Phaseolus species, which is cultivated on small-scale agriculture for the production of immature pods or to obtain dry seeds. However, despite the economic importance and agronomic potential of this species, the runner bean has been little studied from the genetic standpoint. Therefore, the main objective of this study was to characterize ten selected lines of runner bean obtained from Central (Santiago) and Southern  Chile based on morphological and agronomic traits. In addition, the genetic variability of these lines was determined using 12 Inter-Simple Sequence Repeat (ISSR) markers to evaluate the potential of this germplasm for breeding and commercial purposes. As a result, the lines from Central Chile were characterized, and had a higher number of pods per plant compared to the Southern lines, although the size and weight of their seeds were lower. Moreover, a low level of genetic diversity (He = 0.251) was encountered in this population. Finally, this is one of the first studies that generate relevant and novel information on the morphological, agronomic and genetic characterization of the P. coccineus germplasm present in Chile.The runner bean is the third most economically important  Phaseolus, belonging to the Fabaceae family, includes over 70 species, of which five were domesticated in Mesoamerica and have distinct geographical distributions, life histories and reproductive systems [P. coccineus L.) is the third most economically important Phaseolus species worldwide, after the common bean (P. vulgaris L.) and lima bean (P. lunatus L.) [P. coccineus var. albiflorus with white flowers, P. coccineus var. coccineus with red flowers and P. coccineus var. bicolor with both colors (white and red) of flowers [The genus  systems . The (scatus L.) . It is aatus L.) . In addiatus L.) . Based o flowers .Wild populations of runner bean are found from Northern Mexico to Panama, in cool and humid uplands, at altitudes from 1000 to 3000 m.a.s.l. ,6, due tSclerotinia sclerotiorum [Xanthomonas campestris [P. coccineus. However, despite the economic importance and agronomic potential of runner bean, the lack of molecular characterization in germplasm collections restricts its overall utilization, especially as an interspecific hybridization donor species in Phaseolus breeding programs [Runner bean is highly allogamous, and is a source of variability for several agronomic and disease resistance traits for the improvement of the common bean ,8. Some rotiorum  and Xantmpestris  have beeprograms ,15.P. coccineus presents a wide variability in morpho-agronomic traits, and low to moderate levels of genetic diversity in wild and cultivated populations, being little affected by the domestication process [P. coccineus [P. coccineus itself, and therefore there are few breeding programs focused on this species in the world, mainly in Europe [Determining the level of genetic diversity and relationships among genotypes are fundamental steps in any genetic improvement program. Some studies have confirmed that  process ,17,18,19occineus . Howevern Europe .In Chile, there are small home-made vegetable gardens with runner bean grown in the Southern area of the country . AccordiP. coccineus subsp. coccineus and the wild subspecies have hypogeal germination, while P. coccineus subsp. darwinianus has epigeal germination. In this study, six lines  showed hypogeal germination, while four lines  exhibited the epigeal type of germination, which is typical of P. vulgaris [P. coccineus have been described according to the color of the flowers [P. coccineus evaluated, six lines  had white flowers, most of them coming from INIA -La Platina, Santiago, Chile. Orange flowers were only observed in the line 19 . The lines from Pontificia Universidad Católica de Chile (PUC), Villarica, Chile (lines 27 and 28) displayed bicolored flowers. Line 3 presented purple/violet flowers, which were very similar to flowers of the P. lunatus species  [PvTFL1y gene generate plants with a determined growth habit, which could explain the phenomenon that occurred in line 3. The number and the distribution of branches depend on agronomic management practices, environmental conditions and genotype used [P. coccineus that refers specifically to this particular trait.One of the main traits selected during the domestication of legumes is the growth habit, in which plants with few branches, shorter internodes, fewer nodes, reduced twining and a determinate stem ending are desirable . In fact1 (TFL1) ,29. Mutaype used . 70% of Given that runner bean is usually grown for immature green pod production or to obtain dry seeds ,3, the cP. coccineus varieties, in which the var. albiflorus presented white seeds, the var. bicolor had beige seed and brown spots, and the var. coccineus exhibited purple seed and black spots. On the other hand, according to Sinkovic et al. [albiflorus, whereas those from Southern Chile  could correspond to the var. coccineus, given the red color of its flowers and purple seeds. The molecular mechanisms involved in the determination of seed color have been widely studied by bean breeders [P (Pigment) gene and ASPER (Asp) genes are involved in the genetic control of seed color and shininess [pp) generates individuals with flowers and seeds of white color [The appearance of the seed, in particular the color, is the most critical aspect that affects consumer preferences. The light cream-colored seeds with brown spots is the preferred testa color distribution . Six linc et al.  purple sbreeders ,33,34,35breeders . Among trhizobia, which allowed the identification of lines that are more prone to form nodules. Fifty percent of the lines had fibrous (fasciculated) roots , which could be classified as annual lines while the other 50% of the lines had tuberous roots . These lines can be multiplied vegetatively, and they have the potential to re-sprout the following year, being classified as perennial lines  presented nodules. It is possible that lines 3 and 4 were not inoculated with al lines . Outsiden cooked . Since Poot type .r2 obtained from the analysis of the principal components were selected  was higher than 60% for all the traits evaluated (except days to V2). The high determination makes these traits interesting for a runner bean breeding program. However, it is necessary to extend the study to a greater number of environments and repetitions of each line to obtain more precision on the genetic parameters evaluated.The multivariate analysis of ten morphological traits determined three principal components, which may explain 92.07% of the variability between the lines studied. Only the traits with values greater than 0.95 in the sum of the selected . The traP. coccineus evaluated, line 3  presents morphological differences compared to all the lines evaluated, which suggests a hybridization process and a possible heterosis effect that is independent of the genetic distance that exists between the parental lines [p > 0.05) higher than the rest of the lines evaluated, which varied between 1.7 (line 19) to 3.2 (line 1) seeds per pod. This result is in accordance with a previous study by Sinkovic et al. [Phaseolus [H2 = 0.67). Therefore, this trait is a good selection criterion for improving seed yields of Chilean runner bean lines.Even though there is a close genetic relationship between the Chilean lines of al lines . In thisc et al. , who indc et al. . Therefoc et al. ,41. Addihaseolus ,43, whiccoccineus is the one with the longest pods [With respect to the shape of the pod, line 3 had the longest pod 16.5 cm), which was significantly higher than the other lines (10.3 to 13.5 cm). The pods of lines 19 and 20 are significantly shorter with pod lengths of 10.7 and 10.3 cm, respectively . Our res cm, whicest pods ,44. Therp < 0.05). Lines 1, 3, 4, 6 and 7 had long beak pods whose values oscillated between 1.5 and 2.0 cm, while lines 8, 19, 20, 27 and 28 gave pods with short peaks, and their values varied between 0.7 and 0.52 cm , number of effective alleles (Ne), Shannon index (I), Nei’s gene diversity  were estimated to assess genetic diversity levels in the lines of Phaseolus spp. evaluated  was 0.367. In addition, similar levels of genetic diversity were identified between lines from Central and Southern Chile. In the group of P. vulgaris together with P. lunatus, 65.6% of the loci were polymorphic, and the h and I values were 0.214 and 0.327, respectively.The number of different alleles  and pervaluated . The anaP. coccineus [He = 0.50) and landraces (He = 0.54) accessions using SSR markers. In contrast, Guerra-García et al. [He = 0.118–0.208), and cultivated (He = 0.134–0.199) Mexican populations using SNP markers. Similarly, low and moderate levels of genetic diversity have been encountered in European accessions of P. coccineus  and Southern Chile (UACH and PUC-Villarrica).There is little information about the genetic diversity and structure of the wild and cultivated accessions of occineus . For exaoccineus  estimatea et al.  estimate67–0.43; ,18). TheK) was 4 . Although line 8 belongs to Central Chile (INIA-La Platina), it has agronomic traits similar to the lines from Southern Chile, indicating its possible genetic origin. The P. vulgaris varieties were all grouped in the same group, while the P. lunatus line was classified in a different group  was 4 A. The lint group B.P. coccineus germplasm present in Chile. Line 3  presents some morphological  traits different from the rest of the lines evaluated, suggesting a hybridization process and with a possible heterosis effect. Finally, it was determined that the evaluated P. coccineus lines show moderate morphological, agronomic and genetic variability, which allows this specie to be a candidate for genetic improvement programs due to its culinary properties, or to improve some traits in other Phaseolus species.In conclusion, although the findings of this study should be taken with caution due to the low number of lines and replicates evaluated, this is one of the first works which generate relevant and novel information on the morphological, agronomic and genetic characterization of the Phaseolus coccineus L.) from different research centers  were obtained and available for the development of this work (P. lunatus (LU), and four cultivars of P. vulgaris  collected from the Navidad County, located in the O’Higgins Region in Chile, were included.A total of ten lines of runner bean  in a ratio of 1:2. The experimental design was a randomized complete block design with six replications. The irrigation was applied according to stages of development and growth of each line. The fertilization was carried out at the time of transplanting with 200 kg N/ha, 100 kg of P2O5/ha and 60 kg of K2O/ha [Thrips spp.) and whiteflies (Bemisia spp.) was controlled in a traditional way with insecticide applications in the greenhouse. Lines 1, 3, 4, 6 and 7 were harvested on 5 January 2016, while lines 8, 19, 20, 27 and 28 were harvested on 20 April 2016.The runner bean lines were sown in a greenhouse belonging to the Faculty of Agronomy and Forest Engineering (PUC-Santiago) on 3 June 3 2015, and 60-day-old seedlings were transplanted into 25-liter pots using as substrate a mixture of perlite and peat , using the descriptors least affected by the environment. The phenological traits evaluated