Cerastoderma glaucum has been investigated to clarify the complex phylogeographic pattern found in previous studies, to localize major genetic breaks, and to discuss their origin and maintenance.Coastal lagoons form an intriguing example of fragmented marine habitats. Restricted gene flow among isolated populations of lagoon species may promote their genetic divergence and may thus provide a first step toward speciation. In the present study, the population genetic structure of the lagoon cockle The Atlantic and Mediterranean coasts, including the Baltic, North Sea, and Black Sea.C. glaucum individuals from 14 populations were genotyped using restriction site‐associated DNA sequencing (RADseq). The genetic diversity, divergence, and structure were analyzed using genome‐wide single nucleotide polymorphisms (SNPs). Phylogenetic relationships were inferred under a coalescent model using svdquartets.A total of 204 C. glaucum that are separated by many genetic barriers: one lineage in the Aegean–Black Sea region, one in the Ionian Sea, and the last one widely distributed from the Western Mediterranean to the Baltic Sea. The nested branching pattern displayed on the species tree largely agrees with the likely scenario of C. glaucum postglacial expansion from the Mediterranean to the Baltic Sea.The RADseq approach allowed inferring phylogeographic relationships with an unprecedented resolution. Three deeply divergent lineages were identified within The genetic differentiations between geographically separated lagoons proved to be strong, highlighting the evolutionary influence of these naturally fragmented habitats. The postglacial expansion created complex patterns of spatial segregation of genetic diversity with allele frequency gradients in many outlier loci, but also discrepancies between the nuclear and mitochondrial genetic markers that probably arose from genetic surfing of mitochondrial variation. For instance, Mejri, Arculeo, Ben Hassine, and Lo Brutto  proposedOn the other hand, such complex pattern and recent phylogeographic events are not easily deciphered and require high‐resolution genetic markers to get the required information, and to compensate for the heterogeneity of genetic variation across the genome  is widely distributed from the Atlantic coast of Norway to the Mediterranean Sea  as in previous mitochondrial, microsatellite, and EPIC studies  comprehensively reconstruct the phylogeographic relationships among populations, (b) describe divergence patterns across the genome, and (c) localize major genetic breaks and discuss their origin and maintenance.Accordingly, the existence of a strong genetic structure within 22.1Cerastoderma glaucum from 14 previously analyzed samples  from the Atlantic coast of Portugal, Sylt, and Danish fjords have been picked for RADseq analysis. Samples were selected based on DNA quality and quantity, in order to reach 10–17 individuals per population. DNA libraries were prepared from old DNA samples or new extractions from tissue stored in ethanol . DNA extraction has been performed using GeneJET Genomic DNA Purification Kit (Thermo Scientific) following the manufacturer's instructions. The DNA concentration was measured using the Quant‐iT PicoGreen kit (Invitrogen). DNA samples were ranked according to concentration of double‐stranded DNA and divided into six quality groups (libraries) and normalized to the lowest concentration within each group. The RAD libraries were prepared following the protocol outlined in Etter, Bassham, Hohenlohe, Johnson, and Cresko (SbfI high‐fidelity restriction enzyme (New England Biolabs). The predicted number of RAD loci for this enzyme was 15,435, assuming a 1.34 GB genome size . Ligation reactions were then prepared to attach 32  or 53  different barcoded P1 adapters . The barcodes were 6 or 5 bp long and differed by at least two bases. To increase the amount of DNA in low‐concentration libraries, we adjusted the volume of digestion–ligation products for multiplexing from 20 μl per individual in the highest concentration library (beginning at a DNA concentration of 15 ng/μl) to 60 μl per individual in the lowest concentration library (DNA concentrations of around 2 ng/μl). After multiplexing, the samples were sheared in a Covaris sonicator for 75 s. Sonification efficiency and final quality of the libraries were checked on an Agilent Bioanalyzer. Final amplification in 16 PCR cycles was carried out using 1.5 µl of RAD amplification primers. All purification steps were performed using Macherey‐Nagel NucleoSpin Gel and PCR Clean‐up columns, except for the two last ones (before and after PCR reaction), for which the Agencourt AMPure XP beads have been used. Two initial libraries have been sequenced at Institut Pasteur de Lille, France, and the four final libraries at McGill University and Génome Québec Innovation Centre, Canada, on an Illumina HiSeq .In the present study, the same set of samples as used in previous studies by Tarnowska et al.  and Sromd Cresko  with som2.2pyrad v. 3.0.63 software pipeline . The maximum proportion of individuals with shared polymorphic site in a locus was set to 50%. The remaining parameters were kept to default values. pyrad output was analyzed in the software r v. 3.3.1 . In a separate run of the pipeline with the same parameters, C. edule individuals were included and set as an outgroup. This setting allowed to input for C. edule individuals only the loci common to both species, and thus to root the phylogenetic tree.Demultiplexing, filtering, and clustering of sequence reads were performed with e Eaton, . Compares Eaton, . Either s et al.  were fol2.3pyrad vcf output files were converted into the Genepop format using pgdspider version 2.0.1.5  were calculated in “poppr” v. 2.2.0  method of Benjamini and Hochberg  produced more loci per individual, but as a consequence, these loci were shared by less individuals Figure . To asseClustering analyses resulted in a dataset containing 5,127 RAD loci for a total of 77,944 SNPs. The average number of SNPs per RAD locus was 15.2, ranging from 1 to 50 Figure . A total3.1p < 0.0001). The mean number of alleles per locus and the number of private alleles per population also decreased from the Black Sea and Eastern Mediterranean to the Baltic Sea  to 0.434 . In the population structure analysis, a model with K = 6 was found to fit best to the data, as both cross‐entropy criterion (from “LEA” package) and Bayesian information criterion (from “adegenet” package) converged on this number of suggested clusters . The major part (22%) of the genetic variation that was not attributable to variation within individuals  was partitioned across geographic regions, whereas only 8% of the variation was accounted for by differences among populations nested within the geographic regions , and the coefficient of determination R2 equaled 0.686. Geographic distance explains more genetic variance in Atlantic populations  as compared to Mediterranean Sea populations  from Tarnowska et al. .On the coalescent‐based species tree, the Aegean–Black Sea cluster of c Figure . All rel44.1FST value of 0.27 estimated from SNPs . These observations are in accordance with the hypothesis that homoplasy is likely to affect microsatellites when populations are distantly related , but the location of major phylogeographic breaks varied with the marker used  and Mediterranean populations separately , explained a slightly lower proportion of variance (22% vs. 23%). Thus, there must exist a significant proportion of SNPs that distinguish even the youngest split, between the Baltic and North Sea clusters. Furthermore, investigation of allele frequencies at the most differentiated loci  is indeed smaller than for between‐clusters comparison , despite a larger geographic distance. Within the Western Mediterranean cluster, genetic differentiation was not correlated with geographic distance , but the number of pairwise comparisons among four populations was probably too small to provide a reliable estimate of correlation. Interestingly, a finer geographic structure was visible within most clusters, but not within the Aegean–Black Sea clusters  and Constanta (Black Sea) populations was nevertheless the lowest one in the present dataset , the southern ice limit in Europe was probably located at 43°N (northern Spain) (Frenzel, Pécsi, & Velichko, The population structure within ty Table . These oC. glaucum range expansion model along Atlantic coast and can nicely explain the unexpected geographic disjunction found in the mtDNA analysis (Tarnowska et al., C. glaucum forms continuous populations along a large part of the coastal zone, but not in the North Sea nor along the Atlantic coast, where tidal amplitudes are high, and suitable habitats are thus scarce (Boyden & Russell, C. glaucum are moreover marked by temporal variations in population size and frequent extinctions (Labourg & Lasserre, It has been shown that new mutations arising at the front of a range expansion can occasionally travel with the wave of advance, be carried over long distances, and reach very high frequencies in the newly colonized areas (Excoffier & Ray, C. glaucum seems to be spatially segregated because of the stochastic processes associated with a recent colonization of the Atlantic coast, and this can explain the poor resolution and discordant patterns found in previous studies. Mitochondrial surfing is rarely reported in the literature, but our scenario is similar to the one described by Streicher et al. (Micrurus tener. As noticed by the authors, circumstances under which mitochondrial surfing can be detected are rare: Divergent haplotypes need to be maintained in sympatry before range expansion, and the dispersal capability of species has to be limited.Mitochondrial DNA can be more prone to allele surfing than nuclear DNA because of its lower effective population size. However, if the scenario of mitochondrial DNA surfing on the wave of expansion is true, we also should find some nuclear alleles which behaved in a similar way. The spatial distribution of successfully “surfed” alleles should form clines with frequency increasing in the direction of the range expansion (Travis et al., r et al.  for the C. glaucum together with its fragmented habitat could lead to a longer persistence of this structure. However, a clear‐cut separation of mitochondrial haplogroups was also observed among geographically close populations in Western Mediterranean (Tarnowska et al., In species with high dispersal ability, population differentiation that can arise during a range expansion is expected to be transient (Excoffier, Foll, & Petit, 4.3Pomatoschistus tortonesei (Mejri et al., Syngnathus abaster (Sanna et al., Atherina lagunae (Trabelsi et al., C glaucum, because genomic differentiation was found to be highly heterogeneous across the genome.Several mechanisms may cause genetic differentiation between marine species populations such as vicariance processes, caused by historical barriers, oceanographic discontinuities, local adaptation, and limited dispersal capabilities Palumbi, . The spaC. glaucum is too high to be easily explained solely by local adaptation (Bierne, Welch, Loire, Bonhomme, & David, C. glaucum from Northern Europe, the French Atlantic coast, and the Western Mediterranean (Brock & Wolowicz, An elevated level of genetic differentiation at specific loci is commonly interpreted as a consequence of divergent selection (e.g., Nosil et al., C. glaucum habitat means that the environmental conditions it experiences during the year can be