Although evolutionary transitions from sexual to asexual reproduction are frequent in eukaryotes, the genetic bases of such shifts toward asexuality remain largely unknown. We addressed this issue in an aphid species where both sexual and obligate asexual lineages coexist in natural populations. These sexual and asexual lineages may occasionally interbreed because some asexual lineages maintain a residual production of males potentially able to mate with the females produced by sexual lineages. Hence, this species is an ideal model to study the genetic basis of the loss of sexual reproduction with quantitative genetic and population genomic approaches. Our analysis of the co-segregation of ∼300 molecular markers and reproductive phenotype in experimental crosses pinpointed an X-linked region controlling obligate asexuality, this state of character being recessive. A population genetic analysis (>400-marker genome scan) on wild sexual and asexual genotypes from geographically distant populations under divergent selection for reproductive strategies detected a strong signature of divergent selection in the genomic region identified by the experimental crosses. These population genetic data confirm the implication of the candidate region in the control of reproductive mode in wild populations originating from 700 km apart. Patterns of genetic differentiation along chromosomes suggest bidirectional gene flow between populations with distinct reproductive modes, supporting contagious asexuality as a prevailing route to permanent parthenogenesis in pea aphids. This genetic system provides new insights into the mechanisms of coexistence of sexual and asexual aphid lineages. Asexual lineages occur in most groups of organisms and arise from loss of sex in sexual species. Yet, the genomic bases of these transitions remain largely unknown. Here, we combined quantitative genetic and population genomic approaches to unravel the genetic control of shifts towards permanent asexuality in the pea aphid, which conveniently shows coexisting sexual and asexual lineages. We identified one main genomic region responsible for this transition located on the X chromosome. Also, our population genetic data indicated substantial gene exchange between these reproductively distinct lineages, potentially leading to the conversion of some sexual lineages into asexual ones in a contagious manner. This genetic system provides new insights into the mechanisms of coexistence of sexual and asexual lineages. While sexuality is the dominant reproductive mode in metazoans, parthenogenesis - the development of an embryo from an unfertilized egg - occurs in most branches of the animal kingdom  The ancestral life-cycle of aphids is cyclical parthenogenesis Acyrthosiphon pisum. This species conveniently shows CP lineages  and OP lineages , and these two types of lineages locally co-occur in regions with intermediate climate conditions Here, we combined QTL and genome scan approaches to decipher the genetic bases of reproductive mode variation in the pea aphid We produced F1 crosses between males of an obligate parthenogenetic lineage (L21V1) and sexual females of two cyclically parthenogenetic lineages (JML06 and LSR1) . Five F2By contrast with the 61 F1 progeny which all produced sexual females (hence were classified as CP) segregation of reproductive phenotype was observed among the five F2 families . Contrasop1/op2” (CP”). Hence these individuals were either op1/CP, op2/CP or CP/CP . Since chromosomes in male pea aphids do not recombine op1 allele was transmitted to its grandchildren. Conversely, the op2 allele was inherited from the F1 mothers, which themselves inherited the whole op2-bearing X-chromosome from their OP grandfather. Recombination of the op2-bearing X-chromosome in the F1 mothers allowed reducing the region controlling reproductive modes between markers T_128012_2_G (34.8 cM) and T_126075_3_Y (49 cM) on the op2-bearing X chromosome  and D_111865_3 (48.5 cM) on the op1-bearing X copy  or an OP (6 lineages) phenotype  and looked at the alleles inherited by F2 individuals. In three F2 families  at the α = 0.01 threshold . Among these four outliers, T_111491_2 was also identified as outlier under balancing selection in the populations from CP-selecting environment  when ARLEQUIN analyses were performed among populations assumed to share the same reproductive mode  revealed four loci having excessive genetic differentiation  but linkage was not absolute  . A second hypothesis is that the production of sexual females depends on a threshold concentration of some unknown factor . Under this assumption, minor environmental variation could have drastic effect on reproductive phenotype determination around the concentration threshold. We tested for the presence of a second QTL (first hypothesis), and found no statistical support for it. Yet, power to detect such an additional QTL was low  so we cannot at the moment disentangle these two hypotheses. Nevertheless, the mostly single-locus recessive inheritance of obligate parthenogenesis in the pea aphid is in line with the few similar studies which showed that the transition from sexual to asexual reproduction is determined by a small number of loci We have shown here that a key ecological trait – the variation in reproductive mode – was controlled by one main genomic region in the pea aphid. This ∼9 cM-wide X-linked region was identified by two independent and complementary approaches: the co-segregation of molecular markers and phenotypes in experimental crosses and a large scale population genomic survey (genome scans). Interestingly, 100% of phenotypic variation was explained by the genotype at the candidate locus in five crosses  . Two hypop1 and op2) at the identified QTL and the phenotype of homozygotes op2/op2 and op1/op1 significantly differs . This indicates that at least two independent mutations in the same region are involved in the loss of sexual reproduction. Remarkably, the genome scan demonstrates that the region identified by the QTL approach also shows signatures of divergent selection between populations under different selective regimes for reproductive mode. This indicates that the QTL identified with three laboratory clones is also involved in the control of reproductive mode in wild populations originating from a large-scale geographic area (populations were collected up to 700 km apart). These population genomic data give further insights into the transitions from CP to OP. In particular, the occurrence of outliers in the QTL region, combined with their low genetic diversity in OP- compared to CP-selecting environments, reveal that only one or a few mutations leading to the OP phenotype have reached high frequencies in OP-selecting environments (otherwise this genomic region would not have been identified as FST-outlier). Outside the candidate region, populations from CP- and OP-selecting environments are weakly differentiated and show highly correlated levels of genetic diversity along chromosomes, suggesting important gene flow.Transitions from cyclical parthenogenesis  to obligate parthenogenesis (OP) in aphids probably occur through loss-of-function mutations leading to an inability of lineages to produce females in response to the environmental cues that normally trigger the sexual phase. Hence, any mutation  that disrupts the pathway leading to the production of sexual females might be responsible for this transition. In theory, these loss-of-function mutations could occur repeatedly in the same gene, or on different genes involved in the same molecular cascade, these genes being either neighbours or scattered on the genome. Herein, the OP grand-parent used for QTL mapping harbours two distinct alleles (op/CP). These heterozygous CP lineages may produce OP progenies  that would survive if they encounter mild winter environments. Some minimal amount of gene flow can maintain a low genetic differentiation between populations from OP- and CP-selecting environments at the scale of the genome since divergence for neutral loci at a migration drift equilibrium is prevented when Nm>1, N being the effective population size and m the migration rate op alleles are relatively frequent in CP populations, CP lineages will regularly produce new OP lineages . If such OP linages are generated frequently, low differentiation between populations from OP- and CP-selecting environments along the genome is expected, except at the candidate region. This scenario is however less parsimonious than the former. First, it requires very frequent production of OP lineages by CP ones in order to prevent genomic differentiation between these two compartments likely to result from the strong clonal fluctuations  typical of asexual populations op alleles in CP populations should be invoked to maintain these alleles. Yet, we know that op alleles are associated with a cost in CP selecting environments  and therefore their frequencies are expected to decrease under these conditions. Our data are thus best explained by bidirectional gene flow between populations of distinct reproductive modes and support the hypothesis of contagious asexuality in wild pea aphid populations.The most likely scenario to explain these genomic patterns of differentiation involves bidirectional gene exchanges between CP and OP lineages: Let us consider that the rare males produced by OP lineages successfully mate with sexual females from CP (as it is the case in laboratory conditions and presumably into the wild), producing CP offspring heterozygous at the candidate region (op/op genomic region), some of the alleles evolved under the asexual regime might then reintegrate an OP lineage. Hence, contagious asexuality has the potential to combine the beneficial effects of sex , is still unknown because scaffolds from the pea aphid genome sequence are not yet ordered on chromosomes op alleles, a hypothesis that deserves further investigations.To conclude, here we combined population genomics and quantitative genetics to identify the genetic bases of a key trait for aphid adaptation to climate - the loss or maintenance of sexual reproduction. We found this trait to be controlled by one main genomic region located on the X chromosome. The widespread geographical distribution of a few alleles associated with obligate asexuality suggests that these alleles might be particularly advantageous for OP lineages, and might have outcompeted previously established Vicia faba (broad bean) because it is a universal host for all known host races of the pea aphid species complex Medicago ssp. LSR1  produces only sexual individuals . Contrastingly, L21V1  produces only parthenogenetic females (89%) and a few males (11%). LSR1 and JML06 are therefore classified as cyclical parthenogens (CP) and L21V1 as obligate parthenogen (OP). Crosses between males from the OP and  females from the CP lineages were performed. For this, one L4 larva from each of the three grandparent clones was moved to a new broad bean plant and transferred to a climatic chamber with a 12 h photoperiod (18°C) to trigger the production of sexual females and males in CP lineages  Vicia faba) to generate a F1 family  that present contrasted reproductive phenotypes. These three F0 lineages were chosen based on their ability to produce or not sexual females under standard