were days to emergence, germination type, two-leaf stage (V2), four-leaf stage (V4), flower bud, flower color, height of branches and type of growth; for traits related to pods, the length and width of the pod, number of pods per plant, length of pod peak, number of seeds per pod and color and shape of the pod were evaluated; for traits related to seeds, the weight of 10 random fresh seeds per plant, as well as color, width, length, shape and color of the hilum were evaluated; and regarding the roots, the color, type and presence or absence of nodules were evaluated. All traits were evaluated based on the six replicates per line.Morphological traits were analyzed according to “TG/9/5” guidelines of the International Union for the Protection of New Varieties of Plants . Total DNA was diluted to 70 ng/mL using ultra-pure water  for each sample, which were stored at −20 °C until further analysis. The integrity of the extracted DNA was checked by 2% agarose gel electrophoresis in TAE 1X buffer , stained with Gel Red™ and exposed to UV light to visualize genomic DNA.Genomic DNA was extracted from young leaves of one individual from each line. The individuals were selected according to those that met the highest number of morphological traits evaluated and were representative for each line. DNA was extracted using a modified CTAB (trimethyl ammonium cetyl (hexadecyl) bromide) method , and qua4, 0.75U Taq DNA polymerase, 0.2 mM dNTPs, and 0.47 µM of each primer. The PCR amplification was performed using a VeritiTM Thermal Cycler , and utilizing the following program: hold at 95 °C for 1 min, and then 35 cycles of PCR at 95 ℃ for 20 s, annealing (Tm) at an appropriate temperature for each marker for 30 s, and 68 °C for 5 min, followed by a final extension for 15 min at 72 ℃. Finally, the PCR products were analyzed by gel electrophoresis in 2% agarose , stained with Gel Red™ and exposed to UV light to visualize PCR products. The 100 bp molecular weight marker  was used to estimate the molecular weight of the DNA fragments  was performed in the InfoStat program [For the morphological and phenological analyses, the traits were grouped according to whether they were qualitative or quantitative. The Shapiro–Wilk  and Levene (homoscedasticity) tests were performed to evaluate the analysis of variance (ANOVA) assumptions. A principal component analysis (PCA) was performed using the Past3 program  to deter program  to evaluPhaseolus spp. lines evaluated was estimated through the number of different alleles (Na), number of effective alleles (Ne), Shannon index (I), Nei’s gene diversity (h) and percentage of polymorphic loci (P) using the POPGENE 32 program [i [For molecular analysis, the presence or absence of a given band was scored as 1 and 0, respectively (Supplementary File 1). The genetic distances among the individuals were calculated using GenAlEx 6.5 software , which w program . Nei’s g locus i .P. coccineus, P. vulgaris and P. lunatus) to infer the most probable number of genetic groups. The number of subpopulations (K) was set from 1 to 5 based on admixture, and correlated allele frequencies models, and ten runs per K were conducted separately. Each run was performed with 1,000,000 Monte Carlo Markov Chain (MCMC) replicates and a burn-in period of 10,000 iterations. A step of 100 iterations was used to avoid autocorrelation. The true K value was inferred using the method proposed by Evanno et al. [®.Nei’s genetic diversity is a simple measure of genetic variability, and is used when the samples are small . A prelio et al. , and it


Drosophila melanogaster gene fezzik. The derived variant at this site is at intermediate frequency in many worldwide populations but absent in populations from the ancestral species range in sub-Saharan Africa. We collected and genotyped wild-caught individuals from a single European population biannually over a period of 5 years, which revealed an overall difference in allele frequency between the sexes and a consistent change in allele frequency across seasons in females but not in males. Modeling based on the observed allele and genotype frequencies suggested that both sexually antagonistic and temporally fluctuating selection may help maintain variation at this site. The derived variant is predicted to be female-beneficial and mostly recessive; however, there was uncertainty surrounding our dominance estimates and long-term modeling projections suggest that it is more likely to be dominant. By examining gene expression phenotypes, we found that phenotypic dominance was variable and dependent upon developmental stage and genetic background, suggesting that dominance may be variable at this locus. We further determined that fezzik expression and genotype are associated with starvation resistance in a sex-dependent manner, suggesting a potential phenotypic target of selection. By characterizing the mechanisms of selection acting on this SNP, our results improve our understanding of how selection maintains genetic and phenotypic variation in natural populations.Understanding how genetic variation is maintained within species is a major goal of evolutionary genetics that can shed light on the preservation of biodiversity. Here, we examined the maintenance of a regulatory single-nucleotide polymorphism (SNP) of the X-linked  Genetic variation is an essential component of evolution and is shaped by neutral and selective forces that drive changes in allele frequency, leading to the maintenance, loss, or fixation of individual genetic variants over time. How variation is maintained over extended periods of time remains a topic of intense interest and ongoing study in evolutionary biology . ElucidaBalancing selection can maintain polymorphism in a population through several, nonmutually exclusive mechanisms. Perhaps the best-known form of balancing selection is overdominance, which is also known as heterozygote advantage because it refers to situations where the heterozygote has a higher fitness than either homozygote. It has been predicted that overdominant selection may be common when there is frequent adaptation, as large-effect adaptive variants may overshoot the fitness optimum in their homozygous state but meet the optimum in their heterozygous state . GeneticFor selection to act upon a genetic variant and alter its frequency, the variant must affect an organismal phenotype that, in turn, must affect fitness. Gene expression variation underlies much of the phenotypic variation that is observed within and among populations and species . DrivingDrosophila melanogaster gene fezzik (fiz) is located on the X chromosome and predicted to have oxidoreductase activity  than in sub-Saharan African populations  located 67 bases upstream of the start codon (referred to here as position 67) was found to have a major effect on fiz expression . Other cosmopolitan regulatory variants in the fiz enhancer appear to have been fixed by positive selection in a selective sweep that occurred before position 67 became polymorphic, as position 67 is variable within an otherwise fixed haplotype spanning the fiz enhancer  dependent upon the developmental stage and genetic background, although phenotypic dominance estimates were generally in line with parameter estimates from our model. Indeed, these results suggest that variable dominance plays an important role in shaping allele frequency dynamics and this may help maintain polymorphism at position 67. Furthermore, we identified a novel association between starvation resistance and fiz expression. This association was sex-dependent, with increased female starvation resistance associated with the high-expression, derived G variant, and increased male starvation resistance associated with reduced fiz expression, suggesting that genetic variants underlying starvation resistance may be sexually antagonistic.In this study, we examined allele and genotype frequencies over the course of 5 years in a derived, European fiz expression and was found to segregate among isofemale lines derived from cosmopolitan populations (Drosophila Population Genetics Consortium (DrosEU) ; thus, this imbalance is unlikely to be due to an unequal sex ratio immediately following reproduction. Instead, it may be a result of females’ increased attraction to food sources as potential oviposition sites resulting in more frequent trap visits in comparison to males. Alternatively, an increased rate of mortality in males before the time of sampling could also lead to their underrepresentation in our collections.The SNP at position 67 was previously identified experimentally as having a large effect on ulations . To bett(DrosEU) . Data fr(DrosEU)  online.  5 years  online. P = 0.046), because the sample sizes were consistently larger for females. In females, the G variant tended to increase in frequency between June and September and decrease between September and June . Thus, there was no evidence of the polymorphism being maintained by overdominant selection, which could result in an excess of heterozygotes.The frequency of the G variant was higher in females than in males for eight of the ten collections, with the largest difference being 19% (in September 2020) and the average difference being 6% . The difand June A and C. and June A and C, and June . There wand June A and C aand June . Thus, wand June . Note, hIn order to identify a plausible selection scenario that could explain the observed SNP frequency dynamics at position 67, we fit a model with viability selection to our observed data  assumingOur model predicts that, overall, selection was generally sexually antagonistic. Whenever the fitness of GG females was greater than 1 , the relative fitness of G males was below 1, and vice versa  online. Our model is based upon allele and genotype frequencies estimated from finite samples, which can be subject to sampling error. In order to determine how uncertainty in our allele and genotype frequency estimates translates into uncertainty in our parameter estimates, we computed confidence intervals for our parameter estimates in two ways: 1) We assumed that the observed frequencies are the true frequencies and sampled 1,000 new data sets assuming a binomial distribution for males and a multinomial distribution for females and estimated the parameters for each data set  online; To better understand how our full model compares with simpler models when uncertainty is taken into account, we calculated Akaike’s information criterion (AIC). Models with lower AIC are considered to be better. A general rule of thumb is that models with differences in AIC (ΔAIC) less than 2 are equivalent, models with ΔAICs between 4 and 7 are clearly distinguishable; and models with ΔAICs greater than 10 are distinctly different ; howeverIn order to determine whether the observed dynamics and parameter estimates could maintain a stable polymorphism, we used our parameter estimates to iterate the dynamics for a large number of seasons by repeating a season cycle consisting of seasons 1–8, where season 9 was left out to balance the number of “summer” and “winter” seasons  two strains from a population in the Netherlands (henceforth NL), and 2) two strains from a population in Munich, Germany (henceforth MU). Briefly, for each genetic background, a derived GG and an ancestral CC variant