similarly extreme in the entire Mediterranean Sea. This species is characterized as being highly euryhaline and eurythermic (Ansell, Barnett, Bodoy, & Massé, C. glaucum populations is more likely the consequence of endogenous genetic barriers, created by genetic incompatibilities than a product of local adaptation. The endogenous genetic barriers are likely to impede neutral gene flow in a substantial proportion of genome (Barton & Hewitt, Contrary to the Atlantic–Baltic sector, there is no clear association between ecological variables and allele frequency shifts at outlier loci in the Mediterranean Sea. Strong genetic divergence at outlier loci between the Ionian Sea and Aegean–Black Sea clusters is at odds with the close geographic proximity of Aegean and Ionian Sea basin. Furthermore, the lagoon‐like character of C. glaucum. The cockles present a high variability in terms of morphology, which has led some authors to propose numerous species and subspecies which are currently treated as synonyms of C. glaucum or C. edule (Grossu, Cerastoderma lamarcki and a Mediterranean C. glaucum had been suggested based on immunoelectrophoretical, karyotype, and chromosomal DNA differences (Brock, C. glaucum, as a major phylogeographic break had been found in the Eastern Mediterranean (Mariani et al., C. glaucum also constitute isolated entities. Thus, the “Cerastoderma glaucum complex” appears as a particularly intricate example, illustrating both the sampling variation biases introduced by the analysis of a limited number of loci and the important evolutionary role of fragmented lagoon habitats.It should be noted that there have been a variety of hypotheses concerning taxonomical divisions within  Grossu, . The subs Brock, . More reNone declared.M.W., D.F., R.L., and P.F. contributed to the ideas and project design. L.S. and D.F. performed the experiments and analyzed the data. L.S. led the manuscript writing. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Heritability is the most commonly used measure of genetic contribution to disease outcomes. Being the fraction of the variance of latent trait liability attributable to genetic factors, heritability of binary traits is a difficult technical concept that is sometimes misinterpreted as the more-easily understandable concept of attributable fraction. In this paper we use the liability threshold model to describe the analytical relationship between heritability and attributable fraction. Towards this end, we consider a hypothetical intervention that is aimed to reduce the genetic risk of the disease for a specified target group of the population. We show how the relation between the heritability and the attributable fraction depends on the disease prevalence, the intervention effect and the size of the target group. We use two real examples to illustrate the practical implications of our theoretical results. Measuring the genetic influence on human diseases is one of the most important topics in medicine and epidemiology. This is commonly done in terms of the heritability, routinely reported using studies of siblings and twins  liability L, which captures all genetic and environmental risk factors that influence the disease risk. Let G and E be scalar measures that summarize these genetic and environmental risk factors, respectively; we hereafter refer to G and E as the genetic and environmental ‘risks’. The liability model assumes thatL exceeds a threshold Falconer  developeThe epidemiological literature distinguishes between the point prevalence (i.e. the proportion of the population that has the disease at a given point in time) and the lifetime prevalence (i.e. the proportion of the population that develops the disease at some point during life). Here, we are interested in ever experiencing the disease and we are thus interested in the lifetime prevalence.G for which the liability falls below the threshold Before proceeding, we emphasize an important feature of the liability threshold model. Because the model is additive, it is, for any given value G and E are independent and normally distributed with mean zero and variances G and E are linearly related to L as in  proportion of disease cases prevented by the intervention. For instance, suppose that the factual prevalence is 5%, and that the intervention reduces the prevalence to 1%. The proportion of prevented disease cases is then equal to G in the liability model. This is supposed to capture the aggregated impact of variation over the whole genome on the disease, which cannot be ‘eliminated’ in any meaningful sense. We can, however, think of hypothetical interventions that aim at manipulating the genetic risk in other ways, e.g. by changing its distribution or shifting it by a fixed constant. Thus, we use the standard definition of the AF in  prevalence p arises only because of shifted liability levels within this target group. Thus, without loss of generalization we can express the numerator in  and stroke  for AMI and 0.4 percentage points (i.e. from 4% to 3.6%) for stroke. Such a genetic risk reduction corresponds to an intervention effect of Figures entclass1pt{minimaThe examples of AMI and stroke are marked in Figs. Breast cancer is one of the most common cancers in women, with a lifetime prevalence of 8.1% and a heritability of 31%, estimated from Swedish twin data  has a large intervention effect, but can only be given to a limited target group. Compared to BPM, tamoxifen has a smaller intervention effect but can be given to a larger target group. In this example, we observed how the AF is larger for the tamoxifen intervention compared to the BPM intervention despite its lower intervention effect since the target group is larger.Two examples have been used to illustrate how our results can be used to understand the relationship between the heritability and the AF for different scenarios. The first example is an intervention with blood pressure and cholesterol-lowering medication to prevent AMI and stroke. In this example we have considered the target group, We are not the first to use the AF to measure the effect of genes. However, virtually all applications that we are aware of define the exposure as a single SNP/gene  and the genetic and environmental risks are independent. Regarding the first assumption, it is reasonable to assume that the genetic and environmental risks are normally distributed, since we are considering complex traits that depend on the accumulated small contributions from many different genetic and environmental factors. The second assumption of mainly additive effects from the genetic and environmental factors has been debated to a great extent (Hill et al. In this article, we have conceptualized ‘genetic interventions’ as all interventions that modify the genetic risk. These can be pure genetic interventions, such as gene therapy, or interventions that target the mechanisms by which the genes exert their effect, such as bilateral prophylactic mastectomy. Thus, by ‘genetic’ we do not necessarily mean that the genes are modified per se, but rather that the intervention modifies the circumstances that allow the genetic variants to manifest.Throughout, we have defined the target group as those at the highest genetic risk. As suggested by one of the reviewers, target groups may in practice also be defined in terms of environmental risk for disease. In Appendix


However, further backcrosses were not always detectable with backcrossed-Western roan individuals (46.7–60%), backcrossed-East, Central and Southern African roan individuals (28.3–45%) and double backcrossed (83.3–98.3%) being incorrectly classified as non-admixed. Our study is the first to confirm ongoing hybridization in this within this iconic African antelope, and we provide recommendations for the future conservation and management of this species.Biological diversity is being lost at unprecedented rates, with genetic admixture and introgression presenting major threats to biodiversity. Our ability to accurately identify introgression is critical to manage species, obtain insights into evolutionary processes, and ultimately contribute to the Aichi Targets developed under the Convention on Biological Diversity. The current study concerns roan antelope, the second largest antelope in Africa. Despite their large size, these antelope are sensitive to habitat disturbance and interspecific competition, leading to the species being listed as Least Concern but with decreasing population trends, and as extinct over parts of its range. Molecular research identified the presence of two evolutionary significant units across their sub-Saharan range, corresponding to a West African lineage and a second larger group which includes animals from East, Central and Southern Africa. Within South Africa, one of the remaining bastions with increasing population sizes, there are a number of West African roan antelope populations on private farms, and concerns are that these animals hybridize with roan that naturally occur in the southern African region. We used a suite of 27 microsatellite markers to conduct admixture analysis. Our results indicate evidence of hybridization, with our developed tests using a simulated dataset being able to accurately identify F1, F2 and non-admixed individuals at threshold values of  Factors such as climate change, habitat fragmentation, and environmental degradation are influencing the distribution and abundance of species, often in ways that are impossible to predict . Thus, ahttps://www.cbd.int/sp/targets/). The ability to accurately identify introgression is critical to the management of species [Anthropogenic hybridization and introgression are major threats to species conservation  is one of Africa's most iconic large antelope species. It has a sub-Saharan range, is a water-dependent species, and prefers savanna woodlands and grasslands. Ansell [H. e. bakeri, H. e. charicus, H. e. cottoni, H. e. equinus, H. e. koba and H. e. langheldi. However, subsequent genetic studies by Matthee and Robinson [H. e. bakeri) for both the mtDNA control region and eight microsatellite markers. Both the mtDNA control region and microsatellite data provided strong support for a separation between the West Africa population (corresponding to the H. e. koba subspecies) and those from East, Central and Southern Africa . Although some differentiation between East, Central and Southern African roan antelope was evident from mtDNA data, different subspecies did not form monophyletic groups, with no differentiation observed for microsatellite data. The placement of the two specimens from Cameroon (corresponding to the H. e. charicus subspecies) were unclear, and the small sample size precluded robust analyses. Based on these results, Alpers et al. [Roan antelope  by Vaz Pinto [® DNA Sample Preparation Kit  was used to create a paired-end library followed by sequencing on the MiSeq™ sequencer  using 2 x 300 bp chemistry . Library construction and sequencing was carried out at the Agricultural Research Council Biotechnology Platform . FastQC version 0.11.4 [We selected nine cross-species microsatellite markers  that were previously characterised in sable antelope  following the manufacturer’s protocols. Polymerase Chain Reaction (PCR) amplification was conducted in 12.5 μl reaction volumes consisting of AmpliTaq® DNA polymerase  forward and reverse primers (0.5 μM each), and 50 ng genomic DNA template. The conditions for PCR amplification were as follows: 5min at 95°C denaturation, 35 cycles for 30s at 95°C, 30s at 50–62°C  and 30s at 