sex-inducing conditions (i.e. short photoperiod with 12 h light) Six F1 lineages (three per cross) were then chosen to produce the next F2 generation . All F1 ndmin to 200 so that no information from males meioses was used to locate QTLs . QTLs were detected based on likelihood-ratio (LR) along chromosomes. LR values corresponding to a significance level of 0.05 for each chromosome were empirically determined from 1,000 simulations under the null hypothesis of the test (i.e. no QTL). Genome-wide significance levels  were computed to account for multiple testing . The drop-off method implemented in QTLmap was applied to obtain 95% confidence intervals of the QTL location. Similarly to the reduction of x-LOD when using LOD scores, the maximum LR value was reduced by 3.84 (corresponding to a Chi2 distribution with one degree of freedom for p<0.05) to determine a threshold. Region boundaries were then defined by the LR locations crossing this threshold upstream and downstream of the peak LR The three grand-parents, the six F1 parents and the 229 F2 individuals from families 3 to 7 were typed at 401 microsatellite loci . We therefore performed an additional F2 cross (cross 8: X2_25♂×X1_3 ♀) to recombine the X-chromosome bearing the op1 allele . We also crossed X6_2♂×X3_4♀  to produce homozygous individuals at this X chromosome region (i.e. op2/op2) to assess the dominance status of the different alleles in the candidate region . Since these two crosses were performed after we had identified the genomic region controlling reproductive mode variation, only a subset of individuals were phenotyped , chosen accordingly to their genotype at 8 microsatellite markers in the genomic region of interest .The QTL approach led to the identification of a single genomic region, located on the X chromosome, which controls reproductive mode see . Yet, inA. pisum individuals were sampled from the same plant species (Medicago sativa) to prevent confounding effects of plant or reproductive mode specialization on genetic divergence Pea aphid individuals were collected in alfalfa fields from six sampling sites . All A. CTF, which accounts for the geographical structure within populations (three populations originate from OP-selecting environments and the three other from CP-selecting environments). 100 000 coalescent simulations were performed conditionally on the multilocus estimate of CTF at the 436 microsatellite loci, assuming 50 groups and 100 demes per group. The observed data from each locus were compared with the simulated distribution, and a particular locus was classified as a significant outlier if it fell outside the 99% confidence envelope. We focused here on loci putatively involved in divergence between populations with contrasted reproductive mode; hence, we considered in this analysis only the loci falling above the upper confidence limit. As we were interested in identifying outlier loci involved in the variation of reproductive mode, and not in adaptation to local environmental conditions, we checked that the outliers identified from this global analysis (in which the two types of populations were included simultaneously) were not classified as outliers  among populations sharing the same reproductive phenotype. To that end, we ran two independent analyses for the detection of outliers within populations sharing the same reproductive mode. We also checked that the outcomes of genome scan analysis were not affected by the inclusion of markers with >10% missing data (58 loci). Since the confidence interval was similar when including or not markers with >10% missing data and because our aim was to screen the genome with the highest number of markers, we present only the analysis based on the whole dataset (436 markers).To detect loci that depart from neutral expectation, and which are therefore potentially involved in reproductive mode variation, we used a hierarchical method Figure S1op1, op2, CP1, CP2, CP3 and CP4) is shown with a different colour. The upper haplotype (in the F1 to F3 generations) corresponds to fragment of chromosome inherited from the father . The lower haplotype was inherited from the mother (recombination might occur). Numbers correspond to the size of the allele at each microsatellite marker. Loci not successfully genotyped are indicated as 0 (in grey). Crosses 8 (F2) and 9 (F3) were performed after the identification of the candidate region, with the aims of further validation and to investigate the dominance of the two OP alleles (op1 and op2). Hence, only a subset of loci surrounding the QTL was genotyped in these two crosses . These crosses confirmed previous results and allowed to further narrow down the candidate region to the portion of chromosome located between markers 116879_10 (38.9 cM) and D_111865_3 (48.5 cM) based on individuals X8_52 and X8_96). We inferred the putative genotype of each individual at this candidate locus  by minimizing the number of recombination. In cases where recombination occurred between the markers flanking the candidate region, we considered the phenotypic data to determine the most probable genotype assuming that only op1/op2 and op2/op2 genotypes lead to obligate parthenogenesis . When the phenotype was not informative  the two possible genotypes are mentioned . Yet only the genotype corresponding to the left region (38.9 cM) is kept in Phenotype and genotypic data from the 4-generation pedigree. For each of the 263 F0, F1, F2 or F3 lineage, we show the percentage of sexual females (SF), males (M) and asexual females (AF) produced when the lineage is placed in environmental conditions known to induce the production of sexual forms in cyclically parthenogenetic (CP) lineages. The fecundity  and the number of individuals that died before reaching the adult stage (i.e. that were not phenotyped) is also given. These values represent average over three replicates for each lineage . Lineages were classified according to the production of sexual females . We also show for each individual its genotype around the genomic region that contains the candidate locus for the production of sexual females . Genotypes were phased and each of the different grandparental haplotype ((DOC)Click here for additional data file.Figure S2CTF) among six wild populations (∼18 ind/pop) experiencing selection for CP (those collected in North-east France and Switzerland) and selection for OP (those collected south-west France) as a function of heterozygozity for each of the 436 microsatellite loci estimated with ARLEQUIN 3.5. In this hierarchical analysis, populations were grouped according to reproductive strategy (three OP and three CP populations). The line represents the 99th quantile of the neutral envelope. Black dots: non outlier loci; yellow, orange and red dots represent outliers at α = 0.1, 0.05 and 0.01, respectively. Locus name is shown for the four 1% outliers.Genome scans of wild OP and CP populations to identify outlier markers. Genetic differentiation ((TIF)Click here for additional data file.Figure S3Genetic diversity along chromosomes. Expected heterozygosity calculated over OP populations (pink line) and CP populations (black line) along chromosomes on a 15-cM sliding window is shown. The black bar shows the location of the 95% CI of the QTL for reproductive mode variation.(TIF)Click here for additional data file.Table S1Predicted functions of genes located on the three scaffolds that lay within the candidate region. Putative functions were inferred from comparisons to the NR peptide database  and based on the identification of protein domains using Interproscan 5 (Interpro database version of January 2014). Genes in bold are those referred to in the discussion. Scaffolds GL350218, GL350005 and GL350062, respectively, carry microsatellite markers D_116808_1 (located at 38.9 cM on the X chromosome), 116879_10 (39.1 cM) and D_111865_3 (48.5 cM), respectively.(DOC)Click here for additional data file.Table S2List of markers used in genetic mapping/QTL and genome scan approaches. Primer sequences are given in (DOC)Click here for additional data file.Table S3Medicago sativa host plant. CP: cyclical parthenogenesis, OP: obligate parthenogenesis.Geographical origin of populations used in the genome scan approach. The number of individuals used in the analyses is shown. All individuals were collected on (DOC)Click here for additional data file.


Grapholita molesta is one of the major pests of stone and pome fruit species in Brazil. Here, we applied 1226 SNPs obtained by genotyping-by-sequencing to test whether host species associations or other factors such as geographic distance structured populations of this pest. Populations from the main areas of occurrence of G. molesta were sampled principally from peach and apple orchards. Three main clusters were recovered by neighbor-joining analysis, all defined by geographic proximity between sampling localities. Overall genetic structure inferred by a nonhierarchical amova resulted in a significant ΦST value = 0.19109. Here, we demonstrate for the first time that SNPs gathered by genotyping-by-sequencing can be used to infer genetic structure of a pest insect in Brazil; moreover, our results indicate that those markers are very informative even over a restricted geographic scale. We also demonstrate that host plant association has little effect on genetic structure among Brazilian populations of G. molesta; on the other hand, reduced gene flow promoted by geographic isolation has a stronger impact on population differentiation.The oriental fruit moth  Interactions among herbivorous insects and their host plants define most of the dynamics of phytophagous insect populations, which correspond to ca. of 43% of recognized insect species   are a classic example of this prediction (Bush Spodoptera frugiperda (J. E. Smith), based on ecological, genetic and physiological differences (Pashley Cydia pomonella (L.), based on divergent biological responses and oviposition behavior adaptations related to larval host plants  (Lepidoptera: Tortricidae) is, together with C. pomonella, one of the major pests in Brazil of stone and pome fruit species, particularly those in the Rosaceae   Salles . The putG. molesta  in the main regions of occurrence of this species in Brazil, applying for the first time the genotyping-by-sequencing approach of simultaneous discovery of single-nucleotide polymorphisms (SNPs) and individual genotyping . DNA was eluted in 100 μL AE buffer and stored at −20°C. Final concentrations and 280/260 and 260/230 ratios were estimated with a NanoDrop UV spectrophotometer . DNA amount per sample was normalized to 20 ng/μL.Genotyping-by-sequencing libraries were constructed using standard protocols  needed to ensure that the probability (α) of misclassifying heterozygotes as homozygotes was ≤0.05 . Loci under disequilibrium were pruned from the data matrix. Posterior analyses were carried out using loci and individuals that conformed to the above-mentioned parameters. File conversions, to allow the use of various population genetics software, were accomplished using PGDSpider v. 2.0.5.1 . Any locus with FST higher or lower than the neutral distribution (outlier) is considered a candidate for being under selective pressure  all loci and (ii) loci identified as being under selection using the program Lositan. This approach was