isofemale strain from the same population were crossed to each other in both directions and F1 offspring were then mated with each other within each cross of each background. We then measured each trait in F2 females, which consist of heterozygous and homozygous individuals that have been reconstituted in a mixed genetic background of the two parental strains. We measured fiz expression, larval volume, adult body weight, wing length, wing area, and wing load index and calculated the degree of dominance, h , suggesting that within a single trait, there are significant differences in the effect of the allele at position 67 dependent upon the genetic background and developmental stage. It should be noted, however, that this test cannot distinguish between allelic effects and dominance. In order to test for differences in dominance, within each developmental stage and genetic background, we calculated individual dominance values for each heterozygote by rescaling heterozygote expression based on the means of the homozygotes for the respective stage and genetic background and tested for an effect of developmental stage and genetic background on dominance. We detected a significant effect of genetic background and the interaction of developmental stage and genetic background on dominance , we first tested for an effect of the interaction between the allele at position 67 weighted by the estimated degree of dominance and developmental stage as well as the allele at position 67 weighted by the degree of dominance and genetic background on gene expression using an analysis of variance (ANOVA) utilizing all our gene expression data. We detected a significant effect of the interaction of both developmental stage and genetic background with genotype on gene expression , with the genetic background in which this occurred depending upon the trait under examination , suggesting that there are significant differences in the effect of the allele at position 67 dependent upon the genetic background and trait under examination. This test, however, cannot distinguish between allelic effects and dominance. In order to test for differences in dominance, within each developmental stage, trait, and genetic background, we calculated individual dominance values for each heterozygote as described above and tested for an effect of the examined trait and genetic background on dominance. We could not detect any significant differences in dominance , that is, the time until 50% of individuals are dead, male LT50 increased by 5% when fiz expression was knocked down (60.9 vs. 57.8 h in the control line), whereas female LT50 changed by less than 0.05% (84.2 vs. 83.9 h in the control line).The effect of th sexes , suggesth, and similar to expression and body size and proportion phenotypes . Sex, fiz genotype, and genetic background all had a significant effect on starvation resistance . Interestingly, we also detected a significant effect of the interaction between sex and fiz genotype (P = 1.24 × 10−12), suggesting that sex is an important modulator of the effect of fiz genotype and the associated fiz expression level on starvation resistance. Although it is not strictly defined as a life-history trait, starvation resistance in D. melanogaster constitutes an important component of fitness because of its contribution to survival  greater than 1 million   .Sexually antagonistic selection is thought to be of particular importance in the maintenance of polymorphism , 2014b, fiz expression and phenotype is not necessarily linear and 2) that the interaction between variation at position 67 and genetic background influences the effect of fiz expression on final organismal phenotype. In other words, the effect of fiz regulatory variants on phenotype is dependent on the other genetic variants present. Consistent with this, a previous study found evidence for trans-acting factors affecting fiz expression segregating in natural populations  from the binding site of any dosage compensation complex component (fruitless gene (FruM), which has three binding sites within the fiz genomic region, including one within the fiz enhancer  are targets of selection. Indeed, our study underscores both the difficulty and the importance of characterizing individual cases of selection in natural populations. The identification of the organismal trait under selection is particularly difficult for genes with pleiotropic effects, especially if the phenotypes themselves are highly polygenic. However, characterizing these individual cases can improve our understanding of how selection occurs in natural populations, how this selection affects organismal phenotypes, and how it can influence levels of standing genetic variation.Although adult starvation resistance is the trait we found to be most consistent with temporally fluctuating, sexually antagonistic selection at position 67 , it is pD. melanogaster strains and wild-caught flies were maintained at 21 °C with a 14 h light:10 h dark cycle on standard cornmeal–yeast–molasses medium unless otherwise stated.All D. melanogaster were sampled from a population in Munich, Germany  twice per year in late June and early September in 2016, 2017, 2018, 2019, and 2020, which corresponds to approximately the beginning and end of the breeding season in Munich. Sampling was performed at the same time each year and season, with 2.5 months (11–12 weeks) between the June and September collections and approximately 9.5 months between the September collection and June collection of the following year. Flies were collected using traps with apple-yeast bait and transferred to individual 35-ml vials containing standard medium. The species identity of all collected males was confirmed by visual inspection of the genitalia under a dissecting microscope in order to ensure that wild-caught flies were D. melanogaster rather than the closely related D. simulans. For each collection, a subset of the wild-caught females  was allowed to lay eggs and species identity was confirmed from male offspring as described above. We did not detect any D. simulans in any of our collections. Collected flies were frozen individually and stored at −80 °C until DNA extraction. For the September 2019 collection, we tested for a deviation from a 50:50 sex ratio by counting male and female offspring for a minimum of 50 eclosed offspring from each of 40 wild-caught females.Wild fiz expression were measured in the F2 offspring of two sets of reciprocal crosses between isofemale lines presenting either an ancestral C or a derived G at position 67. Reciprocal crosses of 30–40 females and 15–20 males were performed for each pair of isofemale lines, 40–50 F1 progeny were allowed to randomly mate, and phenotypes were measured in the F2 generation. Cross sets were performed using either two isofemale lines from Leiden, the Netherlands (NL01 and NL14) or two isofemale lines collected in June 2014 from the same Munich population as described above (MU06.14_17 and MU06.14_18). The Dutch lines were used in several previous studies about expression, phenotype, and/or sequence variation associated with the fiz enhancer  as well as a line containing an empty vector at the same genomic location (ID: 60100), which we used as a control, were obtained from the Vienna Drosophila Resource Center   . These llt males .fiz enhancer regions upstream of a LacZ reporter gene located at cytological band 86F on the third chromosome were generated and described in fiz enhancer as well as in the F1 hybrids of reciprocal crosses between these strains  the number of individuals is low enough that flies could be quickly and easily scored, 2) the density of flies in each vial was low enough for each individual to have an abundance of freedom of movement before and after the death of its conspecifics, and 3) adults are unable to utilize their dead conspecifics as a food source without the presence of larvae to break them down , and vial as factors for F2 flies, or with line and vial as factors for fiz knockdown and control flies. We further performed a meta-analysis of starvation resistance in all backgrounds  using a Cox proportional-hazards model with sex, strain/genetic background, fiz  genotype assuming additivity , vial, and the interaction between sex and fiz genotype as factors. Control fiz expression was most similar to basal fiz expression in natural populations for both sexes ; therefore, control flies were treated as such in our approximation of additivity. For each sex and genotype or strain, LT50 was calculated from the total number of flies dead versus alive at each timepoint using the glm and dose.p functions as implemented in R . The CMH test was applied to two-by-two tables of the G and C allele counts in the June and September collections within each sex across the 5 years, or in males and females across the ten collections. With the bootstrapping approach, we performed random binomial sampling of alleles within each sex and season. For this, we maintained the same sample size for each sex and season as in our observed data but randomly sampled alleles on the basis of their observed frequency in each year (for tests of seasons) or each collection (for tests of sex). For each test, we compared the observed cumulative difference in G frequency between June and September over all years  to those of 10,000 randomizations. The P-values. First, unlike the CMH test P-value, which is derived from a statistical distribution, the bootstrapping P-value is estimated by random resampling. Thus, the P-value will differ each time the test is run. For our data, we find that the run-to-run variation is typically around 1–2% when 10,000 replicates are performed. Second, the CMH test assumes that the allele frequencies in the population remain constant throughout the entire sampling period. This assumption could be violated if there is genetic drift and/or a consistent directional change in allele frequency. For example, if the frequency of the G allele increased monotonically from June 2016 through September 2020, then within each year the frequency would be higher in September than in June. Although this is not the case for our data  or among years (for the tests of seasons).There are two main reasons why the above tests may give slightly different our data A and theour data , it is dh, was calculated as:XGG, XCC, and XCG represent the average phenotypic value of the GG, CC, and CG genotypes, respectively  as well as with dominance categorized according to the types above, but without any assumptions of how each type of dominance affects the data using categorical variables. The results were concordant with results utilizing our estimates of h (For reconstituted F2 females in the MU and NL genetic backgrounds as well as homozygous and heterozygous reporter gene strains, we calculated the degree of dominance for all examined traits. Degree of dominance, ectively . We log tes of h  online. In order to better understand the selective forces acting on and the mechanisms maintaining variation at position 67, we fit a population genetic model to our wild-caught data. We initially considered two models, one with viability selection and another with fecundity selection see  online; Molecular Biology and Evolution online.msab215_Supplementary_DataClick here for additional data file.


