology  implemen values)  were calbshirani .de novo mutations, as it is more likely that initial adaptation to an artificial environment occurs from standing variation. As a result, selection on preexisting genetic variation is expected to lead to immediate evolutionary response, fixation of more alleles of small effect and importantly, generally weaker footprints of selection compared to strong selective sweeps [To evaluate whether the observed patterns of genetic variation were compatible with the effect of strong directional selection, we used an individual-based forward-time simulation environment, simuPOP 1.0.2 , to exple sweeps . FST from Ne = t/(2FST). We then constructed a simple single population split model applying the derived population genetic parameters for each population pair . We subsequently applied forward simulations using the empirical genotype data from wild population and genetic linkage map information to simulate the effect of selection along specific chromosomes on identified outlier loci. We first created an ancestral wild population by using empirical genotype data from the wild progenitor population  by multiplication of existing genotypes and subsequent random mating for three generations. After the split of the ancestral population, we simulated positive selection on the minor allele in a single candidate region per population pair. We assumed that the fitness of three genotypes AA, Aa, and aa in captive environment is 1, 0.7 and 0.4 , while there is no selection acting in the wild populations. Note that we are aware of only one-published single locus selection coefficient for Atlantic salmon  [FST estimator [FST was visualized by creating box plots using SPSS 14.0.Given that the time since foundation of captive/domesticated strains are known and assuming equal population sizes and complete isolation (drift only) after the split, we calculated the effective population size using the estimated onicida)  but seleonicida) –50. We sstimator , by sampU test, all P values < 0.001). Contrary to earlier population genetic studies on natural and farmed Atlantic salmon populations [P values > 0.05; P values < 0.05; θ, between reared strain/wild population pairs was rather low, ranging from ~0.01 in SWE and IRL to 0.039 in CAN but nevertheless, was highly significant . When comparing wild population and reared strain pairs across different type of markers, the mean genetic differentiation estimates were rather similar , indicating that both types of markers are generally affected by a similar kind of evolutionary forces across the genome. Hardy-Weinberg disequilibrium was detected in 35 of 1370 loci by population tests at the 1% significance level before correction for multiple tests (data not shown). Approximately 14 of them are expected to emerge simply because of the large number of tests performed. As expected, SNPs and microsatellites showed different levels of genetic variation  identified nine outlier loci  with substantial evidence (Bayes factor > 3 corresponding to log10(BF) > 0.5) of directional selection according to Jeffreys' interpretation [Altogether, eleven loci were identified as outliers in three wild-reared comparisons (three each in SWE and CAN and five in IRL) at the 99%  g FDIST2 . Those eretation   or alternatively, show large differences in genetic diversity (lnRH) between wild-reared population pairs. Altogether, seven adjacent locus pairs  exhibited evidence for clustering of markers  with high genetic differentiation (FST) along the chromosome . As expected, a considerable proportion of the loci that fall to the tails of the distribution are probably false positives, that is, they emerge because of the large number of tests performed  for the four previously-used neutrality tests: FDIST2, lnRH, clustering of adjacent loci with high erformed . As a reerformed . Among tWe also evaluated whether strong directional selection over a very short period of time from standing variation also affects adjacent genetic markers along the chromosome, using the empirical genotype data from wild population as a source for subsequent forward simulations. We selected three linkage groups/chromosomes that contained previously-identified outlier loci  with the closest adjacent marker 0 cM (CAN), 7.8 cM (IRL), and 33.6 cM (SWE) apart. Overall, strong selection on the minor allele resulted in high differentiation between wild population and reared strain pairs at selected markers, comparable with the empirical data . The hitIn recent years, there has been a considerably increased interest in searching for signatures of natural or artificial selection, both in model and nonmodel organisms –56. In tP < 0.012). Interestingly, the same two genes also showed significant gene expression differences between domesticated- and wild-rainbow trout (Oncorhynchus mykiss) [By combining information from four different neutrality tests, in total, ten genomic regions showed signs of directional selection based on multiple sources of evidence . When se mykiss) . These fWe found that several of our candidate regions overlap with Atlantic salmon QTLs identified for various ecologically and economically important traits in earlier studies. For example, linkage groups 6 (Chrom. 12), 7 (Chrom. 24), and 11 (Chrom. 3) have been shown to harbour QTLs for body weight , 59, conOncorhynchus mykiss) and brook charr , differentially expressed genes between reared strains and wild populations have been shown to belong primarily to similar biological functions such as energy metabolism of lipids and protein synthesis, but the specific genes involved in these biological functions were found to be different [Oncorhynchus kisutch). Our study provides little support for the hypothesis of independent parallel genetic changes at the DNA level in Atlantic salmon from different population pairs as none of the identified candidate regions overlapped in the three wild-population/reared strain pairs. This is in contrast with earlier studies at the gene expression level that have demonstrated the presence of parallel differences in transcription of genes between independent domesticated- and wild-Atlantic salmon . Howeverifferent , 65. Theifferent  who founifferent  who showvice versa; even if polymorphism exists in all strains, when the allele that is linked to the positively selected site is already at high frequency in a particular population, selection does not necessarily induce a strong increase in the level of genetic differentiation and reduction in diversity. It is also possible that several other factors contribute to the population-specific signatures of selection, such as (i) different selective regimes in farmed versus ranched strains, (ii) different selective regimes between separate ranched strains, or, (iii) allele frequency differences between populations/strains. In fact, it is possible that all of these factors contribute to the lack of overlap of outliers because (i) farmed strains experience strong intentional selection for growth, disease resistance, delayed maturity, and other commercially important traits while ranched strains experience inadvertent selection in captivity as well as natural selection at sea , 67;  in order to obtain more comprehensive understanding about molecular mechanisms of adaptation. Species with domesticated/captive strains are likely to remain popular models in this context, providing unique opportunities to identify molecular targets of artificial selection. In conclusion, our results together with other recent studies , 80, 81 Marker and genetic map information of studied SNP and microsatellite loci.Click here for additional data file.