applied to compare the power of discrimination of individuals within populations of likely non-neutral (or adaptive) markers and putatively neutral markers  using the software Arlequin v. 3.5 (Excoffier and Lischer amova was conducted among the following: (i) clusters of sampling localities found using DAPC and NJ and (ii) host plants from which samples were collected . Genetic structure was interpreted from the Φ statistics associated with different hierarchical levels in which variation is distributed  was estimated with putatively neutral loci. Each nucleotide was numerically coded as follows: A = 1, T = 2, C = 3, G = 4, all other characters = 0, and missing data = −9. Run parameters included 25 runs with 500 000 iterations following a burn-in period of 50 000 iterations for K = 1–12, under the ‘admixture ancestry model’ and allele frequencies ‘correlated’. The ΔK of Evanno . The frequency of individuals in each cluster was visualized using the programs CLUMPP v. 1.1.2 . BayeScan recovered 12 outliers, all of them with positive values of The best model fit explaining the data matrix composed of all loci was K2 + G+I (Kimura-2-parameters + gamma parameter + invariable sites). For loci inferred as being under selection with the program Lositan, the best model was K2 + I. Three main clusters were recovered when those outliers were used to construct the neighbor-joining (NJ) topology Fig.C: group P = 0.0081), as the correlation between geographic distances and Slatkin's pairwise linearized distances inferred by the Mantel test explains most of the variation (r2 = 0.5828).DAPC recovered the same clusters achieved by the NJ topology based only on outliers, with the two samples from São Paulo state more distant than other samples within their respective clusters Fig.D. For boamova resulted in a significant ΦST value = 0.19109 (P < 0.001). The hierarchical amova considering host association resulted in a ΦST = 0.20359 (P < 0.001); however, only 5% of the variation was due to host plants, which indicates a small role of hosts in shaping population structure; 15% of the variation arose among populations within groups, and the remaining 80% arose within populations. The hierarchical amova among the three clusters found with DAPC and NJ analyses resulted in a ΦST = 0.25121 (P < 0.001), which is considered a high level of genetic structure; 19% of the variation is among groups, while 75% is within populations.Overall genetic structure inferred by a nonhierarchical K = 2 with the highest likelihood, which indicates two genetic clusters  is close to the value found at a continental scale (FST = 0.219) based on microsatellites   technique  and in Paranapanema, SP (PR_peach), approximately 688 km apart (TableFST among peach samples was 0.293 (SD = 0.187), among apple samples was 0.213 (SD = 0.198), and among peach versus apple samples was 0.223 (SD = 0.192). Similar pairwise FST values among pairs of samples, from the same or different hosts, are in accordance with a weak effect of host plants in the genetic differentiation among populations.Slatkin's  linearizG. molesta, as found for other populations (Kirk et al. Host plant association does not affect genetic structure between Brazilian populations of G. molesta (Hughes and Dorn G. molesta in Santa Catarina until 1982, and since then moths have been trapped annually in the region, and it is now considered an important pest in apple orchards, damaging up to 90% of fruits (Reis et al. Geographic isolation is known to reduce gene flow among populations of phytophagous insects and is a factor usually associated with low dispersal capability (Peterson and Denno This is the first time that SNPs gathered by the genotyping-by-sequencing technique have been applied to infer genetic structure of a pest insect in Brazil. The results we found so far indicate that these markers are very informative even at a restricted geographic scale. Furthermore, the main advantage in using this technique is the opportunity to infer loci under selection, and to test the potential of putatively non-neutral markers to differentiate populations. A further advantage is the promising possibility for annotation and linking of inferred non-neutral markers to important biological functions or biochemical processes, especially if a reference genome is available. Such identification, however, is limited by the availability of reliable annotated genomes, which has improved in recent years due to the popularization of next-generation sequencing, making annotation of outliers loci more feasible in the near future.


Caenorhabditis elegans. These populations were founded with a fixed pair of deleterious mutations introgressed into multiple wild genetic backgrounds and allowed to evolve for 50 generations with a mixed mating system. There is evidence for some recombination between ancestral haplotypes, but selective sweeps seem to have resulted in the fixation of large chromosomal segments throughout most of the genome. In addition, a few new mutations were detected. Simulations suggest that strong selection and low outcrossing rates are likely explanations for the observed outcomes, consistent with earlier work showing large fitness increases in these populations over 50 generations. These results also show clear parallels to population genetic patterns in C. elegans in nature: recent selective sweeps, high linkage disequilibrium, and low effective recombination rates. Thus, the genomic consequences of selection depend heavily on the biology of the organism in question, including its mating system and levels of genetic variation.Experimental evolution studies, coupled with new advances in DNA sequencing technology, have become a powerful tool for exploring how populations respond to selection at the genomic level. Recent experiments in microbes typically have found evidence for multiple novel mutations, which are usually fixed. In contrast, in animal model systems, evolutionary responses seem to involve more modest changes in the frequencies of pre-existing alleles, probably because these populations outcross and are usually initialized with greater levels of standing variation. In this experiment, I used whole-genome resequencing to estimate allele frequencies and look for novel substitutions in experimentally evolved populations of  Only rede novo mutations to drive adaptation, and typically lacking genetic recombination. This design is typical of microbial experiments . These organisms typically also have larger genomes, potentially offering more mutational targets for adaptation, although the population mutation rate (Neμ) often is smaller in absolute magnitude because of the population sizes used. Although the repeatability of genetic changes is difficult to assess in these studies because they often identify candidate loci by looking for shared patterns across replicates, there usually are multiple loci showing a common signal  to what degree have ancestral alleles become fixed, and how much recombination has occurred among these ancestral haplotypes? (ii) How similar are genomic patterns across experimental replicates? (iii) Is there evidence of any novel mutations? (iv) What selective scenarios best explain these outcomes? The results suggest that selection played an important role in generating the observed genomic outcomes, which here highly convergent across replicates and show similarities to evolve-and-resequence studies of isogenic microbial populations as well as outcrossing, genetically variable animal model systems. Moreover, the results also show clear parallels to patterns of genetic variation in natural populations of C. elegans strain CB5362tra-2(ar221)II; xol-1(y9)X carries two mutations (in an N2 genetic background) that together result in an entirely XX population. These worms are hermaphrodites at cooler temperatures (<13°) and males at warmer temperatures (>24°). At the intermediate temperatures used in this experiment (16° and 18°), ancestral populations are a mix of males and hermaphrodites but with a high frequency of intersex phenotypes and strongly reduced fitness  following the manufacturer’s instructions. The pooled ancestor sample and three evolved samples were submitted to the SUNY University at Buffalo Next-Generation Sequencing and Expression Analysis Core for sequencing on an Illumina HiSequation 2000 with single-end, 51-bp reads (1×51). In addition, I downloaded sequence data for wild-type strains CB4856, AB1, MY2, and JU258 from the NCBI Sequence Read Archive ; these were paired-end reads (2x77 and 2x101). I mapped sequence reads to the reference C. elegans genome (WormBase release WS235) using BWA v0.7.5a (Frozen samples of each population were thawed and grown at a permissive temperature (13°) for three to four generations before DNA isolation. For each population, hundreds of worms were washed  v0.7.5a , allowinFirst, I wished to assess, for each position in the genome, which (if any) of the ancestral genetic backgrounds had become fixed. To do so, I identified SNP alleles unique to each of the five founder genetic backgrounds at nucleotide sites with at least 10× coverage in every background by using a custom R script. I then divided the genome into nonoverlapping 200-kb windows and calculated the average frequency of the alleles specific to each ancestral genetic background in each window, in each population.de novo mutations, by identifying nucleotide substitutions that were nearly fixed (>90% frequency) in at least one evolved line but absent from the pooled ancestor and all the founder wild-type genetic backgrounds .Next, I looked for evidence of fixed I used simulations to assess whether the observed outcomes were consistent with various levels of selection, numbers of fitness quantitative trait loci (QTL), and outcrossing rates. The source code for the simulation is archived on Dryad (DOI: 10.5061/dryad.603d2).s, the proportion of genomic positions in which the same allele was “dominant” (had the highest frequency) in all three populations; and (ii) f, the average proportion of sites fixed  in each population. The expected distribution and 95% confidence intervals for each of these two statistics were computed using 200 replicate simulations for each set of parameter values . The observed values of these statistics (generated from the sequence data) were then compared with the expected distributions to generate a p-value for each scenario. A small p-value (< 0.05) for a given scenario would indicate a poor fit for the observed data, whereas a larger p-value for both statistics would indicate that the parameter values are consistent with the experimental results.Simulations were written to reflect the design of the evolution experiment. In each run of the simulation, 20 populations were evolved for 50 generations each. The three populations with the greatest average fitness were selected and used to compute two test statistics: (i) N2, CB4856, AB1, MY2, and JU258), and subsequently expanded to a population size of 1000, with nonoverlapping generations. Each worm in the simulation carried a diploid XX genome, composed of six chromosome pairs of 69−105 “windows” each . Alternative population sizes of 200, 500, and 2000 also were tested and gave similar results .Each simulated population was initialized with 10 hermaphrodite and 12 male worms of each founder genetic