Malaria remains a public health burden especially in Nigeria. To develop new malaria control and elimination strategies or refine existing ones, understanding parasite population diversity and transmission patterns is crucial.Plasmodium falciparum isolates from 633 dried blood spot samples in Nigeria was carried out using 12 microsatellite loci of P. falciparum. These microsatellite loci were amplified via semi-nested polymerase chain reaction (PCR) and fragments were analysed using population genetic tools.In this study, characterization of the parasite diversity and structure of Estimates of parasite genetic diversity, such as mean number of different alleles (13.52), effective alleles (7.13), allelic richness (11.15) and expected heterozygosity (0.804), were high. Overall linkage disequilibrium was weak . Parasite population structure was low .The high level of parasite genetic diversity and low population structuring in this study suggests that parasite populations circulating in Nigeria are homogenous. However, higher resolution methods, such as the 24 SNP barcode and whole genome sequencing, may capture more specific parasite genetic signatures circulating in the country. The results obtained can be used as a baseline for parasite genetic diversity and structure, aiding in the formulation of appropriate therapeutic and control strategies in Nigeria.The online version contains supplementary material available at 10.1186/s12936-021-03734-x. Plasmodium falciparum that contributes to increased transmission rate and spread of resistant parasites [Although the incidence of malaria infections and malaria-associated mortality has reduced in many African countries –3, transarasites . TherefoP. falciparum field isolates. Early molecular studies focused mostly on the use of polymorphic markers, such as merozoite surface protein 1 (msp-1), merozoite surface protein 2 (msp-2) and glutamate-rich protein (glurp) to characterize P. falciparum genetic diversity and structure in Nigeria [P. falciparum parasitaemia as re-infection or recrudescent infection [Molecular techniques play important roles in the analyses of genetic diversity, transmission dynamics, and population structure of  Nigeria –9. Thesenfection , 10, 11.nfection , 12–14, nfection –17. The nfection .msp-1, msp-2 and glurp due to their abundance, putative neutrality and higher levels of polymorphisms [P. falciparum parasite thereby resulting in an increase in the rate of recombination and subsequently, highly diverse population with low linkage disequilibrium [Microsatellite loci have been suggested to be better alternatives to orphisms . This moorphisms . In pastorphisms  .The objective of this study was to investigate the genetic diversity of circulating P. falciparum infections in children aged 6–96 months old. A cohort of children were enrolled from nine sentinel sites of the National Malaria Elimination Program of the Federal Ministry of Health , namely; Numan (n = 48), Bodinga (n = 50), Kura (n = 100), Barkin Ladi (n = 100), Ilorin (n = 58), Ibadan (n = 50), Otuasegha (n = 45), Agbani (n = 100) and Ogwa (n = 82) in Adamawa, Sokoto, Kano, Plateau, Kwara, Oyo, Bayelsa, Enugu and Imo States, respectively.This is a retrospective, cross-sectional, community-based study that is part of a larger drug therapeutic efficacy testing (DTET) study for monitoring anti-malarial efficacy of artesunate-amodiaquine (AA), artemether-lumefantrine (AL) and dihydroartemisinin-piperaquine (DHP) in the treatment of uncomplicated P. falciparum infections.Children aged 6–96 months old were eligible for enrollment in the efficacy study if they had symptoms compatible with uncomplicated malaria such as fever, anorexia, vomiting or abdominal discomfort with or without diarrhea with Filter papers containing dried blood spots (DBS) obtained from 633 children confirmed as malaria positive by microscopy were randomly selected for this study. All DBS samples were collected in 2014  and 2018 . Samples were collected for a duration of three months (July–September) which represents intense malaria transmission season in Nigeria. Two to three drops of finger-pricked blood samples were blotted on 3 mm Whatman filter paper  before treatment initiation (Day 0). The blood samples impregnated on to filter papers were allowed to air-dry properly at room temperature, and DBS were kept in airtight envelopes with silica gel at room temperature until analysed.DNA was extracted from DBS for parasite genetic diversity and population structure studies as previously described . DNeasy P. falciparum microsatellite loci was done using a previously described protocol [Semi-nested PCR amplification of 12 protocol . The 12 protocol . FAM, YAMicrosatellite data was retrieved from the Genetic Analyzer 3500XL and formatted in Microsoft excel version 16.44) as previously described . Subsequ.44 as prThe number of effective alleles (Ne) and number of different alleles (Na) were computed per locus for each State involved in the study using GENALEX 6.5 .Allelic richness (Ar) was computed using FSTAT (v 3.1) as the average number of alleles per locus .The expected heterozygosity (He), a measure of parasite genetic diversity, represents the probability of being infected by two parasites with different alleles at a given locus, was calculated using ARLEQUIN software version 3.11  with the a locus . The He Inter- and intra-population variance was determined with analysis of molecular variance . ΦPT value of zero (0) is considered indicative of no genetic differentiation among populations.The population divergence was measured by calculating the fixation index (Fst) for all pairs of parasite population in each State using the GENALEX 6.5 software. An Fst value between 0–0.05 was classified as little genetic differentiation, 0.05–0.15: moderate genetic differentiation, 0.15–0.25: great genetic differentiation and values greater than 0.25 represented very great genetic differentiation .A Bayesian model implemented in the program STRUCTURE v2.3  was usedSA) was calculated using the program LIAN version 3.5 [Multilocus linkage disequilibrium measured as the standardized index of association .Overall, 329 (51.97%) were male and the mean age of all children included in the study was 48.4 ± 15.8 months. Also, mean enrollment body temperature was 37.5 ± 2.5 °C. Overall geometric mean asexual parasitaemia was 16,219 μLOf the 633 samples considered for analysis, microsatellite amplification was successful in 571 (90.2%). Most (67.1%) samples were multi-clone infections. There were as many haplotypes as the isolates fully genotyped in the dataset . The mean  of different alleles (Na), effective alleles (Ne), allelic richness (Ar) and expected heterozygosity (He) were 13.52 (+ 0.59), 7.13 (+ 0.42), 11.15 (+ 4.34) and 0.804 (+ 0.013), respectively, when all States were considered as a single population. State-wise, the number of different alleles (Na)  ranged from 11.4 to 17.3. Likewise, the number of effective alleles (Ne)  ranged from 6.2 to 8.2 . In both the complete data set (Multi-clone and single-clone infection) and the sub-data set (single-clone infection), LD values obtained in parasite populations from Enugu, Kano, Sokoto and Plateau States were significant (P < 0.01) (Table Non-random associations among loci (multilocus LD) were measured for all complete haplotypes and also those from single infections by calculating the Index of Association (IA1) Table . It is o1) Table . The low1) Table .Table 2LThe AMOVA result (0.039) further confirmed the low parasite genetic differentiation as just 3.9% of genetic variation were observed between study sites. Furthermore, cluster analysis using STRUCTURE confirmed low population differentiation as only three putative parasite clusters; CL 1, CL 2 and CL 3 (ΔK = 14.73) were identified as admixtures in the nine States , which is weaker than those typically reported in regions with low transmission , 23. StuP. falciparum parasite thereby resulting in an increase in the rate of recombination and subsequently, high diverse population with low linkage disequilibrium [In summary, it has been observed that parasites from areas of low malaria transmission   values of microsatellite loci from parasite populations in the nine States.


Anser fabalis fabalis) have halved during recent decades. Since this subspecies is hunted throughout most of its range, the decline is of management concern. Knowledge of the genetic population structure and diversity is important for guiding management and conservation efforts. Genetically unique subpopulations might be hunted to extinction if not managed separately, and any inbreeding depression or lack of genetic diversity may affect the ability to adapt to changing environments and increase extinction risk. We used microsatellite and mitochondrial DNA markers to study the genetic population structure and diversity among taiga bean geese breeding within the Central flyway management unit using non‐invasively collected feathers. We found some genetic structuring with the maternally inherited mitochondrial DNA between four geographic regions (ɸST = 0.11–0.20) but none with the nuclear microsatellite markers . These results could be explained by female natal philopatry and male‐biased dispersal, which completely homogenizes the nuclear genome. Therefore, the population could be managed as a single unit. Genetic diversity was still at a moderate level (average HE = 0.69) and there were no signs of past population size reductions, although significantly positive inbreeding coefficients in all sampling sites (FIS = 0.05–0.10) and high relatedness values (r = 0.60–0.86) between some individuals could indicate inbreeding. In addition, there was evidence of either incomplete lineage sorting or introgression from the pink‐footed goose (Anser brachyrhynchus). The current population is not under threat by genetic impoverishment but monitoring in the future is desirable.The population numbers of taiga bean goose  in their breeding habitat using a citizen‐science approach. Over 2000 non‐invasively collected feathers were genotyped with microsatellite markers, and mitochondrial DNA sequences were analyzed in the 491 individual geese identified. We found very limited population structure, signs of inbreeding, and surprisingly, indications of hybridization with another goose species, the pink‐footed goose (Anser brachyrhynchus).We studied large‐scale genetic population structure of declining taiga bean geese ( The presence of highly divergent subpopulations with low levels of gene flow could warrant a status of separate management units , and with the most closely related species, the pink‐footed goose (Anser brachyrhynchus). For this, we used highly variable microsatellites first to identify individuals and parentage and then to study variation at the population level. In addition, we defined the genetic structure of maternal lineages by sequencing the most variable part of the mitochondrial control region. This region was also used to verify the subspecies  to evaluate the utility of a citizen‐science approach in collecting genetic samples for the elusive taiga bean goose, (2) to assess the broad‐scale genetic structure among breeding taiga bean geese in Finland , (3) to estimate genetic diversity within this population, (4) to evaluate the current effective population size and demographic fluctuations, and (5) to investigate if taiga bean geese hybridize with another subspecies of bean goose breeding in Europe, the tundra bean goose (22.1A. brachyrhynchus) has been controversial  with four subspecies  and 2016–2018 (n = 2127) or from taiga bean geese handled for ringing  in the years 2017–2018 (n = 20) , (2) migrating Russian taiga bean geese hunted from Finland , (3) Norwegian breeding tundra bean geese , and (4) Iceland breeding pink‐footed geese   