Moringa oleifera were carried out with the aid of twelve primers from, random amplified polymorphic DNA marker. The seeds of twenty M. oleifera genotypes from various origins were collected and germinated and raised in nursery before transplanting to the field at University Agricultural Park (TPU). Genetic diversity parameter, such as Shannon's information index and expected heterozygosity, revealed the presence of high genetic divergence with value of 1.80 and 0.13 for Malaysian population and 0.30 and 0.19 for the international population, respectively. Mean of Nei's gene diversity index for the two populations was estimated to be 0.20. In addition, a dendrogram constructed, using UPGMA cluster analysis based on Nei's genetic distance, grouped the twenty M. oleifera into five distinct clusters. The study revealed a great extent of variation which is essential for successful breeding and improvement program. From this study, M. oleifera genotypes of wide genetic origin, such as T-01, T-06, M-01, and M-02, are recommended to be used as parent in future breeding program.The knowledge of genetic diversity of tree crop is very important for breeding and improvement program for the purpose of improving the yield and quality of its produce. Genetic diversity study and analysis of genetic relationship among 20  Moringa oleifera Lam.), a short to medium height tree with luxurious evergreen leaves, was said to have originated from Himalayan tract in northwestern part of India [n = 28 with a distinguished tripinnate leaves having yellow or white petiole streaks [Drumstick tree , random amplified polymorphic DNA (RAPD), and simple sequence repeat (SSR), has gained popularity as a genetic diversity assessment methods of tree and oil seed crops –16. MoleMoringa oleifera from two populations with the aim of studying genetic diversity pattern in relation to their geographical origin, and dissection of germplasms as a means of initiating the breeding programme in the nearest future.Therefore, this research work will study the genetic diversity of twenty new genotypes of M. oleifera collected from six different countries  was added to 10 mg leaf sample and ground with mortar and pestle without liquid nitrogen according to Ferdous et al. [μL of 2X CTAB solution, 100 mM of Tris-HCl, 20 mM of ethylenediaminetetraacetic acid di-sodium salt (EDTA), 1.4 M of sodium chloride (NaCl), 2% (w/v) CTAB, and 1% (w/v) of polyvinyl pyrrolidone (PVP) and 400 μL chloroform: isoamyl alcohol : phenol (24 : 1 : 5%) mixture were then added to the leaf tissue containing the extraction buffer. After mixing through vortex and centrifuge, the supernatant was transferred to another tube. Two-third of volume of isopropanol was added and incubated at room temperature for 10 to 15 min. Centrifuged supernatant was then removed and the DNA pellet was washed using 70% ethanol; afterwards the DNA pellet was air-dried and dissolved into 50 μL TE buffer.Six hundred s et al. . The finμL.The quantification of DNA was carried out using NanoDrop ND-1000 spectrophotometer . The DNA was again requantified by running it through 1% agarose gel electrophoresis with 1 × TAE buffer for 30 min and viewed under UV light after staining it with Midori green DNA stain . The dilution was done with sterile distilled water to ensure that all of the DNA samples have equal concentration of 100 ng/μL of 5X Green GoTaq Flexi Buffer, 3 μL MgCl2 solution (25 mM), 0.5 μL PCR nucleotide mix (10 mM each), 0.2 μL primers (0.4 μmol), and 1.0 U of Taq DNA polymerase were used for 25 μL of PCR reaction including 1 μL DNA template directly used after extraction [According to the company instruction (Promega), 5 traction . In RAPDtraction . The ampThe image of the gel acquired in JPEG format was imported into UVIdoc 99.02 for band scoring. The band sizes were estimated based on DNA ladder (Promega Inc.). The absence and presence of band were scored in a binary model of 0 and 1, respectively. Band scoring was carried out only on those bands that are clear and reproducible and then those that are >50 bp. The data obtained at the end of the scoring was transferred and saved in Microsoft excel sheet.Data of the twelve primers were analyzed to obtain the information on genetic diversity of the 20 moringa accessions . GeneticM. oleifera  were calculated to be 75.73 and 32.70, respectively (ectively . The obsFst = 0.16) was significant at 5% probability level when permutation test was conducted.Furthermore, in order know the source of genetic variation for these Moringa genotypes, RAPD profile was analyzed using AMOVA. This was aimed to partition all the sources of variation existing in the germplasm into two major groups. The result revealed that 95% of the total genetic variation occurred as a result of variation within the population, while variation among the populations accounted for the remaining 5% of the total genetic variance . Also thCluster analysis based on Jaccard's genetic similarity coefficient showed high level of genetic variation among the genotypes from the two countries. The similarity coefficient ranged from 0.38 to 0.89, with T-11 and T-15 genotypes found to have highest genetic similarity (0.89), while T-06 together with T-07 possessed least similarity coefficient .In addition, a dendrogram was constructed using UPGMA cluster analysis to show the genetic relationship among the twenty genotypes from different geographical backgrounds. From this dendrogram, the twenty genotypes were grouped into five major clusters at a coefficient of 0.63. Cluster III, with highest number of members, had 14 genotypes, followed by cluster I (T-01 and T-03) and cluster IV (M-01 and M-04) with two genotypes each. Clusters II (T-07) and V (T-06) have one genotype each and were ranked the least populated clusters . M. oleifera profile classified the twenty genotypes of Moringa into five major groups with two genotypes from Malaysia occupying one group. This grouping pattern corresponds with that of clustering analysis as shown by the dendrogram. Dimension one of the PCA ranges from 0.49 to 0.92, while dimension two ranged from −0.65 to 0.26 and dimension three ranged from −0.65 to 0.68  carried out with RAPD  to 0.68 .Effective and efficient genotyping of any plant species through RAPD requires a careful selection of suitable primer combination in order to get detail and informative result. High level of genetic polymorphism detected by these markers is in agreement with the assumption that outcrossing plant species from natural population will have higher level of genetic diversity when compared to in-breeding plant species. This finding agrees with earlier report on similar out-crossing plant species, such as Jatropha  and otheMoringa and this is very important for successful crossing and improvement programs in future. This observation follows a similar trend with the result of genetic diversity study on 75 accessions from Sudan and Guinea savanna zones of Nigeria, where