72°C, followed by extension at 72°C for 10min in a T100™ Thermal Cycler . PCR products were run against a Genescan™ 500 LIZ™ internal size standard on an ABI 3130 Genetic Analyzer . Samples were genotyped using GeneMapper v. 4.0 software .DNA extractions were performed using the Qiagen DNeasyz = expected heterozygosity adjusted for unequal sample sizes) [P) was calculated with GenAlEx 6.5 [ID was calculated using GenAlEx 6.5 [IS) and average pairwise relatedness was calculated for the reference groups, between individuals within populations was calculated using GenAlEx 6.5 [Understanding the diversity within groups provide valuable information to identify hybrid individuals. To this end, genetic diversity was evaluated for each group separately . MICRO-CHECKER  was usede sizes)  and numbAlEx 6.5 ,35. ArleAlEx 6.5 ,37 was uAlEx 6.5 . AssociaAlEx 6.5 . In addiAlEx 6.5 ,35. FinaAlEx 6.5 ,35 and tAlEx 6.5 . DemerelST comparisons among populations in ARLEQUIN version 3.5 [qi) of the sampled populations to the inferred clusters. Individuals  were assigned to the inferred clusters using an initial threshold of qi > 0.9 [To estimate the degree of genetic differentiation between populations, we performed an analysis of molecular variance (AMOVA) and conducted pairwise Fsion 3.5 ,37 usingsion 3.5 –44 and asion 3.5  was idension 3.5 . The memsion 3.5  with thesion 3.5 . From thqi > 0.9 . PCA forqi > 0.9 .qi-value  would maximize the accuracy of assignment, simulated genotypes were created using HYBRIDLAB [qi > 0.90  were used as parental (P1) populations to create the simulated hybrid genotypes (see [To determine which threshold YBRIDLAB . Genotyppes (see ). A datapes . Read lengths of 2 x 301 bp  were obtained and after trimming, the remaining reads ranged from 180 to 200 bp . A total of 14 unique loci were identified, of these only six were polymorphic and consistently amplified animals from both ESUs .o values lower than Hz values, which may be an indication of the presence of possible null alleles. However, null alleles were only observed in six markers  from the East, Central and Southern African roan group. Significant linkage disequilibrium (LD) was also observed only in the East, Central and Southern African group. These departures from equilibrium may be because of substructure in this group (see [Deviations from HW equilibrium (HWE) were not consistent across populations, with significant deviations from HWE being observed only in the East, Central and Southern African roan populations. In the East, Central and Southern Africa roan population A (Northern Cape Province), 11 loci  deviated from HWE. In addition, two loci (BM3517 and SPS113) deviated from HWE in East, Central and Southern Africa roan population B (North West Province) following Bonferroni correction. These markers indicated significant heterozygote deficit in the respective populations with Houp  in the Western roan group ranged from 0.67–0.72 and unbiased heterozygosity (Hz) from 0.65–0.71 while Ho in the East, Central and Southern African roan varied from 0.57–0.63 and Hz from 0.605–0.609. The overall genetic diversity for the known hybrid and putative hybrid populations were intermediate compared to the Western and East, Central and Southern roan populations. The putative hybrid population indicated higher levels of genetic diversity compared to the known hybrid population. The PID for the 27 loci was 5.5−20, thus the estimated probability of any two individuals by chance alone sharing the same mulitlocus genotype was 1.819 for the 27 loci combined.Genetic diversity for each population is summarized in ST = 0.165, P < 0.001), validating our two reference groups. Principle component analysis similarly revealed a clear separation between the West African versus East, Central and Southern Africa roan (qi) for non-admixed Western roan qi > 0.881 and for non-admixed East, Central and Southern Africa roan qi > 0.883.The final dataset included 27 microsatellite loci that yielded a total of 267 alleles, with the number of alleles ranging from 3 to 17 per locus. A total of 27 alleles were unique to the West African roan group, while 27 were found exclusively in the East, Central and Southern African group . An analica roan  with 9.3ica roan , S1 Fig.qi) was analysed as a continuous variable and the ability of the marker set to accurately assign individuals was determined at four threshold values . STRUCTURE analysis of simulated genotypes generated by HYBRIDLAB indicated that all (100%) of the non-admixed West Africa roan (qi = 0.896–0.984) and non-admixed East, Central and Southern African roan (qi = 0.887–0.986), F1 hybrids (qi = 0.265–0.739) and F2 hybrids (qi = 0.182–0.766) genotypes were correctly assigned at selected thresholds of qi > 0.80 and qi > 0.85 , F1 hybrids, F2 hybrids, F1 BC-Western roan, F1 BC-East, Central and Southern roan, 2x BC-Western roan and 2x BC-East, Central and Southern roan. The threshold of admixture (i > 0.85 . At a seqi = 0.600–0.969) and 45% of BC-East, Central and Southern African roan individuals (qi = 0.528–0.946) being incorrectly assigned as non-admixed individuals at qi > 0.80. The percentage of incorrectly assigned individuals decreases with an increase in the threshold value for both BC-Western roan  and BC-East, Central and Southern African roan individuals   and 98.3% 2x BC-East, Central and Southern African roan (qi = 0.789–0.982) were incorrectly identified as non-admixed individuals. The incorrect identification of double backcross individuals as non-admixed individuals also decreases with an increase in the threshold value for 2x BC-Western roan  and 2x BC-East, Central and Southern roan   . In addi = 0.95) .qi > 0.85 was selected for the analysis of the non-admixed populations, known hybrids and putative hybrids. At this selected threshold, all non-admixed Western, non-admixed East, Central and Southern roan, F1 hybrids and F2 hybrids were correctly identified, whereas some backcross and double backcross individuals were correctly assigned. With an increase in the threshold value a larger percentage of backcross and double backcross individuals would be identified however a higher percentage non-admixed Western roan and East, Central and Southern roan would be incorrectly identified as hybrid individuals. The qi > 0.85 threshold was applied to the real dataset and all non-admixed Western roan and all non-admixed East, Central and Southern roan were correctly identified as non-admixed. In addition, six of the eight know hybrids were confirmed as hybrids, with two hybrids being identified as non-admixed Western roan (qi = 0.9664 and qi = 0.9510 respectively). Analysis of putative hybrids identified four out of 15 animals (27%) as hybrid and 11 (73%) as non-admixed Western roan.Based on the simulation results, the threshold of An increasing number of species experience dramatic declining population numbers globally, with ample evidence suggesting that we are entering a mass extinction event. Although the drivers of these population declines are numerous and varied, the underlying root cause inevitably stems from anthropogenic pressures. Not surprisingly, hybridization and admixture of groups with distinct evolutionary trajectories are increasing, raising concerns about the integrity of a large number of species, especially those that experience disproportionately large human interest. For roan antelope, one of Africa's most spectacular large antelope species, this is certainly the case. Although roan antelope numbers are increasing in South Africa (largely because of protection under private ownership), real concerns exist about their genetic integrity given admixture with West African roan antelope, also for export to neighbouring countries. We discuss our results here, and provide some suggestions for roan antelope conservation in South Africa.q-values close to threshold values strongly suggest that hybrid individuals are viable and fertile; as also suggested from anecdotal evidence from some game farms. Although genetic diversity estimates were moderately higher in the known and putative hybrid individuals, it has previously been reported that F2 hybrids can display reduced fitness as a result of disruption of sets of co-adapted gene complexes by recombination [qi > 0.80 and qi > 0.85. However, the classification of further backcrosses was less accurate at these thresholds with backcrossed and double backcross individuals being incorrectly classified as non-admixed. The use of higher thresholds (qi > 0.90 and qi >0.95) did increase the number of individuals correctly identified as backcross and double backcross individuals, however, this also resulted in an increase in the number of non-admixed individuals being incorrectly classified as hybrids. Based on the simulated results a threshold value of qi > 0.85 was thus selected as all non-admixed Western and non-admixed East, Central and Southern roan, F1 and F2 hybrids would be correctly identified and some backcross and double backcross individuals would be correctly identified. In certain instances, backcrossed and double backcrossed individuals extend beyond the detection power of the current microsatellite marker panel.Using a suite of variable and informative microsatellite markers, we provide evidence of hybridization and introgression between roan antelope naturally occurring in South Africa , and those of West African decent . More pbination ,53, therCanis lupus) that hybridizes with domestic dogs (C. lupus familiaris) indicated that simply increasing the number of microsatellite markers used does not equate to an increase in the number of correctly identified admixed individuals [The minimum number of markers required to accurately and consistently identify backcrosses is currently being debated. Simulation analysis in the grey wolf  suggest that the occurrence of widespread introgression is low, and largely confined to specific game farms.ex situ breeding programmes and agreements and/or animals that could be allowed to be backcrossed to obtain some form of purity, over four or five generations. This might improve genetic variation within the national population, but may not be desirable given that the impact of hybridization on the South African roan full genome is not known. Thus, we recommend the implementation and continuation of strict genetic monitoring for hybridization in roan antelope in South Africa, at least for the foreseeable future. With the microsatellite marker set described here, and using a threshold of qi = 0.85, it is possible to detect F1 and F2 hybrids prior to translocation, thereby reducing and ultimately eliminating Western roan antelope alleles in the indigenous roan gene pools. In addition, management of roan in South Africa would benefit from a national meta-population conservation plan to inform translocations and reintroductions and to effectively monitor genetic diversity and further hybridization events.Animals of West African decent are no longer maladapted to South African conditions and have, over the span of 20 years, adapted to local conditions. The question that needs consideration is whether South Africa should safeguard the genetic integrity and genetic variability of both roan ESUs. If historic occurrence is considered, then all West African animals should be removed from South African populations. However, the South African situation has spawned several S1 Figa) Probability (-LnPr) of K = 1–6 averaged over 5 runs. b) Delta K values for real population structure K = 1–6.(PDF)Click here for additional data file.