during the years 2006–2014  Figure . For gee) Figure , and the n = 7),  NorwegiaGrus grus) were omitted from DNA extraction. The extracted DNA was stored in −20°C.DNA from a calamus and from a blood clot  to screen for quality and to reduce the number of duplicate individuals, as follows. We performed individual identification by visual inspection of the genotypes and by using the “Regroup genotypes” option in the program Gimlet v.1.3.3. Valière, . If one PID) and probability of identity of siblings (PID SIB)  forward primer ASW12‐D3 , number of private alleles (PA), observed heterozygosity (HO), expected heterozygosity (HE), and inbreeding coefficient (FIS) were calculated using the program GenAlEx 6.503  was calculated using the program FSTAT 2.9.4  were placed in the candidate offspring category, females (n = 237) and geese of unknown sex (n = 62) (based on molecular sexing) were placed in the candidate mother category , and males (n = 189) and geese with unknown sex (n = 62) (based on molecular sexing) were placed in the candidate father category . We excluded each individual from being the mother or father of itself. We used the calculated ADO and FA rates (Table r) between goose dyads using the program ML‐relate , ɸST, and Ne (effective population size) that were calculated with the full dataset because the precision of these genetic estimates may suffer with the purging of all siblings  set from 1 to 10. The ad hoc approach of Evanno et al.  and, accordingly, we randomly chose an equal number of samples in each geographical region  as the DAPC could be biased due to unequal samples sizes. Cross‐validation  was used to evaluate the number of PCs to retain. DAPC was also used to compare the outgroup samples and the Finnish population—by performing DAPC first on the outgroups and then importing the “unknown” Finnish population into this same framework using the “predict” function. Pairwise FST‐values were calculated using the ENA correction implemented in the program FreeNA due to the possible presence of null alleles in the data. To test for isolation by distance, we performed a Mantel test implemented in the R package  was estimated with a linkage disequilibrium model , current population size (N), and time of demographic change in generations (T) were resolved from scaled parameters produced by Migraine  by assuming a microsatellite mutation rate of µ = 5 × 10−4 per locus per generation , with Nratio > 1 indicating a population growth and Nratio < 1 indicating a population bottleneck. The statistical significance was evaluated using 95% confidence intervals. If the 95% confidence intervals do not span 1, the results are statistically significant  and 100 individuals in different hybrid classes  using the program HybridLab1.0 . In this second analysis, the aim was to search for admixture with other subspecies or species in the Finnish population.We studied hybridization between the taiga bean goose and either the pink‐footed goose (taiga bean goose x pink‐footed goose) or the tundra bean goose (taiga bean goose x tundra bean goose) by using a simulation study. First, we simulated 100 pure parental individuals . The primers were designed to contain mismatches to Numts  was amplified using primers AdCR1F and AdCR2R  as an outgroup , haplotype (h) and nucleotide (π) diversities, Tajima's D (D), and Fu's Fs (Fs) were calculated using program DnaSP v. 6.12  and aligned with GenBank sequences of bean geese  with a success rate of 84%. After omitting possible redundancy, this amounted to a total of 491 individuals in our dataset. One individual that may have been on spring migration and had pink‐footed goose mtDNA . The sex could not be determined for 62 individuals due to lack of amplification of this marker.Molecular sexing using the HINTZ/W gametologs showed congruent results with the cloacal examination except for one goose out of seven. Based on the cloaca, this goose was assigned as a female while based on molecular sexing it was a male. This sample was replicated and the same result was obtained by molecular sexing. We identified 237 females and 189 males in the Finnish population which was significantly different from equality . We identified 23 candidate fathers and 28 candidate mothers with probabilities over 0.90.Most of the geese dyads studied were unrelated , 38,049 dyads had relatedness values between 0.05 and 0.39 and 131 geese dyads had relatedness values between 0.40 and 0.59 indicating potentially parent–offspring or sibling relationships according to the ML‐relate program. Relatedness values of approximately 0.50 were found for all known parent–offspring and sibling dyads of ringed geese (two sibling dyads and one parent offspring dyad), except for one gosling that could not have been offspring of the parents it was captured with , producing a final dataset of 376 non‐kin individuals.We identified two male geese harboring mtDNA of the pink‐footed goose to have sired offspring with females having taiga bean goose mtDNA. Fifty‐two pairs of full siblings were identified with probabilities of 1.00, these also shared mtDNA haplotypes. Seventy‐two geese dyads showed relatedness values between 0.60 and 0.86, suggesting potential inbreeding. Based on these results, we created a subset of the data that did not contain parent–offspring or sibling relationships or highly related individuals , followed by Lapland  with all regions showing higher expected than observed heterozygosities (Table FIS) ranged between 0.05 and 0.10  of the full dataset including kin was estimated to be 1127.6 individuals  using the linkage disequilibrium method. The Ne estimate based on sibship using the full likelihood method was 1134 individuals (95% CI: 927–1327). No signs of recent population bottlenecks were detected, instead, the Migraine analysis indicated population growth as the Nratio was 7.8 (CI: 1.746–21.24) and the 95% confidence did not overlap with 1. The Migraine analysis indicated a population growth starting from 21,766 to 32,648 years ago  from 601 individuals  to the current census population size of 4701 individuals  . This analysis showed that the simulated pure taiga bean geese and pink‐footed geese could be separated and that some Finnish taiga bean geese showed admixture with pink‐footed geese  between the taiga bean and the pink‐footed goose and between the taiga bean and the tundra bean goose is difficult using microsatellite markers due to the extent of variation within each hybrid category Figures –S4. Howee Figure . No puree Figure . The twoe Figure , arrows.3.7A. f. fabalis mtDNA haplotypes , followed by the haplotype FAB1a/FAB1b/Fa2 (n = 159). The slashes between haplotype names denote identical haplotypes based on the sequenced region. These haplotypes, however, differ based on the whole control region , Fa1 (n = 3), Fa4 (n = 4), and Fa7 . The rarer haplotypes were more localized with haplotype Fa1 only found in Northern Ostrobothnia, Fa4 only in Southern Lapland, Fa7 in only males in two areas in Northern Ostrobothnia and Lapland, and FAB3 only in females from Ostrobothnia . The pairwise ɸST‐values were higher for females  than in males   according to AMOVA.The number of haplotypes was highest in the Northern Ostrobothnia/Southern Lapland region and lowest in the Western and the Eastern Finland/Kainuu regions Table . Haplotyns Table . Pairwisns Table . The pai 0 Table . Thus, t3) Table . In addi4ɸST‐values for females than in males, however, suggested at least some maternal genetic structure. It was unforeseen to find such little genetic structure within such a large geographic area, but the pairing system of geese, in which pair formation occurs already in common wintering or spring staging areas, can explain these results. We found moderate genetic diversity and signs of inbreeding within the Finnish taiga goose population.We did not detect clear population structure within the Finnish breeding taiga bean geese using microsatellite markers. All analyses suggested close to a panmictic population, except the DAPC that indicated slight structuring among Western Finland, Northern Ostrobothnia/Southern Lapland, and Lapland  in the taiga bean goose. This could indicate hybridization between the taiga bean goose and the pink‐footed goose and admixture was also evident in the microsatellite data. In addition, we confirmed breeding of tundra bean geese in the northernmost Lapland and the presence of a vagrant tundra bean goose in Southern Finland. The microsatellite data suggested introgression between the taiga and the tundra bean goose as well.4.1Grus grus) and the whooper swan (Cygnus cygnus), only a few feathers had to be excluded as belonging to other species. This was determined either based on feather morphology or because no PCR product amplified from pristine feathers not identifiable based on morphology. Benefits of citizen‐science approach for this study included the large number of samples and the broad geographic range of sampling.This study proved that a citizen‐science approach to feather collection was highly efficient for the elusive bean goose. About 89% of the samples were collected by a citizen‐science approach, while the rest were collected by persons with an affiliation to a research institute. Even though bean goose feathers, especially the down feathers, can be mixed with other large birds in similar habitats, such as the common crane  were detected and a few of the goose dyads showed high relatedness values (r = 0.60–0.86), indicating potential inbreeding. This was surprising as the detrimental effects of inbreeding to individual fitness, known as the inbreeding depression, are widely documented  compared to other bean goose subspecies , showing no clear nuclear differentiation between the geographic regions  between the Northern Ostrobothnia/Southern Lapland region and all other regions , most males breed close to their natal site, but a minority of the geese undergo long‐distance dispersal  and the Tule goose   studied with single nucleotide polymorphism (SNP) markers showed not only genetic structuring but also genetic exchange between all flyways  and the estimate of the number of breeding individuals is 0.23–0.33. Accordingly, Ne is just about a quarter or a third of the estimated breeding population. Low Ne/N ratios (0.11–0.14) are commonly reported among animals  would be thus 8057–10,255 individuals, a surprisingly large estimate. However, our estimate of Ne from only Finnish samples is perhaps somewhat misleading due to a continuous population unrestricted by national borders.Both the linkage disequilibrium and the sibship method produced similar estimates of the effective population size with 1128 and 1134 individuals, respectively. The Finnish population size is estimated to be 1700–2500 breeding pairs  based on survey data , the tundra bean goose and the pink‐footed goose, using DAPC. As expected, the Finnish geese mostly grouped with the Russian geese Figure , which sIn this study, we showed that the pink‐footed goose mtDNA is widespread  in the Finnish taiga bean goose population Figure . This wa4.5We did not find evidence to divide the Finnish bean goose population into smaller management units or subpopulations as there was no strong genetic structuring within Finland. Therefore, the flyway management units outlined in the International Single Species Action Plan ; Formal analysis (lead); Investigation (lead); Methodology (lead); Visualization (lead); Writing – original draft (lead); Writing – review & editing . Serena Baini: Investigation ; Writing – review & editing . Jeremy B. Searle: Supervision ; Writing – review & editing . Laura Kvist: Conceptualization ; Supervision ; Writing – review & editing . Jouni Aspi: Conceptualization ; Supervision (lead); Writing – review & editing .Supplementary MaterialClick here for additional data file.