six polymorphic primers of RAPD origin gave a total of 42 polymorphic bands [Additionally, high level of genetic differentiation in these two populations as reflected by the genetic diversity parameters, such as Shannon's information index, expected heterozygosity, percentage polymorphism, and others, are pointing to the fact that there is wild variability in these populations of ic bands .M. oleifera populations are in agreement with those of other outcrossing plant species [M. oleifera exhibit moderate Fst value in order to demonstrate low significant genetic differentiation among the populations. However, higher genetic differentiation and diversity were observed within the populations of M. oleifera and this indicates a relatively restricted variability as expected. This pattern of population structure has been previously reported in other out crossing plant species [Furthermore, interaction between various ecological and biological factors, such as genetic drift, gene flow, selection, and mating system, affects the genetic structure of any plant populations . The ove species , 25, 26. species , 28.M. oleifera genotypes from the two populations were seen clustering together in the same group. This shows that there is no any distinct relationship between the geographical origin and the genetic distance as shown in the dendrogram. This finding implies that the genetic divergence within and between these two populations could not be explained by their geographical distance. This finding also means that isolation as a result of distance cannot be said to have been responsible for the divergence observed in these population [Moreover, clustering analysis showing wide range of similarity coefficient showed that, there is high level of genetic variation within the two populations. The pulation .Moringa populations will be a very good germplasm material for the future breeding and improvement of this economically important tree crop. Genotypes that are far apart based on their genetic similarity coefficient  should be selected for future breeding.In conclusion, these findings have proven that genetic divergence is very high in these populations and it can therefore be inferred from the data that the


Arapaima gigas (Cuvier 1817), few data about its dispersal capacity are available. The present study was based on the analysis of microsatellite markers in order to estimate the dispersal capacity of the species on fine, meso, and large geographic scales. For this, 561 specimens obtained from stocks separated by distances of up to 25 km , 100 km , and 1300–2300 km  were analyzed. The fine scale analysis indicated a marked genetic similarity between lakes, with low genetic differentiation, and significant differences between only a few pairs of sites. Low to moderate genetic differentiation was observed between pairs of sites on a meso scale (100 km), which could be explained by the distances between sites. By contrast, major genetic differentiation was recorded in the large scale analysis, that is, between stocks separated by distances of over 1300 km, with the analysis indicating that differentiation was not related solely to distance. The genetic structuring analysis indicated the presence of two stocks, one represented by the arapaimas of the Mamirauá Reserve, and the other by those of Santarém and Tucuruí. The dispersal of arapaimas over short distances indicates a process of lateral migration within the várzea floodplains, which may be the principal factor determining the considerable homogeneity observed among the várzea lakes. The populations separated by distances of approximately 100 km were characterized by reduced genetic differentiation, which was associated with the geographic distances between sites. Populations separated by distances of over 1300 km were characterized by a high degree of genetic differentiation, which may be related primarily to historical bottlenecks in population size and the sedentary behavior of the species. Evidence was found of asymmetric gene flow, resulting in increasing genetic variability in the population of the Mamirauá Reserve.Despite the ecological and economic importance of the  Arapaima gigas) is one of the World’s largest freshwater fish scales, with some adults reaching three meters in length and a weight of 200 kg The arapaima . Details about the locality and number of samples are showed in Fine scale dispersal patterns were evaluated from the analysis of arapaimas captured in lakes separated by distances of no more than 25 km. For this analysis, specimens of 223 arapaimas were obtained from 15 lakes located within the Jarauá sector of the Mamirauá Sustainable Development Reserve in Amazonas, Brazil . The mesSamples of muscle tissue were obtained directly from local fishermen, identified, and stored in ethanol until processing in the laboratory. The fishing is intended for marketing and obeys the legislation, which limits the capture to a minimum size of 1.5 m The genetic material collected from the 561 specimens was isolated using the standard phenol-chloroform and proteinase K protocol a posteriori probability of a given number of stocks based on a Bayesian approach, run in the Structure 2.3.3 program Possible errors in the identification of the alleles were verified using the Microchecker program The correlation between the genetic and geographic distances among populations at each spatial scale was estimated using the Mantel test, run in Arlequin 3.11 A total of 54 alleles were identified for the seven loci analyzed in the 561 arapaima specimens. The number of alleles, allelic richness, and observed and expected heterozygosities are shown in ST) of 0.056, with 4.97% variation between the two sites and more than 90% of the variation within sites.On a meso scale, 11 alleles were privates to the Jarauá sector, and five to Maraã. Genetic differentiation (Fst) was low to moderate for all the pairs of populations analyzed . The AMOp = 0.00  varied from 0.138 to 0.207, p = 0.00 . From thp = 0.00 . The AMOThe comparison of the arapaima stocks located in várzea lakes separated by distances of less than 25 km indicated a high degree of genetic admixture. The observed profile indicated that the animals that occupy isolated lakes during the dry season admixture extensively during other periods. Three principal factors related to the biology of the species probably influence this pattern – lateral migration, the reproductive characteristics of the species, and the dispersal pattern of adults and juveniles. The movement of the arapaimas to the recently-flooded forests during the annual period of inundation Together with this pattern of lateral migration, the reproductive characteristics of the species may also contribute to the marked genetic homogeneity of the populations on a fine scale. The reproductive cycle of the arapaima is intimately related to the hydrological fluctuations of the várzea, and begins at the end of the dry season, when the animals