Epinephelus polyphekadion, the squaretail coral grouper Plectropomus areolatus, and the common coral trout P. leopardus, with similar life histories but distinct spatio-temporal characteristics in their patterns of forming spawning aggregations.Understanding the factors shaping population genetic structure is important for evolutionary considerations as well as for management and conservation. While studies have revealed the importance of palaeogeographic changes in shaping phylogeographic patterns in multiple marine fauna, the role of reproductive behaviour is rarely considered in reef fishes. We investigated the population genetics of three commercially important aggregating grouper species in the Indo-West Pacific, namely the camouflage grouper E. polyphekadion and P. areolatus that assemble in larger numbers at fewer spawning aggregations and in distinctive offshore locations than in P. leopardus which has multiple small, shelf platform aggregations.By examining their mitochondrial control region and 9–11 microsatellite markers, we found an overarching influence of palaeogeographic events in the population structure of all species, with genetic breaks largely coinciding with major biogeographic barriers. The divergence time of major lineages in these species coincide with the Pleistocene glaciations. Higher connectivity is evident in While palaeogeographic events played an important role in shaping the population structure of the target species, the disparity in population connectivity detected may be partly attributable to differences in their reproductive behaviour, highlighting the need for more investigations on this characteristic and the need to consider reproductive mode in studies of connectivity and population genetics.The online version of this article (10.1186/s12862-018-1284-0) contains supplementary material, which is available to authorized users. In the rrelfish , some sprrelfish , 12, andrrelfish . Hence, rrelfish . The latThe evolution of groupers (family Epinephelidae), a commercially important and speciose taxon of considerable biomass in reef ecosystems, was significantly impacted by environmental change associated with glacial cycles, as revealed by a recent historical biogeographic study . These cEpinephelus polyphekadion , the square-tail coral grouper Plectropomus areolatus , and the common coral trout, P. leopardus . These groupers are top predators on coral reefs that feed predominantly on fish [P. leopardus has a restricted distributional range relative to the other two species , surgeonfishes, damselfishes and parrotfishes –29. FishP. leopardus forms multiple small  aggregations for 1–2 months each year and can migrate up to 10 km between spawning sites and home ranges [P. areolatus forms relatively fewer, yet much larger , aggregations for multiple months , with spawning migrations of up to 30 km [E. polyphekadion forms few, large , spawning aggregations for one or sometimes two months a year, with adults known to travel up to 40 km to spawning sites from their home reefs . The latter two species aggregate at shelf edge/drop-off areas, often associated with outer reef channels and close to offshore waters, for a few days around the full or new moon (depending on location) [P. leopardus characteristically spawns on the shelf platform on the full moon in Australia [P. areolatus, may have evolved to favour long distance larval dispersal, which may have weakened the phylogeographic signal in this species.Among our target species, e ranges , while Pto 30 km , 36. By ocation) , whereasustralia . The disP. areolatus to exhibit the lowest level of genetic population structure across its geographic range because of the concentrated release of gametes and more frequent spawning that could have evolved to favour long distance dispersal. By contrast, we expected P. leopardus to have the strongest genetic population structure across its range, being a poor disperser relative to the other two species, due to smaller and spatially scattered aggregations that are not close to offshore waters. To test these hypotheses, we conducted an extensive genetic examination over the entire range of the three species by using the mitochondrial control region and nuclear microsatellite markers. Moreover, since our target species are among the most commercially important reef fish in the Indo-Pacific, and some are already threatened by overfishing, connectivity information would be valuable to improve their management and conservation. We evaluate our findings against comparable population genetics work in other groupers and consider possible management and conservation implications.This study aims to test whether the phylogeographic patterns of the three grouper species (1) are shaped by past environmental changes, and (2) whether disparity in population genetic structure among the species, if any, could be attributable to differences in their reproductive traits. In this regard, we would predict Fin clips or muscle tissues from the three species were obtained from 28 locations throughout the Indo-Pacific Fig. . Samplin2, 200 μM dNTPs, 200 nM of each primer, 1.5 U of Taq polymerase (Amersham) and ddH2O to a total volume of 50 μl. The thermal cycle consisted of an initial denaturation at 94 °C for 3 min followed by 38 cycles at 94 °C for 30 s, 54 °C for 50 s, 72 °C for 1 min 30 s, with a final extension at 72 °C for 10 min. PCR products were purified using Millipore Montage PCR96 Cleanup Kit, following manufacturer’s instructions. Sequencing was performed in the forward direction using an Applied Biosystems 3100 sequencer and followed standard cycle sequencing protocols . Sequence data were edited using MEGA 5.0 [h) and nucleotide diversity (π) [S [A 580-base pair (bp) fragment of mtDNA control region was amplified using primers developed for this study   were asssity (π) . Neutralsity (π) , and Fu’y (π) [S ) were coK) of groups of populations from 2 to the number of populations minus 1; the K value with the highest and significant FCT was regarded as the best representation of the number of distinctive groups for that dataset. SAMOVA was run with and without geographic information, which gave consistent results. To reduce errors in significance calculations due to missing data, selected grouping schemes were subjected to locus-by-locus Analysis of Molecular Variance (AMOVA) in Arelquin 3.5; (2) Pairwise ΦST statistics were computed using Arlequin with 99,999 permutations; and (3) The evolutionary relationships among haplotypes and genetic structure were evaluated and visualized by a minimum spanning network (MSN) analysed using Arlequin and drawn using Gelphi [Population genetic structure was examined by three means: 1) SAMOVA 2.0  was used SAMOVA 2g Gelphi  and Adob− 1 between lineages [Based on the average nucleotide substitution rate of the control region of 10% Myrlineages , the div2, 150 μM dNTPs, 60–240 nM of each fluorescent-labelled  forward primer, 60–240 nM of each reverse primer, 4% Bovine Serum Albumin (BSA) and 1.5 U KAPA2G Fast DNA polymerase in a total volume of 6.25 μl. A touch-down thermal programme was used: initial denaturation at 95 °C for 2 min, followed by 30 cycles of denaturation at 95 °C for 10 s, 12 s at annealing temperature stepping down 0.2 °C per cycle from 62 °C to 56 °C and an extension at 68 °C for 3 s. Subsequently, another 10 cycles of denaturation at 95 °C for 10 s, 12 s annealing at 56 °C and extension at 68 °C for 3 s, were run. PCR products were genotyped using an Applied Biosystems 3100 DNA Analyzer along with GeneScan LIZ-500 (Applied Biosystems) as internal size standard. Allele sizes were analysed using GENEMARKER 2.4.0 . MICROCHECKER 2.2.3 [FST before and after ENA . Correction for null alleles and bootstrapping  was used to determine significance levels. The observed and expected heterozygosities (Ho and He) were estimated for each locus using GenALEx 6.5 [Between nine and eleven microsatellite loci were analysed in the three species examined  SAMOVA was used to analyse the spatial genetic structure as previously described for mtDNA. The maximum proportion of missing data was set at 5%; (3) A discriminant analysis of principal components (DAPC) [Genotypic population structure was assessed using four approaches: (1) GenALEx was used to estimate pairwise s (DAPC)  was empls (DAPC)  under ans (DAPC)  was useds (DAPC) . We thens (DAPC) , 58.E. polyphekadion, 341 P. areolatus and 340 P. leopardus, respectively  ranged from 0.025 ± 0.013 in P. areolatus to 0.131 ± 0.063 in P. leopardus.This study analysed 723-bp, 580-bp and 556-bp segments of control region from 270 E. polyphekadion, P. areolatus and P. leopardus, respectively, were genotyped for the microsatellite loci  in P. areolatus, and from 0 to 0.317 (mean 0.076) in P. leopardus (data not shown). No significant disparity in global FST values computed with and without ENA correction was found  was detected in 15/99, 8/126 and 28/108 cases E. polyphekadion, P. areolatus and P. leopardus, respectively, most of which were due to heterozygote deficiency  and in microsatellites  WIP, including the Maldives and Phuket, and (2) the CIP-EIP, including samples from CK to French Polynesia  and in microsatellites  in mtDNA and microsatellites with all P < 0.001). Population differentiation within the CIP was generally weak  in four of 36 cases and FST - 0.035 to 0.092, P < 0.00001 in six of 15 cases), but CK was a genetically distinct population from