Genetic correlations between traits may cause correlated responses to selection. Previous models described the conditions under which genetic correlations are expected to be maintained. Selection, mutation, and migration are all proposed to affect genetic correlations, regardless of whether the underlying genetic architecture consists of pleiotropic or tightly linked loci affecting the traits. Here, we investigate the conditions under which pleiotropy and linkage have different effects on the genetic correlations between traits by explicitly modeling multiple genetic architectures to look at the effects of selection strength, degree of correlational selection, mutation rate, mutational variance, recombination rate, and migration rate. We show that at mutation-selection(-migration) balance, mutation rates differentially affect the equilibrium levels of genetic correlation when architectures are composed of pairs of physically linked loci compared to architectures of pleiotropic loci. Even when there is perfect linkage (no recombination within pairs of linked loci), a lower genetic correlation is maintained than with pleiotropy, with a lower mutation rate leading to a larger decrease. These results imply that the detection of causal loci in multitrait association studies will be affected by the type of underlying architectures, whereby pleiotropic variants are more likely to be underlying multiple detected associations. We also confirm that tighter linkage between nonpleiotropic causal loci maintains higher genetic correlations at the traits and leads to a greater proportion of false positives in association analyses. Under nm effect . LD may The evolutionary dynamics of pleiotropic versus linked loci in creating genetic correlations are expected to be similar under the assumptions of complete linkage (no recombination) between pairs of loci each affecting a different trait, weak stabilizing selection, weak mutational effects, and large mutation rates . Correlaμ is the haploid per-locus mutation rate, and Under assumptions of weak mutational effects and relatively high mutation rates  appear to be affected by loci that affect other traits, and especially when targeted gene therapy clinical trials are more widespread than ever  to identify causal genetic variants underlying one or more traits. GWAS use the rapid increase in genomic sequencing to find correlations between traits and genotypes, and their success is dependent on the effect sizes of the loci and the distinction between phenotypes. GWAS have had success in associating genetic variants with traits of interest, which have allowed researchers to find the molecular underpinnings of trait change , and mutation regime (Gaussian and house-of-cards). We focus on the relative contributions of selection, mutation, recombination, and migration to the build-up of genetic correlation between traits having different genetic architectures. We show that unless mutation rates are high as in the Gaussian regime, genetic architectures with tight linkage between loci maintain lower equilibrium genetic correlations than pleiotropic architectures. Even when mutation rates are high, other evolutionary forces affecting equilibrium levels of genetic correlation still show a difference between architectures but to a much lesser extent. In addition, we simulate genomic SNP data sets using the different architectures and show that map distances between causative and noncausative quantitative trait locus (QTL) affect false-positive proportions in GWA analyses.a, at a locus are drawn from a Gaussian distribution μ. To better express the difference between pleiotropic and nonpleiotropic loci, we introduce a second parameter θ such that the column vector of mutation effects on traits 1 and 2 can be expressed as:θ represents the direction of the mutational vector in the two-trait plane, and T denotes transpose. The angle θ is uniformly distributed between 0 and I is the two-dimensional identity matrix. In contrast, the mutational input for two nonpleiotropic loci is the weighted sum of two normal distributions along trait 1 or trait 2 with probability x1 affecting trait 1 and x2 trait 2, can be written as:We seek to express the variance–covariance matrix of two quantitative traits as a function of the mutational input at two diploid nonpleiotropic linked loci, which is considered to be equivalent to one pleiotropic locus in Because of the negligible contribution of double mutations for Stabilizing selection on the two traits is represented by a bivariate Gaussian surface with variance The G-matrix (genetic variance–covariance matrix) at mutation–selection balance for a single haploid pair of fully linked loci can be found using the house-of-cards approximation for an infinite population sitting at the trait optimum under stabilizing selection :(8)G=2 a as in (4) and With θ, it is clear that mutations at nonpleiotropic loci cannot generate genetic covariance between the traits because the matrix part of (9) becomes From (9) and the distribution of Nemo , θ is the vector of local optimal trait values , and Unless otherwise specified, each simulation was run with 5000 initially mono-morphic, diploid individuals  for 50,000 generations achieving mutation–selection–drift(-migration) balance in order to observe general patterns of genetic correlation. Individuals were hermaphrodites mating at random within a population, with nonoverlapping generations. Phenotypes were calculated for each of the two traits modeled by summing the allelic values of all loci affecting each trait. Gaussian stabilizing selection was applied and determined the survival probability of juveniles, whose fitness was calculated as e matrix . We expln traits .μ for each haploid locus. Pleiotropic mutation effects are drawn from a bivariate normal distribution with mean and covariance equal to zero, and per-trait variance of To examine the effects of mutational input on genetic correlation between traits, we contrasted two types of loci: loci with small effects and large mutation rates against loci of large but rare effects. These two contrasted architectures correspond to the assumptions of the Gaussian and house-of-cards regime, respectively see . In both μ≫α2/ω2 , 1985. T of 0.1, 0.01, and 0.001. The backward migration rate represents the average proportion of new individuals in the focal population whose parent is from the source population. The local optimum values for the two traits in the source population were set at To examine the effects of migration from a source population on genetic correlation between traits, additional sets of simulations were run with unidirectional migration from a second population  with backward migration rates    on a set of additional simulations of neutral genetic loci linked to the causative QTL. The neutral loci were diallelic with a per-allele mutation rate of v0.98.1) . Results) , can be mapped to trait variation similarly to marker SNPs. Significance was determined with custom-made R-scripts performing linear regressions for each segregating SNP on each trait separately, and adjusting By generation 50,000, when mutation–selection balance equilibrium is reached, the simulations with pleiotropic loci under the house-of-cards regime maintain a higher average genetic correlation than pleiotropic loci under the Gaussian regime, and higher than simulations with linked nonpleiotropic loci under either regime . The sim 0.0001) . The mut 0.0001)  is reachFor the two sets of mutational parameters, under Gaussian and HoC assumptions, the increase in recombination (increased map distance in cM) within the pairs of linked nonpleiotropic loci rapidly decreases the equilibrium genetic correlation between the traits, as expected . StrongeC regime . A simil(ρω=0.5) . At low ll cases . In cont−5, HoC) .A higher migration rate from a source population, whose traits are under correlational selection, leads to higher genetic correlations in the focal population regardless of the genetic architecture . The effe.g., when correlational selection is decreased from 0.9 to 0.06, see t-test: P = 0.023 for P = 0.0085 for In the HoC regime, when mapping neutral SNPs to trait variation, the pleiotropic architecture yields more significantly associated SNPs than the nonpleiotropic architecture, approximately 95 and 70, respectively, out of about 18,000 segregating SNPs . The sigvs 0.03, respectively, see Compared to the HoC regime, the multivariate GWAA on neutral SNPs in the Gaussian regime had less power to detect significant associations, as shown by the lower number of significant SNPs, their LDs with QTLs, and the QTL DR (<1%) . MoreovecorG in Univariate GWAAs on neutral SNPs are less prone than multivariate GWAA to detect marker SNPs associated with trait variation  of 6.5% and 7.4% in the Gaussian and HoC regimes, respectively  should maintain similar equilibrium levels of genetic correlation as a genetic architecture consisting of a lesser number of unlinked pleiotropic loci . Furtherper-nucleotide mutation rates are in the order of t-haplotype mice , small deviations caused by drift may have a large effect on the orientation of the G-matrix at equilibrium, and thus on the resulting genetic correlation among the traits. As shown in Our main result of a dependency of the equilibrium correlation on the mutation regime was not anticipated because the same mutation-invariant expectation of equilibrium genetic correlation  was founi.e., the genomic mutation rate). Therefore, we can generally expect that the total genetic covariance between traits affected by different nonpleiotropic loci increases with The equilibrium genetic correlation maintained at nonpleiotropic loci strongly depends on the mutation rate, the number of loci, and the linkage between them. As argued in the Model section, the distribution of mutational effects on the two traits affected by a pair of nonpleiotropic loci is far from a bivariate normal distribution. This represents a stark departure from ulations .between pairs of nonpleiotropic linked loci, favored mutations need not affect two loci within the same pair to allow correlational selection to keep them in LD and maintain higher genetic correlation at the traits. LD between pairs can thus do the job. This suggests that high genetic correlations can be maintained under strong correlational selection when many causative loci are clustered within small recombination distances throughout the genome. Our simulations suggest a distance smaller than about 0.01 cM (about 10 kb or less is needed) for loci under the house-of-cards regime and weak selection  on multiple traits of interest, they are dependent upon detecting combinations of effect sizes of genetic variants associated with those traits . The suci.e., within gamete allele effect covariance) when compared to pleiotropic loci  despite large differences in genetic correlations . In a previous version of this manuscript  of a protein-coding gene and the degree to which the gene maintains multifunctionality may itself evolve  if allele combinations are not favored by selection or increase it if they are . Without high mutation rates, the ability to create genetic covariance between linked loci is low because there is a low probability of two simultaneous mutations with effects in the same direction. This low probability can be compensated by an increased number of linked loci in clustered architectures. Our results will have implications in the type of underlying architecture we expect to find in multitrait association studies. On the one hand, tighter linkage between causal loci maintains higher genetic correlations, leading to a greater proportion of false positives in pleiotropy tests. On the other hand, pleiotropic loci are overall more likely to be detected, especially when they affect tightly correlated and functionally distinct traits, increasing true DRs. In most cases though, it will remain challenging to distinguish between pleiotropy and linkage, both functionally and statistically. Our analyses suggest that a mix of multi- and univariate GWAA approaches can help weed out spurious pleiotropy.https://zenodo.org/record/5469293 and code for simulations can be found at https://sourceforge.net/projects/nemo2/files/Publications-Code/ChebibGuillaume-PleiotropyOrLinkage-Genetics-2021/The data for this study will be made available online through Zenodo online repository at GENETICS online.iyab159_Supplementary_Figures_TablesClick here for additional data file.iyab159_Supplementary_DataClick here for additional data file.