background . The worm’s phenotypic sex was then chosen randomly based on the user-specified outcrossing rate.For each generation n+1 were generated one-by-one from the worm genotypes in generation n. To generate each offspring genotype, the first parent was selected randomly, weighted by relative fitness. If that parent was a hermaphrodite, the offspring genotype was generated by self-fertilization; if it was a male, a hermaphrodite mother was selected randomly, again weighted by relative fitness. Gamete genotypes were then generated by simulating meiosis. Each tetrad in the simulation underwent a single crossover during meiosis, mirroring the single crossover per chromosome pair observed in C. elegans .File S1 for details on how models describing the locations of QTL were generated based on observed outcomes). Finally, fitness values for QTL alleles were assigned such that the optimal genotype  would have the maximum relative fitness assigned earlier. For example, if the maximum relative fitness was 2.0, with two diploid QTL with equal effects, then the fitness value for each QTL would be 21/4 ≈ 1.189; the fitness of the optimal genotype  would therefore be 1.1894 ≈ 2. I also tested a null model with no fitness QTL .QTL were modeled by first selecting a value for the maximum relative fitness; five possible values for maximum relative fitness were tested: 1.1, 1.5, 2, 4, and 10. Then, for each maximum relative fitness value, various scenarios for the number and distribution of QTL were tested, based on the observed genotypes . By using the same analysis pipeline with the publicly available sequence data for strains CB4856, AB1, MY2, and JU258, I obtained ~30x coverage for each of the wild-type ancestral genetic backgrounds. Raw sequence files generated in this study are available from the NCBI Sequence Read Archive under accession numbers SRX658438, SRX658572, SRX658573, and SRX658574. Processed data and analysis scripts are archived in Dryad (DOI: 10.5061/dryad.603d2).I obtained a total of ~140 million single-end, 51-bp reads across the ancestral and evolved samples, resulting in at least ~9x genome-wide coverage for each population .Approximately 558,000 putative SNPs were detected, for an average density of one SNP every 180 bp. Of these, approximately 130,000 were specific to a single genetic background, for an average density of one diagnostic SNP per 750 bp, and 330 diagnostic SNPs per 200-kb window. Thus, power to detect heterozygosity within a given genomic window is fairly good because of the large number of diagnostic SNPs in each window, even though detecting heterozygosity at any individual SNP is low, given the modest sequencing depth. Indeed, substantial polymorphism was seen in the ancestor sample and on most of chromosome I in 16EE6. On the other hand, fixation or near-fixation of a single ancestral genetic background occurred throughout most of the genome in all the evolved lines; moreover, patterns of fixation were qualitatively similar in all three evolved lines . The larnhl-2), and one noncoding mutation each in lines 18EE1 and 18EE2 (File S2 and Table S3 for details). For the ancestral population, chromatograms only showed evidence of the reference allele, while only the mutant alleles were present in the respective evolved populations . These results suggest that the mutant alleles are indeed absent from the ancestral population and fixed in the evolved populations, or at least that the frequency of the minor allele is below the detection limit in each case.I found evidence for three fixed novel mutations in total: one coding mutation in line 16EE6 . Results from simulations with alternative population sizes  were consistent overall (Table S2) in rejecting neutral evolution and supporting modest to large fitness gains for evolved worms, although greater outcrossing rates (5%) were also consistent with observed results in these cases.Simulation analyses strongly rejected a null model of neutral evolution both quantitatively ; xol-1(y9) genotype these populations were founded with results in high rates of infertility and intersexuality at the rearing temperatures used in this experiment, both of these mutant phenotypes are largely suppressed in the evolved lines (Earlier work has shown strong phenotypic changes in these tra-2(ar221); xol-1(y9) genotype and the large fitness recovery observed in the actual populations after 50 generations ; xol-1(y9) genotype used in this experiment ; xol-1(y9) genotype, resulting in low-fitness males at these temperatures, and evolved populations are almost exclusively hermaphrodites ; xol-1(y9) mutant genotype fixed in these populations. First, these mutations are highly deleterious and were fixed, so I expect selection to suppress their effects to be quite strong. Indeed, the nature of the phenotypic recovery seen in these lines—e.g., the suppression of intersexed phenotypes—suggests that some of the increase in fitness in these populations is attributable to compensatory adaptation. Such compensatory evolution is probably common, as evidenced by a number of studies of both laboratory and natural systems ; xol-1(y9) genotype’s deleterious effects are expressed, suggesting compensatory adaptation.In addition, the “ancestor” sequence data provide some suggestive clues. This pooled “ancestor” consisted of g1 worms that had undergone one generation of outcrossing before being frozen, thawed, and subsequently recovering for three to four generations. Thus, there was some, albeit limited, opportunity for selection to act before DNA isolation. During this process, worms were cultured at a permissive temperature (13°), at which the deleterious effects of these mutations are largely absent, so selection to compensate for their effects should have been weak, providing something of a negative control. Consistent with the idea that selection for compensatory adaptation explains allele frequencies in the evolved lines, genomic patterns in the evolved lines are distinct from those of the “ancestral” sample. These differences should be interpreted cautiously because of the small number of generations “ancestral” worms were reared at the permissive temperature and because of the potential for genetic drift in those populations after thawing. Nevertheless, they provide a possible hint that selection acts differently when the C. elegans (Unfortunately, generating a true negative control for this experiment would be difficult. For instance, populations seeded with the same wild genetic backgrounds but lacking the mutations would exhibit very different outcrossing rates, as males are typically maintained at relatively high frequencies in genetically mixed populations of The three evolved lines investigated here were chosen from a larger set of 20 because of their especially strong phenotypic recovery. But if the fitness increase in these lines was driven largely by pre-existing genetic variants, why was the evolutionary response in the other seventeen lines noticeably weaker? First, it is possible that the few detected novel substitutions did actually contribute to the strong response seen in these three lines, or that this strong recovery is caused by additional mutations that were undetected due to low sequencing coverage. An alternative explanation is that the stochastic loss of beneficial ancestral alleles early on in the experiment may have prevented most of the evolved lines from attaining such a robust recovery. Deeper sequencing of all the evolved lines, especially using paired-end sequence reads to help identify structural mutations, would help resolve this issue.C. elegans. As one might expect, genomic outcomes also were intermediate: although there was some recombination, ancestral variants became fixed in large genomic blocks, and there was evidence for fewer novel substitutions than in most microbial studies. This result is consistent with the low outcrossing rates and strong selective sweeps seen in natural populations of C. elegans. These results thus demonstrate the utility of the evolve-and-resequence approach in exploring how patterns of genomic diversity are shaped by factors such as selection, drift, mutation, and mating system.Most other evolve-and-resequence studies have focused on populations sitting at two ends of a continuum: either asexual, initially isogenic populations, or genetically variable, obligately outcrossing systems. In contrast, the populations in this study were seeded with a modest amount of genetic variation and exhibited a mixed mating system, similar to wild populations of


Megalobrama amblycephala) is an important freshwater aquaculture fish throughout China. Because of widespread introductions of this species to many regions, the genetic diversity of wild and natural populations is now threatened. In the present study, SRAP (sequence-related amplified polymorphism) markers were used to assess genetic diversity of blunt snout bream. Three natural populations  and one genetic strain (‘Pujiang No. 1’) of blunt snout bream were screened with 88 SRAP primer combinations, of which 13 primer pairs produced stable and reproducible amplification patterns. In total, 172 bands were produced, of which 132 bands were polymorphic. Nei's gene diversity (h) and Shannon's information index (I) values provided evidence of differences in genetic diversity among the five populations (Poyang Lake>Liangzi Lake>Nanxian>‘Pujiang No. 1’>Yuni Lake). Based on cluster analysis conducted on genetic distance values, the five blunt snout bream populations were divided into three groups, Poyang Lake and Liangzi Lake , Nanxian and ‘Pujiang No. 1’ , and Yuni Lake . Significant genetic differentiation was found among the five populations using analysis of molecular variance (AMOVA), with more genetic divergence existing among populations (55.49%), than within populations (44.51%). This molecular marker technique is a simple and efficient method to quantify genetic diversity within and among fish populations, and is employed here to help manage and conserve germplasm variability of blunt snout bream and to support the ongoing selective breeding programme for this fish.The blunt snout bream ( It was originally distributed in the middle Yangtze River and a few accessory lakes, of which Yuni Lake, Liangzi Lake and Poyang Lake are the major sources The Yangtze River is the world's third longest river system, with more than 3000 tributaries and 4000 lakes Genetic diversity has been estimated in several natural and genetically selected populations of blunt snout bream using morphometrics and isozymes In this study, SRAP markers are used to investigate population genetic diversity and differentiation among three natural populations, a genetically selected strain and a cultured population of blunt snout bream in China. Our aim is to better understand the genetic diversity and integrity of natural populations based upon geography and the genetic affinities of selected strain and cultured populations based upon history. This work contributes to the critical need for germplasm conservation while at the same time supporting the ongoing selective breeding and improvement of blunt snout bream.No specific permits were required for the field studies described here. The sampling lakes and ponds are not privately-owned or protected in any way, and the field sampling