all other populations based on microsatellites . We detected no sign of recent population expansion in the WIP group , while in the CIP-EIP group, Fu’s FS, which is more sensitive to sudden demographic expansion than Tajima’s D, was significantly negative , while Tajima’s D was not significant .SAMOVA computed the highest see Fig. . SAMOVA P. areolatus into three groups: Red Sea (RS), Maldives-CK, and the Western Australia–Pacific Ocean (WAPO), with the WAPO population genetically less distinct from the Maldives-CK population  than from the RS clade . The average genetic distance amongst the WAPO and Maldives-CK group was 1.6% (± 0.6%), and amongst the RS and the rest was 6.2% (± 1.2%) and the molecular clock estimates of divergence time were about 0.16 ± 0.06 MYA and 0.62 ± 0.12 MYA, respectively , all with P < 0.0001  or the WAPO region . Both datasets identified the genetic break between the RS and Maldives  revealed that the RS population was strongly isolated  and that the CK population was genetically distinct from all others . Population differentiation within the WAPO was generally low and insignificant (ΦST - 0.922 to 0.146), with significant ΦST (P < 0.00001) only between Fiji and Rowley Shoals, based on the control region , specifically between West Australian sites and the rest, with P < 0.00001 in 22 of 36 comparisons .The optimal grouping inferred by SAMOVA with the highest P. leopardus, as well as some from East Indonesia and a Taiwanese individual. The other group was divided into two subgroups, ten mutational steps apart. One subgroup harboured mostly individuals from Taiwan and Cebu , while individuals from Palawan (west Philippines) and Indonesia dominated the other subgroup. The Eastern population contained three major groups: one group consisted mostly of Solomon Islands individuals, one harboured exclusively Fijian P. leopardus, and the remaining group contained individuals from the Torres Strait, Great Barrier Reef and New Caledonia. The DAPC analysis of microsatellites  and almost all sites were partitioned, except Torres Strait grouped with the Capricorn Bunker Reefs, Cebu grouped with Taiwan, and Scott Reef grouped with Palau , with almost all sites being partitioned as before, except that three northern Great Barrier Reef sites  formed a single group  and were statistically significant (P < 0.001)  and between Palau and the Great Barrier Reef (microsatellites), as pairwise F-statistics between sites on opposite side of this break were higher  than those on the same side . Both Tajima’s D  and Fu’s FS  were negative but not significant in the Eastern group, while Fu’s FS was significantly negative in the Western group , Tajima’s D was not .The S in many of the grouper lineages from this study. The emergence of multiple land barriers including the IPB at the Sahul and Sunda Shelves [E. polyphekadion and P. areolatus based on the mid-Pleistocene divergence estimated by conventional molecular clock methods (1.41–0.16 MYA). Moreover, the IPB appears to present a dispersal boundary for P. leopardus such that the barrier might have resulted in the divergence of P. leopardus from its sister species P. pessuliferus , which largely inhabited the west to central Indian Ocean, in the Pleistocene [This study identifies phylogeographic signatures of Pleistocene glacial cycles in all three species, with genetic breaks generally coinciding with major biogeographic boundaries - the most prominent one being the IPB see Fig. . As shal Shelves  was one  Shelves , leading Shelves , 62, gob Shelves , angelfi Shelves , gastrop Shelves , prawns  Shelves  and mult Shelves , 67. Theistocene .P. areolatus, causing the divergence of the RS lineage at about 0.63 MYA (mid-Pleistocene) based on the conventional molecular clock. Previous comparative phylogeographic work that focused on examining this barrier found genetic differentiation between the RS and Western Indian Ocean populations in five out of seven reef fish species that exhibit a wide spectrum of biological traits [P. areolatus (this study) and in other fauna [At the western periphery of the Indo-Pacific, the RSB exerted a profound effect on l traits . Similarl traits , crab [6l traits , and stal traits , but notl traits . The RS l traits , 73. Sucer fauna .Cephalopholis argus [E. polyphekadion or P. areolatus. One likely explanation is that the extensive separation (> 2000 km) of suitable coral habitats and the relative absence of stepping stones in the deep ocean (> 200 m) for dispersal between the WIOI and IP, and between RS and WIOI , we obssee Fig.  present E. polyphekadion, which has lower genetic diversity  than other populations. Multiple factors, including geographic isolation, oceanographic currents that influence gene flow, and selection pressure in distinct environmental conditions, may play a role in creating the genetic differentiation in this marginal population of E. polyphekadion [At the eastern boundary of the species’ range (Tuamotus of French Polynesia), we detected subtle but significant isolation of the French Polynesian hekadion , 80. In hekadion , the brohekadion , and thehekadion .P. leopardus in the WIP, with the deepest divergence at the Sahul Shelf , suggesting the possible contribution of the Pleistocene glaciation that resulted in the recurrent emergence of the Sahul shelf land bridge. The Sahul shelf land bridge has been documented as a barrier associated with genetic differentiation for several other tropical marine species, from starfish [P. leopardus  threespot dascyllus species complex, where historical allopatry developed during periods of isolation and subsequent ecological factors facilitated divergence and speciation of these species [Our results revealed multiple genetically differentiated populations of elf Fig. . While tstarfish  to sea sstarfish . Though proaches . This waE. polyphekadion and P. areolatus. In E. polyphekadion, the CK population groups with the IP population in all analyses . However, in P. areolatus, the CK population has a close affinity with the Maldives population in the WIOI, based on the mitochondrial control region, but it is more closely related, albeit also genetically distinct from, the IP populations based on FST, SAMOVA and DAPC analyses of microsatellite data. However, STRUCTURE results were congruent with mitochondrial data. While we cannot fully account for the phylogeographic pattern observed, the genetic differentiation inferred from microsatellite analyses of both species essentially reflects the relative geographic distances between the Maldives, CK and Western Australia, and, more importantly, implies that the CK population of both species was in effect isolated from the majority of CIP and WIP stocks at an ecological time scale.Notably, the affinity of the CK population, which is located at the WIP-CIP boundary, is different between P. areolatus WAPO lineage from the rest was much more recent (late Pleistocene) than divergence of the P. leopardus East and West lineages (early Pleistocene), which might partly explain the weak population structure observed in the former species. Nonetheless, length of evolutionary history alone cannot explain the similarly weak population structure observed in E. polyphekadion, whose WIP lineage also emerged in the early Pleistocene. The much lower dispersal ability of P. leopardus in comparison to the other species is the most probable explanation for the disparity observed. In fact, the smaller distribution range of P. leopardus might be an indication that this species is less capable of long distance dispersal.A number of features could have, perhaps in concert, resulted in the drastically different phylogeographic patterns observed in the three grouper species. A longer evolutionary history could have allowed population differentiation to build up and intensify the genetic signal. The divergence of the E. polyphekadion and P. areolatus in unexploited or lightly exploited areas can involve thousands of reproductive adults at fewer sites, with spawning occurring in reef passages adjacent to the open ocean around the full or new moon periods. In contrast, P. leopardus FSAs usually consist of a few hundred adults [P. leopardus aggregations generate an order of magnitude smaller and much more spatially distributed series of gamete source pulses within an area. Given that the three species have similar PLDs, and that monthly spawning events for all three species are similarly brief, often during times when tidal flows are greatest (full and/or new moon phases), one might expect the immense number of gametes released by E. polyphekadion and P. areolatus FSAs to have a greater probability of long-distance dispersal due to oceanography compared to the lower number of propagules released in dispersed smaller pulses . Moreover, E. polyphekadion and P. areolatus aggregate next to the open ocean where currents can potentially carry gametes long distances from the aggregation area, which contrasts with the inshore/reef platform spawning locations of P. leopardus. The dispersal of a few concentrated larger cohorts could potentially be more strongly influenced by meso-scale oceanographic events than multiple, scattered, smaller cohorts, which would probably be more affected by short-term localized events.The patterns of reproductive output per spawning event at each FSA differ substantially among the three species studied, leading to possible differences in connectivity linked to