Amphitetranychus viennensis (Zacher).Evolution of reproductive isolation is an important process, generating biodiversity and driving speciation. To better understand this process, it is necessary to investigate factors underlying reproductive isolation through various approaches but also in various taxa. Previous studies, mainly focusing on diploid animals, supported the prevalent view that reproductive barriers evolve gradually as a by-product of genetic changes accumulated by natural selection by showing a positive relationship between the degree of reproductive isolation and genetic distance. Haplodiploid animals are expected to generate additional insight into speciation, but few studies investigated the prevalent view in haplodiploid animals. In this study, we investigate whether the relationship also holds in a haplodiploid spider mite, We sampled seven populations of the mite in the Palaearctic region, measured their genetic distance (mtDNA) and carried out cross experiments with all combinations. We analyzed how lack of fertilization rate (as measure of prezygotic isolation) as well as hybrid inviability and hybrid sterility (as measures of postzygotic isolation) varies with genetic distance. We found that the degree of reproductive isolation varies among cross combinations, and that all three measures of reproductive isolation have a positive relationship with genetic distance. Based on the mtDNA marker, lack of fertilization rate, hybrid female inviability and hybrid female sterility were estimated to be nearly complete (99.0–99.9% barrier) at genetic distances of 0.475–0.657, 0.150–0.209 and 0.145–0.210, respectively. Besides, we found asymmetries in reproductive isolation.The prevalent view on the evolution of reproductive barriers is supported in the haplodiploid spider mite we studied here. According to the estimated minimum genetic distance for total reproductive isolation in parent population crosses in this study and previous work, a genetic distance of 0.15–0.21 in mtDNA (COI) appears required for speciation in spider mites. Variations and asymmetries in the degree of reproductive isolation highlight the importance of reinforcement of prezygotic reproductive isolation through incompatibility and the importance of cytonuclear interactions for reproductive isolation in haplodiploid spider mites.The online version contains supplementary material available at 10.1186/s12862-021-01896-5. Reproductive isolation facilitates divergence of closely related groups by restricting gene flow. Therefore, evolution of reproductive isolation is an important process for understanding speciation , 2. MechDrosophila , because mortality among unfertilized, male offspring was negligibly low , therefore, the interaction term was removed from the model. Genetic distance significantly affected [1—(#daughter/#eggs)] ]. [1 − (#daughter/#eggs)] in parent-population crosses varied among combinations of crosses Fig. c. The inF1,37 = 127.381, P < 0.001). As the genetic distance increased, so fraction of dead offspring in the backcrosses increased /#eggs]  [Total reproductive isolation in parent population crosses  was nearly complete at a genetic distance of 0.145–0.210 (99.0–99.9% reproductive isolation) based on an mtDNA genetic marker (COI). This is similar to that in the haplodiploid spider mite, 2–0.210) –43. The A. viennensis , and also than prezygotic isolation in the S. miscanthi species group (genetic distance for 99.0–99.9% isolation: 0.190–0.258) [A. viennensis populations used in this study is completely allopatric whereas populations of S. miscanthi species group used in [A. viennensis was collected from the Palaearctic region ranging from France to Japan, whereas S. miscanthi species group was collected from Japan and surrounding countries [A. viennensis populations experience more isolation-by-distance, reinforcement of prezygotic reproductive isolation would possibly not occur in A. viennensis, resulting in the pattern of slow evolution of prezygotic reproductive isolation. However, the isolation-by-distance might also have caused higher genetic diversity among populations in A. viennensis, which might have promoted genetic incompatibility, and therefore a stronger postzygotic reproductive barrier.Prezygotic reproductive isolation seemed to evolve much slower than hybrid female inviability in A. viennensis. In the crosses with females from Inner Mongolia (CIM), Eastern China (CN), and Korea (K), prezygotic reproductive barrier was found and stronger as the genetic distance was larger. However, in the crosses with females from France (F), Turkey (T), Iran (I) and Japan (J), the relationship between prezygotic barrier and genetic distance was not clear, but viability of F1 hybrids was lower as genetic distance was larger. The presence of these two patterns of reproductive isolation in A. viennensis can be useful to investigate whether prezygotic reproductive isolation in spider mites is often associated with reinforcement or whether other mechanisms affect prezygotic reproductive isolation. It can be thought that Inner Mongolia (CIM), Eastern China (CN) and Korea (K) are in geographical locations where contact with other genetically different groups frequently occur. Otherwise, as we mentioned previously, A. viennensis is thought to originate from Eastern Asia [The reproductive isolation pattern including the strength of prezygotic reproductive barrier showed two patterns among female populations in ern Asia , althougern Asia , 44–47. A. viennensis. Yet, we often found asymmetries in reproductive isolation in this study. For example, hybrid females were partly produced from Iran  × France , but not at all from France  × Iran  . We estimated the genetic distances which complete the reproductive barrier, and discussed about the importance of reinforcement of prezygotic reproductive barrier and the importance of cytonuclear interaction in haplodiploid animals. The findings are significant for understanding evolutionary patterns of reproductive isolation in haplodiploid animals.In this study, we focus on a haplodiploid spider mite and investigated the evolutionary mechanism of reproductive isolation among populations within the species Prunus × yedoensis Matsum., which were placed on a water-soaked sponge in Petri dishes (9 cm in diameter) under constant climatic conditions . We placed the leaves underside up and the perimeter was covered with water-soaked tissue paper. In winter, these populations were kept as diapause females, which were reared from eggs under 15 ± 1 °C and 8:16 light:dark photoperiod. Diapause females were put onto black paper and set into glass vials, which were kept in a refrigerator (ca. 5 °C) for five months from December to April under darkness. We collected Tetranychus kanzawai Kishida from Japan  Cogn., date: January 31, 2008), and used it as the outgroup of phylogenetic analysis of A. viennensis.A list of populations used in the present study is provided in Table c oxidase subunit I gene (COI) of mitochondrial DNA (mtDNA). DNA was extracted from a single female mite from each population by using PrepMan Ultra Sample Preparation Reagent (Thermo Fisher Scientific Inc.). To amplify the fragment of mtCOI region, PCR was carried out using primers given in Additional file 2 + plus, Takara Bio Inc.), 0.14 µl of TaKaRa Ex Taq , 2.88 µl of dNTP mix , 0.72 µl of each primer (10 pmol/ul each) and 27.44 µl of ddH2O. PCR cycling conditions were 3 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 51 °C and 1.5 min at 72 °C, and a final extension at 72 °C for 10 min. In some samples, the fragment was not amplified. Therefore, in these samples, we carried out PCR by decreasing the annealing temperature or increasing the number of cycles. PCR products were purified using MinElute PCR Purification Kit (QIAGEN). The purified products were sequenced using ABI BigDye Terminator ver. 3 Cycle Sequencing Kit (Applied Biosystems) and ABI3130xlGenetic Analyzer (Applied Biosystems).In measures of genetic distance among populations and the phylogenetic analyses, we used the cytochrome A. viennensis and T. kanzawai and the COI sequences for A. quercivorus from previously published data (accession numbers: AB981238 and LC435686) [Obtained sequences of the COI  for C435686)  were aliC435686) . A maximC435686) . As the Kimura 2-parameter genetic distances  were calWolbachia, Cardinium, Spiroplasma and Rickettsia in the mites, we carried out PCR assay for these endosymbionts using primers given in Additional file 2 Solution, 0.2 µl of BIOTAQ DNA Polymerase , 0.4 µl of dNTP mix (10 mM each), 1 µl of each primer (10 pmol/µl each) and 13.4 µl of ddH2O. PCR cycling conditions were 3 min at 95 °C, followed by 36 cycles of 30 s at 95 °C, 30 s at 52 °C and 30 s at 72 °C, and a final extension at 72 °C for 5 min. To be sure, we carried out this check for endosymbiont infections twice by using the same DNA template from each population . We used DNA of Wolbachia-infected Panonychus mori Yokoyama , Cardinium-infected Tetranychus urticae (Koch) , Spiroplasma-infected Tetranychus truncatus Ehara  and Rickettsia-infected Nephotettrix cincticeps (Uhler) [Wolbachia, Cardinium, Spiroplasma and Rickettsia infection, respectively. Distilled water was used as negative control.To detect the presence of  (Uhler)  as positA. viennensis in all combinations and both directions (42 combinations). As controls, intra-population crosses were carried out in each population (7 controls). The number of replicates in each cross combination ranged between 12 and 30  in the mite rearing. To make the area unified, a square (4 × 4 cm) was created by using strings of water-soaked tissue paper. A female in the last molt before adulthood  and an adult male were collected from the mite culture, and placed on the leaf arena. The female and male were allowed to mate and oviposit for five days, then they were removed from the leaf arena. The number of eggs, the offspring survival and the gender of offspring were checked and recorded.stats and MASS [Analyses were carried out with the statistical package R version 3.6.2 . We analand MASS , 62.Additional file 1: Table S1. Number of eggs laid during the first five days of the oviposition period, hatchability, survival rate of immature stages, female ratio of offspring and female offspring per ovipositing female in crosses.Additional file 2: Table S2. Quasibinomial generalized linear models used in the lines in each female population in Fig. Additional file 3: Table S3. Primers used in genetic analyses of Amphitetranychus viennensis and in PCR assay for endosymbiont infection.