form breeding pairs, build nests, spawn and fertilize the eggs The third factor that likely contributes to the observed pattern of genetic diversity is the dispersal pattern of juveniles and adults. The males care for the juveniles during approximately three months This pattern of dispersal of the juveniles to the lakes and channels adjoining must further reinforce the spread of the different alleles across the várzea habitats. In addition, distinct patterns of dispersal of adult arapaimas in the Mamirauá Reserve have been observed. Specimens have been recaptured or harvested at distances of more than 60 km from the locations at which they were marked and released. On the other hand, some specimens were recaptured in the same (or adjacent) lakes in which they had been released five years previously Osteoglossum bicirrhosum) Colossoma macropomum), or even the migratory catfish, such as the dourada (Brachiplatystoma flavicans), which inhabit the major river systems of the region Despite the intense exchange of alleles and an absence of genetic structuring among lakes on a fine scale there is no clear evidence that the arapaimas migrate actively and systematically between different bodies of water. Migration seems to occur at short distances mainly due to lateral migration. However, the pattern of active migration is common in other várzea fish species, such as the silver arowana  of the breeding adults.The results of the present study indicate that the genetic differentiation observed between the arapaimas of the Jarauá and Maraã sectors may be related to three principal factors – the random dispersal of individuals among lakes, the sedentary behavior of the species, and the effects of distance on these populations. As the flood cycle in this type of environment follows an annual pattern, the dispersal of the fishes to neighboring lakes is limited by this temporal dynamic. Given this, the genetic differentiation observed among lakes on a meso scale has arisen slowly and gradually, reaching significant levels when the distances between lakes approach about 100 km. Each annual flood cycle results in the reconstitution of the population of each lake, including both the adults and the juveniles, as described above. The formation of new breeding pairs, associated with the dispersal of the juveniles results in the dispersal of genes, independently of the migration  contributing more to the Mamirauá population (mostly green) than vice versa . This paA. gigas on the genetic structure of its populations should be taken into account for the development of effective conservation strategies. In the present study, the indirect analysis of dispersal patterns based on hypervariable genetic markers indicated the occurrence of gene flow between A. gigas stocks separated by distances of approximately 100 km in várzea floodplain habitats, with intense admixture on a fine scale (distances of up to 25 km). On the other hand, populations separated by much longer distances, of over 1300 km, were significantly different, indicating a major reduction in gene flow.While the arapaima has traditionally been classified as a sedentary fish, no previous studies have analyzed the dispersal capacity of this species over different geographic scales. Together with its dispersal capacity, the effects of the reproductive and behavioral patterns of


Castanopsis sclerophylla is wind-pollinated, with seeds that are dispersed by gravity and rodents. Using microsatellites, we found no significant difference in genetic diversity between pre- and post-fragmentation cohorts. Significant genetic structure was observed in pre-fragmentation cohorts, due to an unknown genetic barrier that had isolated one small population. Among post-fragmentation cohorts this genetic barrier had disappeared and genetic structure was significantly weakened. The strengths of genetic structuring were at a similar level in both cohorts, suggesting that overall gene flow of C. sclerophylla has been unchanged by fragmentation at the regional scale. Fragmentation has blocked seed dispersal among habitats, but this appears to have been compensated for by enhanced pollen dispersal, as indicated by the disappearance of a genetic barrier, probably as a result of increased wind speeds and easier pollen movement over water. Extensive pollen flow can counteract some negative effects of fragmentation and assist the long-term persistence of small remnant populations.Gene flow strongly influences the regional genetic structuring of plant populations. Seed and pollen dispersal patterns can respond differently to the increased isolation resulting from habitat fragmentation, with unpredictable consequences for gene flow and population structuring. In a recently fragmented landscape we compared the pre- and post-fragmentation genetic structure of populations of a tree species where pollen and seed dispersal respond differentially to forest fragmentation generated by flooding.  The spatial distribution of genetic variation can provide perspectives into current and past population dynamics, and hence is of great significance for biological conservation  is a widely distributed monoecious canopy tree of subtropical evergreen forests in southeastern China. It can reach a height of about 25 m, is wind-pollinated, and flowers in April and May. In November, the heavy nuts of C. sclerophylla fall to the ground, from where there is some secondary dispersal by rodents. Previous studies of populations of this species in Qiandao Lake, a fragmented landscape of numerous islands formed when a river was dammed about 50 years ago, have shown that seed dispersal is restricted to within remnant habitat islands, with most seeds being dispersed less than 30 m from their maternal parents . Conversely, pollen flow may be more extensive across the water separating the plant populations than it is in the forest patches, because a large pollen-to-seed migration ratio (162) was estimated for plants within an area of 100 km2 encompassing the lake  Is there a significant difference in genetic structure between pre- and post-fragmentation cohorts at the landscape scale? And (2) based on any changes in landscape genetic patterns, what can be inferred about the effects of fragmentation on patterns of pollen flow in C. sclerophylla?The post-fragmentation changes in SGS we detected at small geographical scales may not necessarily be reflected regionally, because of the predominant role of pollen dispersal in shaping SGS at larger spatial scales. Here, we compare SGS among pre- and post-fragmentation cohorts of 2. Before the lake was formed, forests were more or less continuous in the area. Currently, C. sclerophylla is one of the dominant trees in forests of the southeastern part of the lake, but would have been more widespread previously. According to field observations , a hydroelectric impoundment formed in 1959 by the establishment of Xin'anjiang Dam. When the dam is full, the lake contains 1078 islands