larval dispersal. Spawning aggregations of d adults  at multid adults . Hence, P. areolatus spawn over multiple months while E. polyphekadion and P. leopardus only spawn for one or two months each year, we might predict wider overall dispersal in P. areolatus than in the other two species if oceanographic conditions vary across seasons and thereby spread offspring more widely. More/less extensive larval dispersal may be linked to weaker/stronger population genetic structure, respectively as has been documented in many terrestrial and aquatic (including marine) species [per se, is not a good predictor of population genetic structure [E. polyphekadion and P. areolatus.On the other hand, given that  species , 85. Howtructure  or rangetructure  in the mEpinephelus guttatus and the non-aggregating Cephalopholis fulva [C. fulva is sedentary, spawning over large areas of the shallow reef platform, and may maintain the same territory, forming multiple small mating groups over multiple months in the year [E. guttatus, which aggregates over just a few months each year and at the shelf drop off close to open ocean, may join other aggregations in subsequent (unexamined) years, thereby homogenizing the gene pool, given that E. guttatus can migrate more than 30 km to spawning sites. This proposal, however, does not consider pelagic larval distribution and recruitment as an alternative avenue of connectivity. Consistent with this finding, E. striatus, which forms large spatially and temporally restricted spawning aggregations like E. guttatus in the Caribbean, was also found to exhibit weak population genetic structure across a long distance (~ 1600 km) [Few other studies have examined population connectivity in groupers and considered the possible effect of reproductive mode with similar outcomes to our study. Portnoy et al. tested the hypothesis that aggregative spawning behaviour affects gene flow by examining population genetic structure and connectivity of two groupers: the aggregate spawning is fulva . Over a the year . Additio1600 km) . Results1600 km) .P. leopardus and C. fulva). Reduced population structuring is evident among species that assemble in large numbers at relatively few spawning sites at the shelf edge close to the open ocean  , fully supporting our approach. Nonetheless, further studies on reproductive biology and spawning behaviour of these species are encouraged to advance our understanding of factors shaping population connectivity. Such information could also provide important information for fishery management frameworks for these economically important reef fishes [P. maculatus [P. areolatus [In summary, stronger genetic structuring appears to be closely associated with species that form multiple small spawning groups away from the shelf edge , such sE. polyphekadion and P. areolatus spawning aggregations in Palau, Fiji, Pohnpei, French Polynesia and Indonesia for example, (www.scrfa.org). Our study species are listed as threatened or near-threatened on the IUCN Red List, largely as a result of uncontrolled aggregation-fishing [Groupers are highly sought after and many are also often intrinsically vulnerable to fishing pressure because of their longevity, late sexual maturation, high site fidelity, complex social structure and, for some species, protogynous hermaphroditism –97. As m-fishing . Better -fishing .E. polyphekadion and P. areolatus would need larger MPAs and regional scale management than a species like P. leopardus. Conversely, other management methods may be more practical for the former two species such as seasonal protection during spawning complemented by seasonal sales bans because enforcement may be particularly challenging for offshore sites [P. maculatus [P. areolatus, [The results of this study highlight the importance of population structuring in determining possible management units and the spatial scale that management would need to consider. For example, species with large catchment areas like re sites . Howeveraculatus , and P. eolatus, ). Taken P. leopardus exhibited markedly stronger population genetic structure at various geographic scales within the CIP than the other two groupers, while E. polyphekadion displayed only slightly stronger population structuring than P. areolatus. The populations in the CIP were generally highly connected in the latter two species, and a significant genetic break only occurred among ocean basins. Results from this study and a comparable study in the Caribbean both suggested that stronger genetic structuring appeared to be closely associated with species that form multiple small spawning groups on the shelf platform and away from the open ocean. Our results highlighted the need for more investigations on this characteristic and the need to consider reproductive mode in studies of connectivity and population genetics as well as in more sustainable fisheries management.This study identified phylogeographic signatures of Pleistocene glacial cycles in all three species, with genetic breaks generally coinciding with major biogeographic boundaries, while species-specific reproductive traits determine the magnitude of these phylogeographic signals. As hypothesized, Additional file 1:Table S1. Primers used for PCR amplification of control region sequences. Table S2-S8. Basic information and genetic diversity of control region and microsatellite datasets. Table S9. SAMOVA results. Table S10-S15. Pairwise ΦST and FST of control region and microsatellite datasets. Table S16. Results of linkage disequilibrium test. Figure S1-S6. Results of DAPC analyses. Figure S7. Summary of population genetic structure and reproductive characteristics of five grouper species analysed in this study and Portnoy et al. (2013). (PDF 4110 kb)Additional details of sampling, methodology, and results:


Zizania latifolia, domesticated as the special form of a plant-fungus (the host Zizania latifolia and the endophyte Ustilago esculenta) complex, is a popular aquatic vegetable endemic in East Asia. The rapid domestication of cultivated Z. latifolia can be traced in the historical literature but still need more evidence. This study focused on deciphering the genetic relationship between wild and cultivated Z. latifolia, as well as the corresponding parasitic U. esculenta. Twelve microsatellites markers were used to study the genetic variations of 32 wild populations and 135 landraces of Z. latifolia. Model simulations based on approximate Bayesian computation (ABC) were then performed to hierarchically infer the population history. We also analyzed the ITS sequences of the smut fungus U. esculenta to reveal its genetic structure. Our results indicated a significant genetic divergence between cultivated Z. latifolia and its wild ancestors. The wild Z. latifolia populations showed significant hierarchical genetic subdivisions, which may be attributed to the joint effect of isolation by distance and hydrological unconnectedness between watersheds. Cultivated Z. latifolia was supposedly domesticated once in the low reaches of the Yangtze River. The genetic structure of U. esculenta also indicated a single domestication event, and the genetic variations in this fungus might be associated with the diversification of cultivars. These findings provided molecular evidence in accordance with the historical literature that addressed the domestication of cultivated Z. latifolia involved adaptive evolution driven by artificial selection in both the plant and fungus.Crop domestication is one of the essential topics in evolutionary biology. Cultivated  Cultivated plants are fundamental materials for ensuring the survival and development of human beings . ThroughZea mays) had been domesticated once from its ancestor species teosinte (Zea mexicana) in Mexico (reviewed in Phaseolus vulgaris) had more than two independent domestication events (reviewed in Oryza sativa)    and a fe sativa) . Moreove sativa) . TherefoZizania belongs to the rice tribe , and it is an aquatic genus with a discontinuous distribution between eastern Asia and North America or enlarged young shoots as vegetable   should be screened out for clonal propagation to ensure the stability of agronomic traits of offspring (The genus  America . Of the to Asia) . Becauseer Basin . The culck spots . Once Z.ffspring . The plaffspring . To dateZ. latifolia in China (Z. latifolia had been harvested to offer as tribute to the nobles in the Zhou Dynasty (from 771 to 221 BC). Until the Tang Dynasty (618–907 AD), Z. latifolia was famous for the tender taste of its grains. During the period between the Zhou and Tang Dynasties, the swollen shoots were rarely reported to be used for food. Only in the low reaches of the Yangtze River was it occasionally eaten by local residents. After the Tang Dynasty, people no longer harvested the grains of Z. latifolia. Alternatively, Z. latifolia was domesticated to be an aquatic vegetable  were then performed to infer the domestication history. The pattern of genetic variations for U. esculenta was identified based on ITS sequence analyses. By combining the genetic structures of host and endophyte, the domestication scenario of cultivated Z. latifolia could be unveiled.This study focused on dissecting the genetic structure of Z. latifolia were collected across the species distribution range in China  and 16 Z. latifolia specific microsatellites . All ITS sequences were deposited into GeneBank, with accession numbers MK811211-MK811247.Fst