Lobatus gigas from Pedro Bank yielded low but significant values (FST = 0.009: p = 0.006) and suggested a high magnitude of gene flow indicative of a fit and viable population throughout the bank. Analysis of molecular variance (AMOVA) indicated a 100% variation within individual samples with little variation (0.9%) between populations. In contrast pairwise genetic comparisons identified significant differences between populations located to the south eastern and eastern region of the bank to those in the central and western locations. Bayesian clustering analysis also indicated the likelihood of two population sub-divisions (K = 2) on Pedro Bank. The results provided evidence of a weak but significant population structure which has crucial implications for the fishing industry as it suggests the use of ecosystem based management (EBM) in setting quotas to promote sustainable harvesting of L. gigas within each monitoring zone on Pedro Bank.The queen conch fishery in Jamaica is sustained by Pedro Bank, which is the main harvesting site located approximately 80 km south-west from Kingston. Due to its relative size, Pedro Bank has been subdivided into zones for management purposes by the Fisheries Division and the Veterinary Services Division. Understanding whether these sub-divisions reflect different sub-populations is critical for managing exploitation levels because fisheries management must demonstrate that harvesting does not endanger the future viability of the population as queen conch are on Appendix II of the Convention in Trade in Endangered Species of Wild Fauna and Flora (CITES). This determination is essential for the continued export to international markets such as the European Union. Two hundred and eight samples were collected across the entire Pedro Bank and were genetically characterized using nine polymorphic microsatellite loci. Population structure analysis for  Lobatus gigas, (Linnaeus 1758), is an edible marine gastropod mollusk of the order Mesogastropoda, and one of six species of the family Strombidae found throughout the Caribbean region [The queen conch Linnaeus 758, is an region , fishersn region , 10.L. gigas) to a listing in Appendix II. This requires that all international trade by signatory countries be accompanied by a valid CITES permit, issued by the authorized National CITES Management Authority and endorsed by the Scientific Authority [In response to these events and concerns, the Convention on the International Trade in Endangered Species of Wild Fauna and Flora (CITES), moved queen conch . A total of 208 dro Bank , with 14dro Bank  and an adro Bank . A tissutraction . DNA forAll conch individuals were genotyped for nine microsatellite loci: Sgig1, Sgig2 and Sgig6 , and ConIrregularities in the data set such as genotyping errors caused by the presence of null-alleles and scoring errors due to large allele drop-outs and stuttering were tested for using Micro-Checker version 2.2.3 . FreeNA FST) based on 1,000 permutations were computed in Arlequin version 3.5.2.2 [FIS) and the inbreeding coefficient adjusted for bias (GIS) using GenAlEx 6.502 with probability data based on 999 permutations [Estimates of genetic diversity, including total heterozygosity estimated from observed heterozygosity (Ho) and expected heterozygosity (He), number of alleles per locus, effective number of alleles along with variance in allele frequency , and the hierarchical AMOVA was effected among populations using Arlequin version 3.5.2.2. Pairwise population matrices were calculated using the G-statistics option in GenAlex 6.502., with probability based on 999 permutations. To determine the most probable number of populations clusters (K) and assign each individual sampled to these clusters based on their genotype, a Bayesian assignment method was performed as implemented in STRUCTURE version 2.3.4 . A totalFST = 0.0084 and a corrected Global FST = 0.0083. GenAlEx computations of G-statistics such as FST adjusted for bias GST, and standardized G”ST which further corrected for bias when number of population is small . After false discovery rate (FDR) correction (p < 0.001) only comparisons with Sgig 2 appeared significant. The results showed two significant deviations from Hardy Weinberg Equilibrium (HWE) from different populations (1C and 3), and at different microsatellite loci (Sgig2 and ConchF23) respectively. Analysis in Micro-Checker  suggesteis small , showed IS detected at low levels and was negative at three of the five locations sampled.All nine microsatellite loci were highly polymorphic across all five sampled locations on Pedro Bank. The average number of alleles per locus ranged from 12.4 to 15.1 with 6.7 (± 0.79) being the mean effective number of alleles across all sites . Zone 1 FST = 0.009: p = 0.006). Pairwise FST comparisons ranged from 0.007 to 0.15, with four significant comparisons observed after FDR correction  indicated a 0.9% genetic differentiation among sampling locations, while variation within individuals was 100%. The hierarchical AMOVA  indicaterrection , three orrection  and the rrection  which shThe optimum number of sub-populations (K) of queen conch on Pedro Bank based on the largest value of ΔK using the Bayesian clustering analyses performed in Structure was K = 4 . There wL. gigas sampled, is indicative of a vigorous population. This high genetic diversity is likely due to the large area represented by the bank resulting in spatial variability across the Pedro Bank with little population isolation. These results are expected in large populations, which is consistent with the high conch density 374 conch/ha reported by Morris et al. [This is the first genetic study where all monitoring zones encompassing different depth strata on Pedro Bank were sampled for genetic assessment. Despite the heavy exploitation of queen conch on Pedro Bank, the high genetic diversity values observed within each group of s et al.  from thes et al.  cautionePrivate allelic richness can be used as a simple measure of genetic distinctiveness as it has proven to be informative in conservation genetic studies , 42. SliIS = 0.003 and 0.040) were observed in the most heavily fished areas suggesting some level of relatedness due to removal of alleles from the population. However, the average inbreeding coefficient across Pedro Bank was negligible signifying a population with high genetic variability, which is less related than is expected under the assumption of random mating. Inbreeding can contribute to the failure of a species to recover from exploitation as it increases the frequency of deleterious alleles [Our results  indicate alleles , 50. Des alleles , 46 and  alleles . In fact alleles  noted th-1 [-1 [Kenchington and Heino , regarde-1  or below-1 [-1 . The hig-1 [-1 .L. gigas with an extended planktonic larval stage, which may last up to 8 weeks and hence have a high dispersal potential [ST = 0.009: p = 0.006), across Pedro Bank, suggesting genetic homogeneity which can be maintained by the exchange of genetic material between a few individuals per generation [L. gigas within the Caribbean region by Kitson-Walters et al. [Stegastes partitus (damselfish) by Villegas-Sanchez et al. [Low estimates of population structure are typically found in benthic marine invertebrates such as otential , 56, 57.neration , 58. Sigs et al. , and forz et al. . The latL. gigas density [While many of these processes may contribute to the fine-scale and large-scale spatial variation within the population on Pedro Bank, the results from the pairwise comparisons which indicated that the easternmost group (sampling location 3) was the most significantly different from the other groups in the central and western end, supports the mechanism of genetic variation caused by oceanographic conditions. Surface currents in the Caribbean generally flow towards the west , 61, 62, density , 27, 64. density , found t density  reportedThe neighbour joining (NJ) tree is in congruence with the pattern of genetic structure found , where GST pairwise comparisons.The Bayesian clustering analysis indicated four clusters (K = 4) with an admixture of individuals assigned to all clusters indicative of mixed ancestry with a richer and more complex representation than is traditionally assumed by clustering models where each individual belongs to a unique cluster . This anSample location 3 situated on the eastern side of Pedro Bank (zone 3) has a predominant depth of 11–20 m and considered to be a nursery area due to the substrate type. Here larvae transported by the Caribbean current could settle and metamorphose into juveniles , 39, 69.L. gigas on Pedro Bank have received considerable attention from a wide variety of stakeholders ranging from within Jamaica and the wider Caribbean, to international trading partners concerned with conservation of the species [Over the years the impacts of commercial fishing and overexploitation of  species , 71–73.  species , 39, 64  species  and gene species . However species .Genetic diversity of queen conch on Pedro Bank embodies that of a fit population in all monitoring zones sampled under prevailing environmental conditions, however the central to eastern and southeastern end of the bank comprising monitoring zones 1 and 3 are heavily fished and incidentally comprise dense patches of seagrass beds which potentially act as nursery areas for recruitment to the fishery. These zones are predominantly < 20 m in depth hence the increased exploitation as it is more economical and safer for commercial fishers to fill their quotas in these areas . The deeL. gigas and as such institute appropriate periods for fishing restrictions suitable to the biology of the species. An alternate management measure to closed seasons may be closed areas as monitoring zones can be closed for specific periods to achieve the objectives of a closed season.A closed season to protect the reproductive period is a common management strategy in fisheries. This is particularly important for queen conch because they are susceptible to overexploitation when they aggregate in particular areas to spawn , 55, 75.Without proper enforcement, management strategies are mere suggestions for good practices. Aiken et al.  has lameThe identification of two possible subpopulations on Pedro Bank will assist fisheries managers in regulating harvesting areas and strengthen the certification scheme implemented to facilitate trade with the EU as entire sections of the bank may be closed off to fishing if necessary, and traceability of the individuals landed will be possible. The genetic information elucidated from the study will also strengthen the non-detrimental findings (NDF) required by CITES as traceability of the products can be substantiated along with the data provided for export. Knowing the genetic composition of the stocks on Pedro Bank and being able to trace the final product to the source stock will also serve as a deterrent to IUU fishing , which cL. gigas on Pedro Bank as was seen in previous studies done elsewhere in the Caribbean [L. gigas stocks on Pedro Bank should ensure a viable population continues to exist with the use of proper management tools incorporating all available data into ecosystem management and continued monitoring of the stocks.The results from this study revealed a weak but significant population structure for aribbean , 57, 70.S1 Table(DOCX)Click here for additional data file.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S1 Data(TXT)Click here for additional data file.