activities did not involve endangered or protected species.2) was clipped from each individual without anesthesia. After sampling, the individuals of the selected and cultured populations were returned to their corresponding ponds, whereas the individuals of the three natural populations were transported to the Ezhou fish breeding base of Huazhong Agricultural University (HZAU) for further breeding programme. The clipped fin tissues were preserved in 95% ethanol. Total DNA was extracted from fin tissues using the phenol-chloroform method In total, 234 individuals from five blunt snout bream populations were included in this study. Samples were collected from three natural populations , from one selected strain (‘Pujiang No. 1’ from Shanghai), and from one cultured population (Nanxian County in Hunan Province). Details of the sampled populations are provided in A set of 19 SRAP primers , 0.5 µM each of primers, and 20 ng template DNA. All PCRs were conducted in a PTC-200 Thermocycler under the following conditions: an initial denaturing step was performed at 94°C for 5 min, followed by five cycles of 1 min at 94°C, 1 min at 35°C, 1 min at 72°C, then 35 cycles at 94°C for 1 min, 50°C for 1 min, 72°C for 1 min, followed by a final extension step at 72°C for 10 min. PCR products were separated on a 6% non-denaturing polyacrylamide gel via electrophoresis and silver staining was used to visualize the results.All SRAP PCR reactions were carried out in a final volume of 15 µL containing 1.5 µL 10×PCR buffer, 2.0 mM MgN), percent of polymorphic bands (P), Nei's gene diversity (h), Shannon's information index (I), Nei's unbiased genetic distance (D) and genetic similarity (S) estimates D, a NJ tree with bootstrap support values  was constructed using POPTREE2 FST) was used to evaluate the level of genetic differentiation among populations. Sequential Bonferroni corrections Using a 100 bp DNA marker ladder as a size standard, SRAP size-dependent fragments were scored as absent (0) or present (1) to obtain a binary matrix. Only clear, strong and reproducible bands in the size range of 60 to 1124 bp were scored. POPGENE 1.32 was used to calculate number of polymorphic bands  to determine the population assignment success for each individual fish based on its genetic diversity. The SRAP markers had 140 variable bands, a number too large for the DFA to work with, so we summarised the genetic variation using principal components analysis (PCA). The first 40 eigen vectors, which explained 82% of the variation in the SRAP data set, were used in the DFA. The DFA was based on original group size . The DFA and PCA were carried out using STATISTICA v7 software (StatSoft Ltd).Eighty-eight primer combinations were tested and of these, 13 primer pairs  showed clear, reproducible and polymorphic banding patterns. The number of amplified bands for each primer pair ranged from 8 to 21 (average 13). In total, 172 bands were detected, of which 132 bands showed polymorphism, giving a polymorphic value of 76.7%.N) and the percentage of polymorphic bands (P) were highest in the PYL population , and lowest in the YNL population . Nei's gene diversity (h) across the five populations ranged from 0.090 (YNL) to 0.185 (PYL), and Shannon's information index (I) ranged from 0.137 (YNL) to 0.280 (PYL) (For fish from the five populations (mean ± SD sample size of 46.8±2.17), the number of polymorphic bands (80 (PYL) .FST values ranged from 0.351 between the geographically adjacent natural populations of Liangzi Lake (LZL) and Poyang Lake (PYL), to 0.685 between the Yuni Lake (YNL) population and the genetically selected strain of ‘Pujiang No. 1’ (PJ-1). The differences in genotype frequencies between all pairs of populations were highly significant (P<0.001)  .FST = 0.555, P<0.001)  of hierarchical gene diversity indicated that 44.51% of the genetic variation was explained by within-population variation, whereas 55.49% of the variation was explained by among-population variation, with a significant fixation index (P<0.001) .D) among all populations ranged from 0.087 (LZL-PYL) to 0.270 (YNL-NX), and Nei's unbiased genetic similarities (S) ranged from 0.763 (YNL-NX) to 0.917 (LZL-PYL) (Nei's unbiased genetic distances (LZL-PYL) .The NJ dendrogram constructed using Nei's unbiased genetic distance  showed tFST, AMOVA and NJ analysis, all of which reveal a high level of genetic differentiation among fish from the five populations.Discriminant function analysis (DFA) of the first 40 eigen values from the PCA resulted in 100% correct assignment. That is, all fishes from all populations were correctly assigned to their populations of sampling. The DFA result is consistent with the et al. noted that the cluster of lakes within the middle Yangtze River region requires protection for the preservation of migratory fishes and that there is a need to identify areas of high fish biodiversity to mitigate loss of fish biodiversity in the Yangtze River basin China's freshwater fish diversity is high at 920 species in 302 genera N, percent of polymorphic bands P, Nei's gene diversity h, Shannon's information index I) showed the same order from largest to smallest for the five populations. Both h and I indicated that the highest genetic diversity level was observed in the PYL population, followed by the LZL population, and the lowest was in the YNL population, all three being natural populations of Megalobrama amblycephala. As suggested by Li et al.All four indices  and large yellow croaker (Pseudosciaena crocea) For the cultured (Nanxian) and selectively bred (‘Pujiang No. 1’) populations, the levels of genetic diversity were much lower than within the native PYL and LZL populations. These results are in agreement with investigations on largemouth bass . This surprising result may be due to the degradation of germplasm resources of YNL over a reasonably short period of time. Previously, Li FST is one of the most widely used measures for evaluating the genetic differentiation between/among populations, which can provide important insights into the evolutionary processes that cause the genetic differentiation among populations FST values is to some extent species- or even group-specific, as a general rule FST values of 0–0.05 represent little differentiation, values of 0.05–0.25 indicate moderate differentiation, and values higher than 0.25 indicate very great differentiation among populations FST between the five populations were >0.25, indicative of a high level of genetic differentiation among these five populations. The highest FST value detected (0.685) was between PJ-1 and YNL, suggesting that pronounced genetic differentiation now exists between the selectively bred strain (PJ-1) and the natural YNL population. These findings were supported by our discriminant function analysis (DFA) which was able to assign each and every fish to its population of origin, are comparable to the results reported by 7 strain). The five populations of blunt snout bream clustered into three groups, as revealed by NJ analysis. Two of the three wild populations, PYL and the geographically close population of LZL, clustered together, while the third wild population (YNL) was highly differentiated from the other two wild populations . In addition, we tried to construct a NJ tree based on pairwise FST between populations at the same time. The NJ tree based on pairwise FST was consistent with the results above (data not shown). These results confirm earlier observations that obvious genetic divergence occurred between fish from YNL and LZL or PYL, and no divergence occurred between LZL and PYL et al.In conclusion, the SRAP system has been successfully employed to assess population genetic diversity among domesticated and wild genotypes of blunt snout bream from China. Analysis revealed that cultivated, genetically selected and wild bream germplasm are highly differentiated and that wild lake populations have high levels of genetic diversity. Comparison of the genetic diversity of wild fish to that of cultured and selectively bred fish illustrates the need for greater care managing this important resource so that the aquaculture programme to support this species is not disadvantaged in the future and so that wild populations of fish are protected for both conservation and fisheries purposes.


Blattella germanica) is a widespread urban invader that can actively disperse within buildings but is spread solely by human-mediated dispersal over longer distances; however, its population structure is poorly understood. Using microsatellite markers we investigated population structure at several spatial scales, from populations within single apartment buildings to populations from several cities across the U.S. and Eurasia. Both traditional measures of genetic differentiation and Bayesian clustering methods revealed increasing levels of genetic differentiation at greater geographic scales. Our results are consistent with active dispersal of cockroaches largely limited to movement within a building. Their low levels of genetic differentiation, yet limited active spread between buildings, suggests a greater likelihood of human-mediated dispersal at more local scales (within a city) than at larger spatial scales (within and between continents). About half the populations from across the U.S. clustered together with other U.S. populations, and isolation by distance was evident across the U.S. Levels of genetic differentiation among Eurasian cities were greater than those in the U.S. and greater than those between the U.S. and Eurasia, but no clear pattern of structure at the continent level was detected. MtDNA sequence variation was low and failed to reveal any geographical structure. The weak genetic structure detected here is likely due to a combination of historical admixture among populations and periodic population bottlenecks and founder events, but more extensive studies are needed to determine whether signatures of global movement may be present in this species.Understanding the population structure of species that disperse primarily by human transport is essential to predicting and controlling human-mediated spread of invasive species. The German cockroach ( Human-mediated dispersal has greatly contributed to the spread of invasive and non-native species, distributing them far beyond their native ranges. Although long distance dispersal events, such as human transport, are typically rare, they can be important to population dynamics and the broad scale shaping of population genetic structure in some species Urbanized areas provide many examples of human-mediated invasive species because of the considerable amount of transit between cities, and because cities provide modified environments and substantial resources that tend to favor invasive and non-native species at the expense of natives F-statistics Genetic markers have long been recognized as useful tools for the study of population genetic structure for the inference of past demographic events, including dispersal and gene flow Blattella germanica L.) is a ubiquitous inhabitant of human-built structures and is widely considered one of the most prominent of the structural pests and arguably one of the most successful invasive species on earth. It has been implicated in the spread of numerous human pathogens, including bacteria , suggesting