with an area larger than 2500 mividuals . The disividuals A. All ouividuals A. The poC. sclerophylla individual was allocated into either a post- (age <50 years) or pre-fragmentation (age >50 years) cohort based on a previously established relationship between age and basal trunk diameter . Eight polymorphic microsatellite loci developed in HO) and expected (HE) heterozygosities in each pre- or post-fragmentation subpopulation were estimated using GenAlEx version 6.3 , allele richness (AR) (El Mousadik and Petit FIS) were quantified with FSTAT version 2.9.3.2  as described by Hedrick  among subpopulations within each cohort was evaluated according to the formula proposed by Slatkin and Barton (Departures from Hardy–Weinberg equilibrium (HWE) were tested, and observed  using GenAlEx version 6.3 . Thus, we show the results of eight loci. Genetic variation within both cohorts were similar, with no significant differences in allelic richness (AR), observed (HO) and expected (HE) heterozygosity between cohorts .All eight loci were independent from each other and revealed a high level of polymorphism. The number of alleles per locus varied from 3 to 19 with a mean of 10.88. Significant departures from HWE were detected in populations GM and XS, based on average fixation index values across the eight loci. Pre-fragmentation subpopulation LS and post-fragmentation subpopulation HY were also not at HWE . SignifiF′ST and G′ST among post-fragmentation subpopulations  were much lower than among pre-fragmentation subpopulations , with estimated gene flow among post-fragmentation subpopulations (4.355) twice that of pre-fragmentation ones (2.012). In the pre-fragmentation cohort, genetic differentiations (pairwise F′ST) between subpopulation GM and the other subpopulations (except for DB) were significantly larger than random (F′ST (0.044) and G′ST (0.055) of the pre-fragmentation cohort as a whole (Nm = 5.438).Values of F′ST = 0.048 and G′ST = 0.051; and the post-fragmentation cohort F′ST =0.039 and G′ST = 0.041), but fragmentation had weakened genetic differentiation among the small populations . The exclusion of pre-fragmentation subpopulation GM generated a sharp reduction in F′ST and G′ST values among the small populations (pre-fragmentation cohort: F′ST = 0.031 and G′ST = 0.038). Homogenous patterns of genetic differentiation were detected between groups of large and small populations in both cohorts .There was no clear difference in genetic differentiation between the two cohorts from large populations  in the small and global populations, respectively.All correlograms showed significantly positive autocorrelations in the first distance interval (≤900 m). Among the large populations, values of the autocorrelation index in the first distance interval were at similar levels in pre- and post-fragmentation cohorts . In cont cohorts . Emphasiblog was present in all correlograms  were found between pre and post cohorts from large populations, whereas significant (P < 0.05) heterogeneity was present between pre- and post-fragmentation cohorts among small and global populations  (Homogeneous correlograms (ctively) .P < 0.001), indicating the presence of a genetic discontinuity and two recognizably distinct groups of plants within the overall population. Using the software BARRIER, we confirmed a significant genetic barrier present around pre-fragmentation subpopulation GM  populations, with reduced SGS in the post-fragmentation cohort, although no difference was detected in large populations. Values of F′ST and G′ST among pre-fragmentation subpopulations were much larger than those among post-fragmentation subpopulations in the former two population groups, and spatial autocorrelation analyses also showed significant differences between pre- and post-fragmentation subpopulations at distances ≤900 m and at the global population level. Further analyses indicated that the post-fragmentation reduction in SGS resulted from the removal of a pre-fragmentation genetic barrier that existed around one small island (GM). The overall SGS was therefore not significantly changed by fragmentation in the absence of population GM, indicating that extensive gene flow persisted among remnant populations. This was consistent with some previous studies , and then increased pollen flow carrying pollen from the other cluster to population GM rapidly changed its genetic composition, leading to the removal of the genetic barrier.Genetic barriers are often observed within the ranges of plant species, even when they occupy a continuous habitat, as a result of historical events such as previous fragmentation and recolonization  and the characteristics of this particular anthropogenic disturbance. The open-water area has largely or entirely prevented cross-island migration by the rodents that act as secondary seed dispersers, but has also extended the distances that pollen grains are carried. This can be contrasted with species such as Fraxinus excelsior that rely on wind for both seed and pollen dispersal, which is not negatively affected by fragmentation (Bacles et al. C. sclerophylla that depend on nonvolant animals (e.g., Bacles et al. The reduction in gene flow via seed dispersal in response to fragmentation has not been reflected in similar reductions in pollen dispersal. To the contrary, we have now detected more extensive pollen dispersal in the post-fragmentation cohort. These contrasting genetic responses to the water matrix surrounding the remnant populations reflect the different interactions between the specific pollen and seed dispersal methods of C. sclerophylla landscape-scale genetics have revealed contrasting patterns of seed and pollen dispersal at different scales, showing that this is a useful tool for measuring genetic consequences at an early stage of habitat fragmentation (Segelbacher et al. Landscape genetics methods, such as spatial autocorrelation analysis, are flexible enough to unravel SGS and underlying gene-flow patterns at multiple scales, and so provide insights into genetic responses to changes in landscape configuration (Manel et al. C. sclerophylla can reduce or prevent genetic divergence among fragmented populations at regional scales, help maintain overall genetic variation and thereby increase the viability of small remnant populations by reducing the detrimental influences of inbreeding depression and genetic drift (Ellstrand and Elam C. sclerophylla. The stabilized SGS at both local and regional scales confirms that genetic variation can be sustained in this kind of remnant population because large effective population sizes are maintained, irrespective of fragmentation (Young et al. Extensive gene flow such as we detected in C. sclerophylla. No significant difference in genetic diversity was found between pre- and post-fragmentation cohorts. Although restricted seed dispersal strengthened fine-scale spatial genetic structure at local scales (Wang et al. C. sclerophylla was not significantly changed after fragmentation, suggesting consistent gene flow. Furthermore, the disappearance of a pre-fragmentation genetic barrier provides evidence of enhanced pollen dispersal. Even without seed dispersal between fragments, pollen dispersal can link patchy populations and have a positive effect on genetic diversity. This should favor the persistence of remnant populations.Our results suggest that multi-scale landscape genetic studies are necessary to comprehensively understand the genetic consequences of fragmentation, given scale-dependent seed and pollen dispersal, and their differential responses to fragmentation. Fragmentation has different impacts on pollen and seed dispersal in wind-pollinated and gravity- plus-rodent-dispersed trees such as