based on the data of all loci compared to the estimate based on corrected genotype data for null alleles . The parameters of population genetic variations were estimated by expected heterozygosity (He), observed heterozygosity (Ho) and Wright’s fixation index F using GENALEX 6.5 (Ae) and allelic richness (Ar). Hardy–Weinberg equilibrium departures were tested using exact tests implemented in GENEPOP v4.0 for each population–locus combination. A global test across loci for departures from Hardy–Weinberg equilibrium was conducted using Fisher’s method. Significant deviations were further evaluated using the sequential Bonferroni test.The frequencies of putative null alleles were estimated using the software FREENA . Null alALEX 6.5 . We alsoNe generations or in a recent period. A significant heterozygote excess or deficit due to a bottleneck was assessed by a two-tailed Wilcoxon signed rank test with 1,000 iterations under the assumption of a stepwise mutation model. At the same time, the distribution of allelic frequencies was drawn to identify whether there had been a mode shift away from an L-shaped distribution, indicating recent population bottlenecks.BOTTLENECK  was usedNe. MIGRATE uses coalescent theory to jointly estimate the mutation scaled population size θ (4Neµ) over a long period of time (∼4Ne generations). Three runs were conducted using MIGRATE. First, two shorter runs  were performed and used to verify that the MCMC estimated the parameters correctly. Then, a final run  was performed, and θ values from this final run are reported. The initial run used an estimate of Fst as a starting parameter to calculate θ. Each subsequent run used the ML estimates from the previous run as new starting parameters.MIGRATE  was usedZ. latifolia plants. The genetic relationship between the cultivated and wild plants was also estimated using STRUCTURE 2.3.4 (K = 2 and following the method described by Q) from the cultivated group was >50%, we defined that finding as a sign of cultivar ferality.Principal coordinate analysis (PCoA), as implemented in GENALEX 6.5, was conducted to investigate the genetic divergence between cultivated and wild RE 2.3.4  with K =Fst with GENEPOP 4.0. A PCoA was performed with the wild populations only to investigate the pattern of genetic divergence within this species. The genetic structure was further explored using the Bayesian clustering algorithm implemented in STRUCTURE 2.3.4. The program was given no prior information on ancestral populations and run 10 times for each value of K ancestral populations, under the admixture model with correlated allele frequencies, using 200,000 Markov chain Monte Carlo iterations and a burn-in of 100,000 iterations. We inferred K using the ad hoc statistic ΔK  matrix for all pairs of individuals and populations of wild  MSA 3.0 . UnrooteIsolation by distance (IBD) among the wild populations was tested using the Mantel test implemented in GENALEX 6.5. The significance of IBD values was assessed using 9,999 permutations.Z. latifolia using Approximate Bayesian Computing (ABC) . To reduce the complexity of scenario settings, hierarchical methods were applied following We tested possible models of the demographic history of wild and cultivated accessions to infer the domestication history of cultivated ng (ABC) , which ang (ABC)  was usedWe then used an ABC approach to infer demographical parameters for the best scenario. The prior parameters and designs of the scenarios are given in S), haplotype diversity (Hd), and genetic variation measured by average pairwise differences per base pair between sequences (θ) (θw) from S   and Watt) from S , were ca) from S . Maximum) from S . Gaps weZ. latifolia accessions yielded a total of 138 alleles with 11.46 alleles per locus on average. The mean effective number of alleles (A), allelic richness (Ar), observed and expected heterozygosity (Ho and He), and Wright’s fixation index (F) are listed in He ranged from 0.167 (JXDA) to 0.588 (JSBY) among wild populations. The genetic diversity of cultivated accessions (He = 0.242) was much lower than that of wild accessions (He = 0.432). Deviations from Hardy-Weinberg equilibrium were detected in 90 of 384 comparisons after applying the sequential Bonferroni test, which might be due to inbreeding. The Wright’s fixation indices varied greatly among populations . The outcomes of a Bayesian assignment with STRUCTURE indicated that K = 2 is the best for describing the actual genetic structure of wild populations , K = 2. Instead, the PCoA revealed the genetic differences between ecotypes  was 0.991. The nucleotide diversity (θ) was 0.01, and θw was 0.01.A total of 37 strains of U. alcornii was downloaded from GeneBank and used as the outgroup taxon. Another ITS sequence of U. esculenta (Genebank accession number: JX219372.1) was used as the control. The MP tree is shown in An ITS sequence of Z. latifolia is a typical example that can demonstrate how a plant-fungus complex rapidly evolved under artificial selections. Our results showed that there was prominent genetic divergence between wild populations and cultivated accessions after long-time cultivation. A single domestication event occurred in the lower reaches of the Yangtze River at the Tang Dynasty , then this vegetable was constantly cultivated and bred. The genetic divergence of the plant may be responsible for the differences between the ecotypes of cultivars, and the genetic variations in U. esculenta are likely associated with the diversification of cultivated varieties.The domestication of cultivated Z. latifolia. Compared with its wild ancestor, the cultivated plant usually has an erected plant architecture, broader leaves, and shortened internodes  was even greater than the differentiation among wild populations (Fst = 0.175). This phenomenon is usually observed in highly domesticated species such as wheat, rice or maize  should result in low genetic divergence between wild and cultivated plants, which is contrary to our finding. The unexpected high genetic divergence between wild and cultivated plants could also be attributed to the breeding system of cultivated Z. latifolia. Because cultivated Z. latifolia would occasionally degenerate to the wild phenotype, farmers had to screen out the elite plants as genets every year. Although the clonal reproduction of cultivated Z. latifolia greatly restricted new mutants stemmed from sexual propagation and blocked gene flows from wild accessions to cultivars, the annually strong artificial selection would somehow accumulate mutants, inducing significant genetic divergence between wild and cultivated plants.We found significant genetic divergence between wild and cultivated or maize . Unlike Z. latifolia may be domesticated in the Yangtze River Basin, but they did not provide any convincing evidence. . However, they might have underestimated the genetic diversity of both species due to insufficient genetic markers. In contrast, another study investigated the genetic variations of Z. latifolia restricted to the middle reaches of the Yangtze River by using 10 microsatellites and found a high level of genetic diversity (He = 0.532) (He = 0.497) was detected.= 0.532) . In our F) for the high-latitude populations were significantly greater than 0 . Previous phylogeographic analysis suggested that the genus Zizania originated in North America and then dispersed into eastern Asia via the Bering land bridge during the Tertiary (Z. latifolia indicated that high-latitude populations harbored more abundant haplotypes (He = 0.447) did not show a higher genetic diversity than those in low latitudes .Most of the fixation indices .The genetic assignments and morphological performances of two populations (GDYJ and GXNN) at the leading-edge seemed to be rather special , which implies a weak competitive ability of the infected plants.As an obligate parasitic fungus, Z. latifolia seemed to accelerate the process of achieving symbiosis. On the one hand, the persistent selection force of mankind delayed the development of the fungus and extended the mycolic stage  and the China Postdoctoral Science Foundation Grant (2015M571483).