populations were slightly inbred. Mean number of alleles per locus ranged from 4.62 in the Hickory, NC population to 8.50 in the Gainesville, FL (GFL) population and allelic richness  ranging from 3.36 in the Hickory, NC population to 5.74 in the Puerto Rico (PR) population. Genetic diversity was generally much lower in the B. germanica populations from Eastern Europe and Asia, with only Singapore (SIN) having a level of heterozygosity and average alleles per locus comparable to the U.S. populations. As with the U.S. populations, observed heterozygosities were significantly lower than expected . In all other populations, observed heterozygosity and alleles per locus were as low as 0.42 and 3.25, respectively, despite sample sizes of 30 individuals in each case. While eight of 18 Raleigh populations deviated significantly from Hardy-Weinberg equilibrium, only three of the other 16 U.S. samples differed significantly from the expected frequencies. All Eurasian samples except the Kiev (KUA) and Crimea (CUA) populations, which had the lowest average number of alleles per locus in this group, tested highly significantly (P<0.001) for departure from Hardy-Weinberg equilibrium.Levels of genetic diversity were highly variable among the 34 U.S. and Puerto Rico samples with observed heterozygosities ranging from 0.46 in the Hickory, NC (HNC) population to 0.76 in the LS-B Raleigh, NC population . ObserveP<0.0001). In the 15 cases in which populations were not significantly differentiated, 12 involved populations in Raleigh; in five cases it involved apartments within the same building  and in two cases it involved apartments in the same complex but different buildings (DR-X was not significantly differentiated from DR-Y or DR-Z). The remaining three cases involved population CCA , which was not significantly differentiated from three populations in Raleigh: DR-X, DRD-Y and CS-A.Pairwise tests of differentiation showed that in all but 15 of the 780 comparisons, populations were significantly differentiated following Bonferroni correction  in mean pairwise FST values across our six spatial scales  increased from 0.028 (0.023) and 0.026 (0.023) within apartment buildings and apartment complexes, respectively to 0.050 (0.002) among locations within Raleigh, to 0.106 (0.006) across U.S. populations using only a single population from Raleigh (DR-X), and to 0.206 (0.006) when considering only comparisons between continents. However, mean FST was significantly higher within Eurasia, with a value of 0.259 (0.018). Within the U.S. , the least differentiated pair of populations (FST = 0.025) was Norfolk, VA (NVA) and Raleigh, NC (DR-Y), and Baton Rouge, LA (BTL) and Raleigh, NC (CS-A), whereas the most differentiated pair of populations (FST = 0.350) was Hickory, NC (HNC) and Gary, IN (GIN). Within Eurasia, the least differentiated pair of populations (FST = 0.181) was TRU  and TIR , whereas the most differentiated pair of populations (FST = 0.335) was MRU  and TIR . Comparing the U.S. and Eurasia, the least differentiated pair of populations (FST = 0.053) was Raleigh, NC (DRD-Y) and Singapore (SIN), whereas the most differentiated pair (FST = 0.391) was Hickory, NC (HNC) and Crimea, Ukraine (CUA).An ANOVA test found significant differences , although the slope of the regression was only 0.007  as shown in TCS found a maximum of eight connection steps within a probability limit of 95% (P = 0.954), with one haplotype not connecting to the others within this limit (not shown). Haplotypes varied greatly in their frequency and geographic ranges, and no geographic pattern of B. germanica mitochondrial diversity was detectable with this network. Given the low degree of sequence variation, no further individuals were sequenced for this gene.A total of 66 individuals from 34 populations were sequenced for the 16S mtDNA gene. The sequences have been deposited in GenBank (accession numbers KJ937895-KJ937960). Only three sites were variable within the 385 bp region examined (0.8% uncorrected sequence variation), and the average nucleotide difference was only 2.63. A parsimony haplotype network computed in FST was lowest among samples collected from the same apartment building and among populations from different buildings in the same housing complex, although the average values were not significantly different from those among populations at the within-city level. Results of the Bayesian analysis revealed a greater level of structure within 10 km within the city of Raleigh than was evident from F-statistics alone, placing each set of apartments from the same building into the same genetic clusters, but each set formed a different cluster. In addition, all of the apartment samples collected from separate buildings in the same complex  were clustered together except for sample DRD-Z which grouped together with samples from Texas and Minnesota.Using a combination of standard measures of genetic differentiation and Bayesian clustering methods, we characterized the genetic structure of German cockroach populations at various spatial scales from within buildings to across continents. Our results show increasing levels of genetic differentiation among apartment infestations at greater spatial scales, but also reveal a complex structure that reflects human-mediated dispersal across both short and long distances. Genetic differentiation as measured by Taken as a whole, our results show little genetic differentiation among samples collected from different apartments in the same building, whereas there was evidence of slightly greater levels of differentiation among populations from different buildings in the same apartment complex. These results are consistent with the limited active dispersal range of this flightless cockroach B. germanica using allozymes and RAPDs did not find evidence of population clustering in two French cities located approximately 900 km apart At the within-city scale, we found a mosaic pattern of genetic differentiation. Bayesian analysis detected four genetic clusters in the city of Raleigh, with nearly all of the populations from four of the six apartment complexes clustered together with populations from more distant locations, including California and Puerto Rico. This heterogeneous mix of apparently similar and dissimilar populations within a city is consistent with limited active dispersal between buildings and extensive human-mediated dispersal through movement of infested materials within the city as well as between more distant locations. While we found some clustering of populations within the city of Raleigh, previous genetic studies of FST-values were relatively high . Nonetheless, the degree of genetic differentiation within Eurasia was greater than that between the U.S. and Eurasia.Levels of pairwise differentiation among the six Eurasian populations were higher than those among U.S. populations. This may be due in part to the fact that the Eurasian samples represented a larger geographic area than the samples from the U.S., although in the two cases where there were samples from the same country within Eurasia, pairwise FST values within the U.S. were significantly lower than those between continents (n = 6) and represented a very large geographic region and several different countries. In addition, we found little divergence in global samples for the mitochondrial 16S gene, and no geographical pattern of haplotype diversity. These results suggest that there is some historical connectivity between populations around the world, a finding consistent with the centuries-long global movement of this species We were able to detect very little population genetic structure differentiating German cockroach populations as a whole between the different continents. We did find that mean ntinents , and whiRattus rattus) across the globe B. germanica, but mtDNA diversity in the original source populations was low to begin with and human transport of cockroaches has homogenized global populations. We found only 0.8% sequence variation in the 16S gene across 34 populations. Similarly low mtDNA variation (0.5–0.8% nucleotide diversity) has been found in both native and introduced ranges of the invasive Formosan subterranean termite Coptotermes formosanusB. germanica, which are likely to be located in eastern Asia One possible explanation for the weakness of the global genetic structure is that a human-adapted lineage of German cockroaches was rapidly spread by human activity from a single source population with limited haplotype diversity, resulting in a relative lack of diversity on a global scale. This would be similar to the proposed expansion of a low-genetic diversity “ship rat” lineage of the black rat  based on the same eight microsatellite loci used here and with similar sample sizes  between pairwise FST-values and geographic distance among 21 bed bug populations spanning the eastern U.S. from Florida to Massachusetts. Significant isolation by distance has also been reported in the black rat (R. rattus) in the U.S., which was likely introduced to the U.S. about the same time as B. germanica (mid 1500 s), but not in the Norway rat (R. norvegicus), which was introduced to the U.S. in the 1700 s Unlike our study, Booth et al. C. lectularius, a species that has undergone very recent global resurgence. Using microsatellite markers, Booth et al. B. germanica populations within a building. Alternatively, unique features of their respective mating systems might facilitate admixture and homogenization among cockroach propagules and not among bed bugs.Our results show similarities and differences to both the small-scale and large-scale genetic structure of FST of 0.179 between populations of C. lectularius in two apartment buildings in the same complex, whereas we found an average FST of only 0.026 among B. germanica populations located in different buildings in the same apartment complex. At spatial scales at the level of the city and above, bed bug populations show much greater differentiation than do German cockroach populations, with average pairwise FST-values among populations in the eastern U.S. of 0.68 FST = 0.11 among B. germanica populations across of the U.S we found here. And, as mentioned above, weak but significant isolation by distance across large spatial scales has been found in both species. It is likely that the bed bug resurgence in the U.S. over the last 15–20 years has originated from multiple highly diverse source populations, with extensive propagule pressure and influx of new genotypes still occurring today. In contrast, German cockroaches have associated with the human-built environment in North America without interruption for >500 years. Global trade and transport likely have increased the frequency of long-distance dispersal events, facilitated gene flow, and homogenized populations, resulting in little global genetic structure in the German cockroach.Genetic differentiation among populations in different buildings within the same apartment complex appears to be greater in bed bugs as well. Booth et al. F-statistics, Bayesian clustering methods and isolation by distance did find a signal of greater connectivity of populations within a single city  compared to connections among other cities in the U.S., and of populations within the U.S. relative to populations between continents, suggesting a