Megastigmus schimitscheki (Hymenoptera: Torymidae) in southeastern France. We characterized the diapause strategy of M. schimitscheki using records of emergence from diapause in 97 larval cohorts, and we conducted a temporal population genetic study on a natural invasive wasp population sampled during ten consecutive years (1999–2008) using polymorphic microsatellite markers. We found that M. schimitscheki can undergo a prolonged diapause of up to five years and displays two main adult emergence peaks after two and four years of diapause. Such a bimodal and atypical pattern did not disrupt temporal gene flow between cohorts produced in even and in odd years during the period of the study. Unexpectedly, we found that this wasp population consisted of two distinct genetic sub-populations that strongly diverged in their diapause strategies, with very few admixed individuals. One of the sub-populations displayed both short and prolonged diapause (2 and 4 years respectively) in equal proportions, whereas the other sub-population displayed mainly short diapause. This study provided empirical evidence that prolonged diapause phenotypes can substantially contribute to reproduction and impact temporal genetic structures. Prolonged diapause is likely to act as both demographic and genetic refuges for insect populations living in fluctuating environments.Many animal species experiencing spatial or interannual fluctuations of their environment are capable of prolonged diapause, a kind of dormancy that extends over more than one year. Such a prolonged diapause is commonly perceived as a temporal demographic refuge in stochastic environments, but empirical evidence is still lacking of its consequences on temporal population genetic structures. In this long-term study, we investigated how a particular pattern of prolonged diapause may influence the temporal population genetics of the invasive seed-specialized wasp  In species living in seasonal and stochastic environments, life cycle traits are strongly selected to respond to the spatial and temporal heterogeneity of abiotic and/or biotic environmental factors Variability in diapause duration and its evolutionary consequences have been documented in various model systems such as insects Megastigmus schimitscheki Novitzky (Hymenoptera: Torymidae), which faces interannual fluctuations in its food resource and displays a particularly atypical pattern of prolonged diapause.From a genetic standpoint, it is generally assumed that prolonged diapause may help promote the maintenance of genetic diversity, as late-emerging individuals may not experience demographic or selection events similar to early-emerging ones Megastigmus genus  exhibit an extended diapause, which can be interpreted as a response to the dramatic annual variation in seed production characterizing most of their conifer host species M. schimitscheki is an obligate predator of true cedar (Cedrus spp.) seeds. It was introduced in southeastern France in the early 90s in cedar seeds imported from Cyprus C. libani Barrel in the Near East  and C. brevifolia Henry in Cyprus M. schimitscheki have been observed to date solely in southeastern France. M. schimitscheki progressively invaded most of the planted C. atlantica stands in France, even showing a competitive advantage over its direct and closely related resident competitor M. pinsapinis (Hoffmeyer) C. atlantica is considered a masting tree species M. schimitscheki produces one larval cohort per year. At year n, adult wasps emerge between April and June from seeds released by mature cones during the fall of year n–1. Females lay their eggs directly inside cedar ovules by inserting their ovipositor through the young cone scales, the larval instars develop within the seed by consuming the female gametophyte and the seed embryo entirely. At the end of summer, mature larvae enter diapause and first adult emergences occur only during the spring of year n+2 due to an 18-month cone maturation period, but a fraction of the larvae can extend their diapause and emerge at years n+3, n+4 or n+5 Cones and seeds of conifers are exploited by approximately 400 species of phytophagous insects worldwide M. schimitscheki by estimating propensities to SD and PD within larval cohorts sampled throughout its current French distribution; (ii) assessing the temporal genetic structure of one of these natural wasp populations and testing whether or not prolonged diapause can lead to its temporal genetic substructuring; and (iii) assessing how PD phenotypes could contribute to the genetic pool of this population. Using eight polymorphic microsatellite markers, a temporal population genetic approach was developed on the oldest natural invasive population of M. schimitscheki in its introduced area, which has been sampled during 10 consecutive years and for which the diapause phenotype (SD or PD) of emerging individuals was identified. We showed that M. schimitscheki consistently displays an atypical prolonged diapause strategy that may substantially influence the temporal genetic structure of its populations.In the present long term study, we aimed at: (i) characterizing the diapause strategy of the introduced populations of Megastigmus sp. were sampled as diapausing larvae within C. atlantica seeds. A cohort was defined as all the individuals that were produced the same year because Megastigmus species are univoltine Cohorts of C. atlantica can be parasitized by the 2 closely related species M. schimitscheki and M. pinsapinisCollected cones were further individually disarticulated in the laboratory for manual seed extraction. Once extracted from cones, infested seeds were identified in each sample (site and year of collection) using numerical X-ray radiography , then stored until adult emergence in rearing boxes outdoor in the Luberon cedar forest site 15, . Seeds oM. schimitscheki (M. schimitscheki. The predicted variable was the arcsine square root transformation of proportions of emerged adults. Both the duration of diapause (from 2 to 5 years) and the sex of adults were considered as fixed effects. Using the R-3.0.0 program Megastigmus species exploiting the same host display similar diapause strategies, adult emergences