greater history of gene flow and migration within each successively smaller spatial scale. On the other hand, cluster analyses provided evidence that long distance human-mediated dispersal of B. germanica frequently occurs in the U.S. resulting in a patchwork of local populations sharing recent ancestry intermixed with unrelated populations. We found evidence that there is greater movement of cockroaches among cities in the U.S. than in Eurasia but no clear pattern of global genetic structure from the regions we sampled. Mitochondrial DNA variation was very limited and therefore uninformative in revealing population structure. More extensive studies including a broader geographic range, particularly eastern Asia which is the likely geographic origin of B. germanica, should provide greater insights into the global genetic structure and dispersal history of this species.We were able to detect very little global genetic structure in the German cockroach, which is probably attributable to a combination of many centuries of human transport and a lack of migration-drift equilibrium. However, traditional In order to address gene flow and differentiation at several spatial scales, cockroach samples were obtained for various geographic scales: among apartments within apartment buildings, among buildings within apartment complexes, among locations within a city, among cities across the U.S., and global . CockroaB. germanica for U.S. and global analysis were provided by colleagues; these samples represented 16 additional cities within the continental United States, Hawaii, Puerto Rico, and six international cities. Each sample represented a single collection from a single apartment at a single point in time. All samples were stored at −20°C in 95% ethanol until DNA extraction.An additional 24 samples of Bg-1D5, Bg-A7, Bg-B12, Bg-CO4, Bg-D05, Bg-D9, Bg-F7, Bg-G7, Bg-wb-1 A, Bg-wb-2 A) according to the polymerase chain reaction (PCR) procedures outlined by Booth et al. Bg-wb-1 A and Bg-D9 were later removed from the analysis due to a high frequency of null alleles as detected by the program MICRO-CHECKER DNA was extracted from the legs and thorax of each German cockroach using the Gentra Puregene DNA extraction kit . Each cockroach was amplified for 10 microsatellite loci , and expected and observed heterozygosity were calculated using FSTAT version 2.9.3.2 2 test as implemented in GENEPOP FST values FST values across U.S. populations we used only a single population from Raleigh (DR-X). Confidence intervals for global FST values were provided by bootstrapping over loci. Pairwise FST values were compiled for all comparisons within each spatial scale and variance between scales was tested by ANOVA using JMP Pro 9.0.0 . Mean FST values for each scale were compared using a Tukey-Kramer test.To determine if populations were significantly differentiated from each other, we conducted tests of genotypic differentiation for all pairs of populations using the exact χn = 6; LS-A, CR-A, CS-A, DR-X, DRD-X, FC-X). Geographic distances were loge transformed and genetic distances were transformed to FST/(1-FST), following Rousset Using a Mantel test implemented in GENEPOP, a test of isolation by distance was conducted to determine whether genetic differentiation and geographic distance were correlated across the U.S. The test was first conducted using all populations from within the continental U.S., including all 18 populations from within Raleigh (3 populations from each of 6 apartment complexes). Distances between apartment complexes in Raleigh ranged from 1.3–9.1 km, from 15–45 km for the three Los Angeles area populations, and from 130–3600 km for comparisons between cities. The Mantel test was performed a second time with comparisons of all populations using only a single population from each Raleigh apartment complex  was performed using BAPS 6.0 Two randomly selected individuals per population were selected for preliminary mtDNA analysis and a 385 bp of the mtDNA 16S gene was sequenced. PCR was conducted using primers LR-J-13017 and LR-N-13398 DnaSP version 4.50.3 Aligned sequences for all global German cockroach populations were analyzed using


Plumbago zeylanica and ascertain whether these variations are dependent on geographical conditions; and (ii) evaluated genetic differentiation and population structure within the species. ISSR and RAPD markers were used to study the genetic structure and variation within and among populations collected from 13 widespread regions in India. High levels of genetic diversity and significantly high genetic differentiation were revealed by both the markers among all studied populations.Knowledge of the natural genetic variation and structure in a species is important for developing appropriate conservation strategies. We aimed to examine (i) the patterns and levels of morphological and genetic variability within/among populations of  Plumbagozeylanica remains unknown, we aimed (i) to examine the patterns and levels of morphological and genetic variability within/among populations and ascertain whether these variations are dependent on geographical conditions; and (ii) to evaluate genetic differentiation and population structure within the species. A total of 130 individuals from 13 populations of P. zeylanica were collected, covering the entire distribution area of species across India. The genetic structure and variation within and among populations were evaluated using inter-simple sequence repeat (ISSR) and randomly amplified DNA polymorphism (RAPD) markers. High levels of genetic diversity and significantly high genetic differentiation were revealed by both the markers among all studied populations. High values of among-population genetic diversity were found, which accounted for 60 % of the total genetic variance. The estimators of genetic diversity were higher in northern and eastern populations than in southern and western populations indicating the possible loss of genetic diversity during the spread of this species to Southern India. Bayesian analysis, unweighted pair group method with arithmetic average cluster analysis and principal coordinates analysis all showed similar results. A significant isolation-by-distance pattern was revealed in P. zeylanica by ISSR  and RAPD  analysis. The results obtained suggest an urgent need for conservation of existing natural populations along with extensive domestication of this species for commercial purpose.Knowledge of the natural genetic variation and structure in a species is important for developing appropriate conservation strategies. As genetic diversity analysis among and within populations of  An understanding of the patterns of genetic variation within and among populations of medicinal plants is essential for devising optimum genetic resource management strategies for their conservation, sustainable utilization and genetic improvements. Natural populations of medicinal plant species are extensively exploited due to their heavy demands. In such cases, long-term survival as well as semi-domesticated nature of many medicinal plants depends on the maintenance of sufficient genetic variability within and among populations to accommodate new selection pressures exerted by continuous environmental changes . GeneticPlumbago and other Plumbaginaceae family members in India inspite of their high economical benefit  and inter-simple sequence repeats (ISSR) markers which are widely used because of low cost, easy access and high polymorphic nature. Unlike SSRs these markers do not require prior knowledge of genome sequence. Despite limitations regarding reproducibility of RAPD, combination of ISSR and RAPD markers has been used for understanding population genetic diversity and structure in a number of species (P. zeylanica using RAPD and ISSR markers to (i) evaluate the wide-range genetic structure of 13 populations selected to cover its distribution across India, (ii) infer relationship between latitude and the components of genetic variation in P. zeylanica populations, (iii) compare the population genetic structure in P. zeylanica populations using two dominant markers and (iv) provide necessary information for developing conservation strategies for this endangered medicinal shrub.We focused on large-scale population genetic analysis of No national permissions were required for this study as it did not involve critically endangered or protected species. No specific permissions were required to access the study sites; the collections were made on public lands.P. zeylanica were randomly selected from each population site covering an area of 50 km and collected during the month of October–December 2010. Data on the coordinates and altitudes of all the population sites are presented Supporting Information—Table S1] and ΔK were retrieved from the STRUCTURE HARVESTOR software  represented that the first two components, which had eigenvalue higher than 1, denotes the total of 77.6 % of whole phenotypic variability, contributing to all the variables to the morphological diversity of sampled populations Supporting Information—Fig. S3 and Table S2] at the species level (Tables P. zeylanica (H) = 0.03 and 0.05, Shannon's index (I) = 0.04 and 0.07).In 13 populations of ) Tables . These vP. zeylanica population using both ISSR and RAPD markers , indicating that the populations were subjected to genetic isolation. Similar results were also reported in many other medicinal and endemic species such as Torreya jackii . The sampling sites chosen in this work were geographically widely separated  and this may account for the high genetic diversity and gene differentiation observed in this species. However, some of the sampling sites (Kolli and Coimbatore) were separated by ∼150 km, and the probable causes underlying genetic diversity of these populations could not be attributed to geographical separation alone. From the present study it is revealed that the populations of P. zeylanica from different eco-geographical regions of India were grouped into two major clusters, one with purple trichomes and other with semi-transparent trichomes on sepal, this is also supported by both ISSR and RAPD data  when compared with the natural forest represented by Bhandardara population . This could be attributed to a reduction in the gene pool and increased gene differentiation in fragmented forests, which arises due to the presence of fewer individuals in the population and induced inbreeding (Sinopodophyllum hexandrum (GST = 0.62) (Podophyllum hexandrum (GST = 0.75) (Saruma henryi (GST = 0.69)  (Dactylorhiza hatagirea (GST = 0.25)  (Taxus fauna (HS = 0.12) (Changium smyrnioides (HS = 0.11)  . Plant sP. zeylanica is not known, but the species is widely distributed in the different eco-climatic zones of India, which is suggestive of a broad genetic base  of growth and morphological variables.Figure S4. Regression of genetic diversity (A: He and B: PPB) on latitude (oS) for ISSR (a and b) and RAPD (c and d) marker data and (C: He and D: PPB) of P. zeylanica populations.Figure S5. Unrooted dendrogram from the neighbor-joining analysis of ISSR markers.Figure S6. Unrooted dendrogram from the neighbor-joining analysis of RAPD markers.Figure S7. Principal component analysis (PCoA) using ISSR and RAPD data.Figure S8. Types of growth habit observed in P. zeylanica populations.


Callinectes sapidus was evaluated along 740 km of the Western South Atlantic coast. Blue crabs are the most exploited portunid in Brazil. Despite their economic importance, few studies report their ecology or population structure. Here we sampled four estuarine areas in southern Brazil during winter 2013 and summer 2014 in order to evaluate diversity, gene flow and structure of these populations. Nine microsatellite markers were evaluated for 213 adult crabs, with identification of seven polymorphic loci and 183 alleles. Pairwise FST values indicated low population structure ranging from -0.00023 to 0.01755. A Mantel test revealed that the geographic distance does not influence genetic (r = -0.48), and structure/migration rates confirmed this, showing that even the populations located at the opposite extremities of our covered region presented low FST and exchanged migrants. These findings show that there is a significant amount of gene flow between blue crab populations in South Brazil, likely influenced by local current dynamics that allow the transport of a high number of larvae between estuaries. Considering the elevated gene flow, the populations can be considered a single genetic stock. However, further information on population size and dynamics, as well as fishery demands and impacts at different regions, are necessary for harvest management purposes.Population connectivity in the blue crab  Understanding the population structure of commercially valuable species is extremely important for identifying stocks, defining fishing boundaries, and managing exploitation of fishery resources ,2. The dPenaeus monodon [Carcinus maenas [Callinectes sapidus [Genetic markers are commonly used to evaluate the degree of connection among populations of marine invertebrates, since the minute size of these organisms during their larval phase hinders direct observations of dispersal between areas ,5. Micro monodon  and the s maenas , as well sapidus . This in sapidus .The blue crab is a marine-estuarine crustacean ,14 that C. sapidus larvae.The eastern coast of Brazil in influenced by two main ocean current systems: the Brazil Current (BC) and the Malvinas Current (MC) . The BC Anchoviella lepidentostole in the southeast and the pink shrimp Farfantepenaeus paulensis in South Brazil, when it is commonly caught using banned fishing gear [C. sapidus in the Western South Atlantic [Blue crabs represent an important commercial and recreational asset valued at approximately US$185 million worldwide in 2013 , and areing gear . DespiteAtlantic . Such stIn this context, the present work aimed to evaluate the genetic diversity and connectivity of blue crab populations in Western South Atlantic. Since coastal ocean currents are likely to influence larval transport and gene flow between areas, we tested the hypothesis that there are seasonal differences in gene flow between populations due to variations in currents.This work was approved by the evaluation committee of the Biological Oceanography Master Program of the Universidade Federal do Rio Grande-FURG. According to Normative Instruction 154/March 2007, all capture, tagging, sampling and transport of biological samples of wild animals for scientific purposes must have approval from Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) SISBIO committees. This study was approved by ICMBio, and conducted under SISBIO license #40765–1.Adult blue crabs were sampled along 740 km of the cost of Santa Catarina (SC) and Rio Grande do Sul (RS) states, in South Brazil, at four areas: Itajaí and Laguna (SC), Tramandaí and Lagoa dos Patos (RS) . SamplesIndividuals were captured using nets and traps at Lagoa dos Patos and Itajaí, or purchased directly from local fishermen after morphological identification at Tramandaí, Laguna and Itajaí. Sampling within regions was opportunistic, but always conducted inside the estuary in order to adequately represent the population. Muscle tissue was removed from the pereiopods, placed in absolute alcohol, and stored at -20°C temperature until laboratorial analyses.Genomic DNA was extracted using standard proteinase K digestion and Phenol:Chlorophorm:Isoamyl Alcohol method . QuantifCallinectes sapidus (12) were amplified through Polymerase Chain Reaction (PCR) , 0.06 μM forward primer labeled with an M13 tail, 0.3 μM reverse primer, 0.24 μM universal fluorescent-label M13 primer, and ultrapure water to complete a total volume of 10 μL [Nine microsatellite loci previously described for on (PCR) . Each PCof 10 μL .TM (Biotium).Amplification conditions were the same for all primers, with variations only in the annealing temperature . PCR conMicrosatellite loci were genotyped on an ABI 3500® (Applied Biosystems) capillary sequencer, and allele sizes obtained with the use of 600LIZ® internal size standard (Applied Biosystems). For genotyping optimization, samples labeled with different dyes were combined; therefore, each plate well contained PCR products of a same individual amplified with labeling dyes 6-FAM™, VIC®, NED™ and PET®. Results were visualized and molecular weight of alleles determined with GeneMapper v 4.1 (Applied Biosystems).FIS) [Identification of null alleles and correction of allelic frequencies were performed with MICRO-CHECKER 2.2.3 . Allele FIS) , were obFIS) . SignifiFST estimates and Analysis of Molecular Variance (AMOVA) were used to compare genetic differentiation among groups by season (winter and summer). All FST significances were determined after a Bonferroni correction (α < 0.001). Due to insignificant FST (α < 0.001) between seasons, winter and summer samples of each population were grouped and genetic structure inferred only between populations. Isolation by distance was evaluated by a Mantel test following Rousset [FST calculated across all loci. All pairwise FST, AMOVA and the Mantel test were run in ARLEQUIN 3.0 [Pairwise  Rousset  using thQUIN 3.0 .Population clustering was done using a Bayesian approach in STRUCTURE 2.3.4 , assuminγab = θb.Mab, where γ is the effective number of migrants, θ the populations size and M = m/μ, where m is the immigration rate and μ is mutation rate [To estimate the migration rates between populations, a maximum likelihood approach based on the coalescent method was implemented in MIGRATE 3.6  using deion rate ,44. MigrSeven of the nine analyzed microsatellite loci were polymorphic, displaying high levels of allelic diversity. Loci CSC-074 and CSA-092 were monomorphic in all locations, and were therefore excluded from all analyses. A total of 183 alleles were detected over the seven polymorphic loci at the four sampled areas, with a mean number of alleles per locus of 26.1. Locus CSA-073 presented the highest number of alleles  while locus CSA-121 displayed the lowest allelic diversity, with four alleles .Genetic variability was similar among samples. Two tetranucleotide microsatellite loci, CSA-121 and CSC-094, displayed lower allelic diversity than the remaining polymorphic loci. The five dinucleotide microsatellite loci  were highly polymorphic and all displayed private alleles to some populations. The frequency of private alleles between locations was low .Expected heterozygosity (He) per locus varied from 0.1374 (locus CSC-094 at Tramandaí) to 0.9558 (locus CSC-007 at Laguna), and observed heterozygosity (Ho) ranged from 0.0882 (locus CSC-094 at Tramandaí) to 0.9444 (locus CSA-073 at Itajaí). Mean expected heterozygosity of all loci was 0.5891, 0.4410, 0.5414 and 0.6148, and mean observed heterozygosity was 0.5419, 0.4538, 0.4392 and 0.5749 for Lagoa dos Patos, Tramandaí, Itajaí and Laguna, respectively. Loci CSC-007, CSC-004, CSC-001 and CSA-073 showed deviations from HWE. In this manner, among the 28 locus-area combinations , significant deviation from HWE was observed for a total of eight groups (α < 0.001) .FIS) varied from -0.0646 at locus CSA-121 to 0.6771 at locus CSC-001 (both at Tramandaí), and mean FIS for all areas was 0.1116  (The inbreeding coefficient (s 0.1116 . As note< 0.001) . TherefoFST values varied from -0.00023 to 0.01755, and global FST was 0.00682, revealing very low structuring between the analyzed populations. Highest FST was observed between Tramandaí and Laguna (0.01755), which also indicates low, albeit significant (α < 0.001), differentiation (Pairwise ntiation . The AMOBayesian posterior probabilities indicated that the sampled populations were grouped into two clusters (K = 2), with all individuals presenting almost equal probabilities of being assigned to both clusters . This onγ = 24.19), and lowest from Tramandaí to Itajaí (γ = 2.90) (γ = 3.44) was observed from Lagoa dos Patos to Itajaí, wich are the two populations located at the extremities of our study area range, separated by 740 km.Estimated migration rates showed highest number of migrants from Lagoa dos Patos to Tramandaí ( = 2.90) . TramandCallinectes sapidus from four populations in the Western South Atlantic presented relatively high genetic diversity, with 183 alleles found in 213 individuals over the seven considered loci. However, this diversity was lower than the one reported by Steven et al. [et al. [Callinectes danae at the same region studied in the present work [Callinectes danae in southern Brazil through allozymes analysis. Lower diversity and heterozygosity could be a result of continuous gene flow due to larval dispersal between areas, leading to shallow structure and admixture of C. sapidus populations at the studied region.n et al.  for blue [et al. , as wellent work . Deviatient work  for CallFST = 0.00682. This estimate is below the FST value reported for other C. sapidus populations in the Atlantic, even those separated by smaller distances than the 740 km considered in this work. Yednock et al. [FST of 0.09460, indicating a higher structure at this region when compared to the Western South Atlantic. Low structure and high gene flow was also shown through Bayesian clustering, number of migrants between populations, and AMOVA results, allowing us to infer a certain degree of panmixia among populations [FST between Tramandaí and Laguna (0.01755), wich despite being significant (α < 0.001) is still quite low. Lagoa dos Patos and Itajaí, located at the two margins of our studied region, were not significantly structured (FST = 0.00373). These areas are separated by over 700 km and it would be expected that gene flow between them be limited, since migration is generally higher between geographically proximal areas [Overall differentiation between blue crab populations in southern Brazil was very low with ulations . Pairwisal areas ,48.Penaeus paulensis (currently Farfantepenaeus paulensis) shrimp post-larvae disperse from the coast of Santa Catarina to Rio Grande do Sul from the end of winter to the end of summer. The same process could be responsible for C. sapidus larval transport between these areas, since blue crab females lay eggs in the open ocean from the end of spring to autumn [Blue crab life history includes an oceanic larval period with seven or eight zoea followed by a megalopa phase, lasting around 31–69 days ,49. Thero autumn . This coet al. [C. sapidus larvae from Rio Grande do Sul to Santa Catarina. Rodrigues et al. [C. sapidus larvae ecloded at the end of summer and autumn to estuaries located northwards, reinforced by southwestern winds and the Malvinas Current [Callinectes sapidus populations along the southern coast of Brasil.The dynamic behavior of the Lagoa dos Patos plume can also influence larval flow along South Brazil. Marques et al.  showed ts et al.  found ov Current . This coCallinectes sapidus populations are highly connected throughout this region. High gene flow and low population structure were evidenced and attributed to dispersal by coastal currents, which present different directions and intensities according to season and likely transport blue crab larvae between areas. Information on stock structure is fundamental for determining impacts of exploitation on genetically separate stocks, and consequently establishing effective fisheries management policies. Considering the elevated gene flow between the blue crab populations along southern Brazil, they can be considered a single genetic stock. However, further information on population size and dynamics, as well as fishery demands and impacts at different regions, are necessary for harvest management purposes.This work is the first to infer, through genetic markers, the population structure of blue crab populations along the Southern Brazil coast. The results obtained in this work clearly show that