were similarly recorded for all M. pinsapinis individuals that emerged from the same seed lots as M. schimitscheki. In M. pinsapinis populations, thelytokous parthenogenesis generates strongly biased sex-ratios in favour of females: less than one out of 4000 sampled individuals is a male in this species M. schimitscheki only, a fraction of the emerging individuals were further genotyped.Adult emergences were recorded over 6 consecutive years in each of the 97 larval cohorts of itscheki . BecauseM. schimitscheki were estimated in the Mont Ventoux population , 1.8 µL RNAse-free water, 5 µL QIAGEN Multiplex PCR Master mix 2x , and 0.02 µL of each primer forward and reverse of 2 µM. The PCR program was 95°C 15min, 94°C 30s, 57°C 90s, 72°C 90s for 30 cycles, final elongation at 72°C for 10min and 4°C for 1 min to stop Taq polymerase in an Eppendorf thermocycler.Total genomic DNA was extracted from the entire body of each insect in individual 200 µL tubes, each containing a solution of 10% chelex resin 100 and 6 µL of 10 mg/ml proteanase K 2O), combined with 8 µL of GeneScan500 Size Standard (10 µL GS500 (−250Liz) +900 µL Formamide), denatured at 95°C for 3 minutes and then injected into the sequencer. The microsatellite profiles were analysed in GeneMapper® software version 4.1.Electrophoresis of PCR products was performed on an ABI 3730XL sequencer as follows: 2 µL of PCR product for each individual was diluted  and suggested that >95% of the individuals of a cohort produced at a year n will emerge at year n+2 and n+4, while those issued from a cohort produced at year n+1 will mainly emerge at n+3 and n+5. Consequently, we tested if such a bimodality in adult emergence could result in a disruption of gene flow between cohorts that were produced in even years  and those produced in odd years  at the Mont Ventoux site. The temporal genetic substructuring of this population was thus tested using Wright’s One goal of this study was to clarify the role of PD phenotypes in the temporal gene flow at Mont Ventoux. When possible in our samples, SD and PD phenotypes were distinguished among all the genotyped cohorts to define the following 12 groups of individuals: 1999-SD, 2000-PD, 2001-SD, 2002-SD, 2002-PD, 2003-SD, 2004-SD, 2004-PD, 2005-SD, 2006-SD, 2007-SD, 2008-SD and 2008-PD. This grouping was done so we could investigate: (i) the respective contributions of the successive parental SD and PD cohorts to the genetic pool of this population during the study period, and (ii) a potential link between parents and progeny that were characterized by the same phenotype (SD or PD). In this regard, progeny related to the cohorts produced at a year n and emerging at n+2 (SD) and n+4 (PD), while SD and PD parents related to the cohorts emerging at n but being produced at n–2 and n–4, respectively.Pairwise Fst were estimated using these 12 groups of individuals to test for genetic differentiation between progeny and SD or PD parents and between SD and PD phenotypes. We also performed assignment tests using all individuals of all cohorts on the basis of their multilocus genotypes using the Bayesian inference method implemented in STRUCTURE 2.3.3 M. schimitscheki spread over a maximum of five years in each cohort, i.e., adult emergences from cohorts produced at a year n (1999–2007) could be recorded at n+2 (2001–2009), n+3 (2002–2010), n+4 (2003–2011) and n+5 (2004–2012), indicating that a fraction of each cohort could have a diapause prolonged by 1, 2 or 3 years . As expected, there was a significant effect of diapause duration on emergence proportions . Adult emergences displayed a clear bimodal pattern as wasps mostly emerged after two and four years of diapause , while very small fractions of each cohort emerged after three and five years  , such a bimodal pattern was conserved among the 97 studied cohorts  of individuals emerged after the obligatory 2-year diapause.Emergences of  3 years . The diactively) . Althoug cohorts . To asseugh time . CompareM. schimitscheki sampled at the Mont Ventoux site during the period 1999–2008 displayed the typical bimodal pattern of adult emergence from diapause: the mean emergence percentages after 2-year and 4-year diapauses were estimated at 67.6% (SE = 7.9) and 29.2% (SE = 7.8), respectively  for all 9 loci. There was significant LD between loci MS1-110 and MS3-91 in each cohort. The locus MS1-110 was thus removed from the analysis to achieve unbiased and robust results. The HWE tests revealed that none of the ten cohorts x eight loci combinations showed significant departures. All loci were polymorphic in all sampled cohorts (1999–2008) . The estThere was statistical support for population bottlenecks at Mont Ventoux in cohorts produced in 1999, 2001 and 2005 as sign tests under the TPM and SMM hypothesis and the mode shift test were all significant . These tM. schimitscheki between 1999 and 2008. In particular, 1999 and 2004 cohorts were as much differentiated from 2-years earlier and 2-years later cohorts (to which they directly relate through SD) as from other cohorts.The genetic differentiation between pooled genetic data from ‘even’  and ‘odd’  cohorts was low and non significant (Fst = 0.2%), suggesting that the bimodal pattern of adult emergence did not influence the temporal genetic structure of the population. The temporal genetic structure of the Mont Ventoux population was also analyzed using pairwise Fst between cohorts. The matrix of pairwise Fst obtained with the correction for the presence of null alleles is given in The matrix of the pairwise Fst divergences between the 12 groups of individuals of the SD and PD phenotypes is given in tructure on the same data set to infer the temporal genetic structure of the Mont Ventoux population on the basis of individual assignments. Following the method of Evanno et al. We further used SCurculio elephas, 61, 35 and 4% of individuals emerge after one, two and three winter diapauses, respectively Megastigmus genus have a prolonged diapause, which can last up to five years in several cases M. schimitscheki can spread over five years with two consistently major emergence peaks at the second and fourth years, while only a small fraction of a cohort (less than 5% of emerging wasps) emerges at the third and fifth years. Prolonged diapause in M. schimitscheki also contrasted with that of M. pinsapinis, a closely related sympatric wasp species M. schimitscheki.Following the seminal ecological and evolutionary concepts that were developed for seed germination strategies in desert annual plants C. atlantica stands generally follows a 3- or 4-year cycle with two consecutive years of moderate to high seed production and one or two years with an almost null seed production M. schimitscheki was found to invest more in prolonged diapause than M. pinsapinis, this trait is likely to reflect their adaptation to the Cedrus genus C. atlantica is the host of M. pinsapinis in both its native (North Africa) and introduction (southern France) areas, one should note that native populations of M. schimitscheki in Cyprus did not co-evolve with C. atlantica but exclusively with the endemic Cyprus cedar C. brevifoliaC. brevifolia in Cyprus, but both high population fragmentation levels and adverse environmental conditions are thought to generate longer intervals between mast years than in C. atlantica, i.e. four to seven years M. schimitscheki and M. pinsapinis populations could then reflect different host-parasite associations in their respective native areas. Alternatively, as diapause duration is likely to be a multigenic heritable trait M. schimitscheki were likely founded from an extremely restricted number of individuals and probably had to face substantial interannual variations in food supply in the early stages of their establishment M. schimitscheki in its native area is currently unknown, ascribing a selective interpretation for the diapause pattern observed in France is still questionable. Work is currently in progress to characterize the diapause strategy of native M. schimitscheki populations, which may help unravelling the potential effects of phylogeography, demography and/or selection on this trait in France.In seed-specialized wasps feeding on conifers, prolonged diapause is viewed as an adaptive response to spatial and temporal heterogeneity of food supply that concomitantly reduce competition for a limited resource M. schimitscheki estimated a mean frequency of prolonged diapause of 0.16 (±2.1) between 1999 and 2008. Accordingly to Mahjoub and Menu Adequate life-history traits may be a key to the establishment and persistence of introduced populations M. schimitscheki may have experienced significant reductions in population size at the Mont Ventoux site during the period of the study. Significant population bottlenecks were found only in the cohorts produced in 1999, 2001 and 2005. The use of a rarefaction procedure allowed unbiased estimates of allelic richness and provided the greatest statistical power to detect differences in variation despite a small sample size in some M. schimitscheki cohorts. Recent theoretical work suggests that false bottleneck signals may be generated in a population that used to be large and structured M. schimitscheki in France, its low genetic diversity due a strong founder effect at introduction and the absence of a clear spatial genetic structure since its introduction C. atlantica at Mont Ventoux between 1999 and 2008, the three population bottlenecks detected there may reflect several low seed availability episodes that led to occasional M. schimitscheki population collapses. Another possible source of these bottlenecks may relate to the single introduction of M. schimitscheki at Mont Ventoux with a severe founder effect M. schimitscheki population may have progressively reached a sufficiently high effective size to be less susceptible to population collapse, and/or prolonged diapause may act as a genetic refuge promoting temporal gene flow in such a fluctuating environment.We investigated whether M. schimitscheki sampled for ten consecutive years at the Mont Ventoux site, we aimed at determining the possible impact of this wasp’s diapause strategy on its temporal genetic structure. We firstly tested whether adult emergences occurring principally after two and four years of diapause may result in significant gene flow disruption, i.e., significant genetic differentiation, between cohorts that were produced in even years  and those produced in odd years . A non significant Fst value (0.2%) suggested that the consistent bimodal pattern of adult emergence did not disrupt gene flow during this period. Additionally, we did not detect any clear pattern of temporal genetic differentiation between all cohorts using pairwise Fst estimates over the 1999–2008 period (Fst<5%). Three main hypotheses may be formulated to explain these results. First, annual immigration events into the Mont Ventoux population could have prevented local temporal population differentiation M. schimitscheki from Mont Ventoux (the likely introduction site) has been biased towards the south-east, mainly due to strong regional prevailing winds travelling towards the south-east  M. schimitscheki to be too recent in France (approximately ten generations) to show any detectable within-population genetic differentiation due to the diapause strategy. In this regard, it will be of critical interest to conduct similar studies within populations with longer histories such as those of the native area in the Middle East .Focusing on an invasive natural population of While prolonged diapause has been well described as a demographic refuge against environmental stochasticity M. schimitscheki at the Mont Ventoux site indicated a strong structuration of this population resulting from two distinct genetic clusters and an extremely low frequency of admixed individuals  under variable conditions of resource supply could probably help shedding some critical light on these aspects.Although this study could not provide sufficiently strong evidence for this, it may raise critical questions regarding the determinism of prolonged diapause in this wasp and probably other insect species living in fluctuating environments. Indeed, the physiological basis and the factors governing the different stages of the prolonged diapause process  remain poorly documented. Some studies suggested that the larval prolonged diapause corresponds to physiological processes that are independent of those underlying short (obligatory) diapause and that may be governed by other environmental factors M. schimitscheki populations would thus help clarifying the prevalence of PD phenotypes contributing to temporal gene flow.In this study, we described a particularly atypical pattern of prolonged diapause, which is likely to constitute both a demographic and a genetic refuge for invasive forest insects facing interannual fluctuations in resource supply. Propensity to prolonged diapause was estimated from infested seed lots maintained under natural climatic conditions for six consecutive years in rearing boxes, instead of laying on the ground in the forest litter as it occurs in fully natural conditions after cone disarticulation. This procedure favoured an optimal expression of the diapause strategy in the sampled wasp populations, but also allowed diapausing larvae to escape natural post-seed dispersal mortality factors such as pathogens, seed predators or seed decay. This may have led to an overestimation of the frequency of individuals surviving prolonged diapause in field conditions. We were however able to emphasize the genetic influence of PD phenotypes in the field, but further work is needed to assess the survival cost of prolonged diapause Microsatellite data: DRYAD entry doi: 10.5061/dryad.c1g08.Figure S1(DOCX)Click here for additional data file.Table S1(DOCX)Click here for additional data file.Table S2(DOCX)Click here for additional data file.