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Several C/EBPβ putative binding sites were previously predicted on the STAT3 promoter in mammals. However, the molecular regulation of C/EBPβ on STAT3 and their effects on cell proliferation and apoptosis remain virtually unexplored in GCs. Using porcine GCs as a model, the 5′-deletion, luciferase report assay, mutation, chromatin immunoprecipitation, Annexin-V/PI staining and EdU assays were applied to investigate the molecular mechanism for C/EBPβ regulating the expression of STAT3 and their effects on the cell proliferation and apoptosis ability. We found that over and interfering with the expression of C/EBPβ significantly increased and decreased the messenger RNA (mRNA) and protein levels of STAT3, respectively. The dual luciferase reporter assay showed that C/EBPβ directly bound at −1397/−1387 of STAT3 to positively regulate the mRNA and protein expressions of STAT3. Both C/EBPβ and STAT3 were observed to inhibit cell apoptosis and promote cell proliferation. Furthermore, C/EBPβ might enhance the antiapoptotic and pro-proliferative effects of STAT3. These results would be of great insight in further exploring the molecular mechanism of C/EBPβ and STAT3 on the function of GCs and the development of ovarian follicles in mammals.Previous studies suggest that signal transducer and activator of transcription 3  Ovarian dysfunction causes the reproductive failure and infertility in female mammals. The main functions of ovaries are to produce mature oocytes that can propagate the species ,2 and sySTAT3) protein has been suggested to be involved in folliculogenesis [STAT3 expressed highly in porcine GCs [STAT3 is 93% and 90% homologous to humans and mice, respectively [STAT3 expresses higher in adult ovaries than in fetal ovaries [STAT3 is activated by the epidermal growth factor [P450scc and follicle-stimulating hormone receptor mRNA abundance to regulate the biological functions of GCs [STAT3 enhances the early apoptosis rate of mGCs [STAT3 in ovarian GCs can impair fertility with significantly fewer litters and smaller litter size [STAT3 is seldom investigated in porcine GCs.In mammals, the signal transducer and activator of transcription 3  plays an essential role in female reproduction [C/EBPβ mRNA is specifically and rapidly induced by luteinizing hormone in GCs [C/EBPβ results in reproductive defects in female mice [C/EBPβ knockout causes subfertility with the absence of corpus luteums in 70% and the reduction expression of Ptgs2, Star, and Cyp11a1 in mice [C/EBPβ in GCs for mammalian folliculogenesis. However, the functions of C/EBPβ on cell apoptosis and proliferation remained virtually unexplored for ovarian GCs.Much evidence has suggested that CCAAT/enhancer binding protein beta . We hypothesized that C/EBPβ might play a cis-acting regulatory role in transcription of STAT3 and thus regulate the function of GCs in mammals. In this study, using porcine GCs as a model, the molecular mechanism regarding the regulation between C/EBPβ and STAT3 was first identified, and then its biological functions were explored for cell apoptosis and proliferation.Previous studies report that  rat GCs , and morAll experiments in the present study were performed in accordance with the guidelines of the Animal Care and Use Committee of South China Agricultural University Guangzhou, China .STAT3 (upstream 2 kb) were download from NCBI for human [C/EBPβ. The putative binding sites of C/EBPβ concurrently predicted by all of those four tools were used for further analysis. The putative binding sites of C/EBPβ on the promoter of STAT3 in humans, mice, and pigs are shown in The promoter sequences of or human , mouse [or human  and pig or human . TFBIND or human , Biobaseor human , Jaspar or human  and Reseor human  were use®  high fidelity enzyme to obtain the STAT3 promoter of 2575 bp. Primers are presented in STAT3. Then we used T-STAT3 as a template and designed five different upstream primers to amplify deletion fragments. The longest deletion fragment was named P1. The location of each deletion fragment of STAT3 was P1 (−2199/+375), P2 (−1532/+375), P3 (−1035/+375), P4 (−587/+375) and P5 (−167/+375). We used the same method to obtain plasmids of each deletion fragment containing MIuI and XhoI cleavage sites  and previous study [The genomic DNA of porcine ovary tissues was used as a template. PCR was performed using PrimerSTARge sites . Simultaus study , we use 2 flasks and cultured at 37 °C under 5% CO2 in DMEM  containing 10% fetal bovine serum , 100 IU/mL penicillin, and 100 μg/mL streptomycin.Porcine ovarian GCs were cultured as previously described . BrieflySTAT3 is presented in GCs were cross-linked until the cell density reached 70%. Then we discarded the original culture medium in the flask and the cells were sequentially treated with formaldehyde, glycine, PBS-Halt Cocktail and centrifuged to collect the cell pellet. The chromatin immunoprecipitation (ChIP) assay  was carried out according to the previous ChIP protocol . ChIP prGAPDH mRNAs were used as endogenous controls, and the fold changes of STAT3 and C/EBPβ were calculated using the 2−ΔΔct method. The primer sequences are listed in At least three wells per group were collected for extraction of total RNA. Total RNA was extracted using TRIzol reagent  and then reverse-transcribed using a RevertAid First Strand cDNA Synthesis Kit  for mRNAs. The relative expression levels of mRNAs were quantified using Maxima SYBR Green qRT-PCR Master Mix (2×)  and THUNDERBIRD SYBR qPCR Mix  in a LightCycler Real-Time PCR system. The expression levels of Cell proliferation assays were performed using Cell-Light Edu Apollo 567 In Vitro Kit . GCs were seeded into 48-well plates at one day prior to transfection. When the cells reached 30–50% coverage of one well, pcDNA3.1-C/EBPβ, pcDNA3.1-STAT3, pcDNA3.1-Control, C/EBPβ-siRNA, STAT3-siRNA or siRNA-NC were transfected into the cells at different final concentrations for 48 h. The specific steps are: the Edu solution was diluted 1:1000 with cell culture media to prepare 50 μM Edu medium, add 100 μL of 50 μM Edu media to each well for 2 h, discard the culture medium, add 100 μL of cell fixing solution (80% acetone) to each well for 30 min at room temperature, wash twice with PBS, add 100 μL of penetrant (0.5% TritonX-100 in PBS) to permeabilize the cells and rinse once with PBS, add 100 μL of 1 × Apollo Staining Solution and incubate for 30 min at room temperature in the dark, discard staining solution and add 100 μL DAPI per well incubate for 30 min at room temperature in the dark, then add PBS to take pictures under microscope.5 cells/well) were cultured in triplicate in 6-well plates at one day prior to transfection. When the cells reached 30–50% coverage of one well, pcDNA3.1-C/EBPβ, pcDNA3.1-STAT3, pcDNA3.1-Control, C/EBPβ-siRNA, STAT3-siRNA or siRNA-NC were transfected into the cells at different final concentrations for 48 h. The cells were then harvested, washed twice with ice-cold PBS, and resuspended in 500 μL of binding buffer. Next, 1.25 μL of Annexin V-FITC was added in the dark for 15 min at room temperature, then 1000× g centrifugation for 5 min at room temperature to remove the supernatant. The cells were gently resuspended with 0.5 mL precooling 1 × solution, and 10 μL of PI  were added. Last, the cells were analyzed in a flow cytometer  using the FITC signal detector (FL1) and phycoerythrin emission signal detector (FL2). All experiments were performed at least three times. Cells in the lower right quadrant are annexin-positive/PI-negative early apoptotic cells. The cells in the upper right quadrant are annexin-positive/PI-positive late apoptotic cells.Cell apoptosis assays were performed using an Annexin V-FITC Apoptosis Detection Kit  according to the manufacturer’s instructions. Briefly, GCs . The molecular weight of STAT3 was 91 kDa. Protein concentrations were determined using a BCA Protein Assay Kit , and equal amounts of protein were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk in PBS containing a percentage of Tween-20 for 1 h, and then incubated with a primary antibody against hamartin  overnight at 4 °C. An anti-GAPDH antibody  was used as an internal control. After incubation with secondary antibodies for 1 h at room temperature, antibody-bound protein bands were visualized using an ECL-PLUS Kit . The gray scale values of the bands were calculated using ImageJ software, which was free downloaded from NIH. The relative protein expression level of STAT3 was normalized by β-Actin values. At least three replicates were conducted for each group.The cells were harvested and analyzed for their expression levels of total t-test (two-tailed). * indicates p < 0.05; ** indicates p < 0.01.All experiments were repeated at least three times independently. Data were expressed as means ± standard deviation (SD) of repeated experiments. Statistical analyses were carried out using R software. The significance of differences in means between two groups was analyzed by using Student’s C/EBPβ were first built to explore the effects of C/EBPβ on the expression of STAT3  and protein (p < 0.05) levels of STAT3. Three C/EBPβ-specific small interfering RNA (siRNA)  and a negative control (siRNA-NC) were transfected into GCs to evaluate the inhibition efficiency for C/EBPβ  and protein (p < 0.05) levels of STAT3. These observations indicated that C/EBPβ could positively regulate the mRNA and protein amounts of STAT3 in porcine GCs.The overexpression plasmid and small interfering RNA (siRNA) of of STAT3 . We foun1-C/EBPβ A, and thr C/EBPβ D. As shoC/EBPβ were predicted on the promoter of STAT3 in pigs  in porcine GCs (p < 0.01). Moreover, pcDNA3.1-C/EBPβ significantly increased the relative luciferase of P2 (p < 0.01). These results suggested that C/EBPβ directly bound at −1397/−1387 of STAT3 to positively regulate the transcription of STAT3 in porcine GCs.To validate reporter C. We foureporter D. Moreovcine GCs E, and siSTAT3 on the apoptosis and proliferation, pcDNA3.1-STAT3 or STAT3-siRNA was transfected into porcine GCs  was significantly lower than group 2 (pcDNA3.1-C/EBPβ + STAT3-siRNA), and group 3 (pcDNA3.1-STAT3 + C/EBPβ-siRNA) was significantly lower than group 4 (STAT3-siRNA + C/EBPβ-siRNA). These results indicated that C/EBPβ might enhance the antiapoptotic effect of STAT3 in ovarian GCs. For the proliferation rate of porcine GCs (C/EBPβ + pcDNA3.1-STAT3) was significantly higher than group 2 (pcDNA3.1-C/EBPβ + STAT3-siRNA), while group 3 (pcDNA3.1-STAT3 + C/EBPβ-siRNA) was significantly higher than group 4 (STAT3-siRNA + C/EBPβ-siRNA). These results suggested that C/EBPβ might enhance pro-proliferation effects of STAT3 in ovarian GCs.PcDNA3.1-ectively . For theectively A, group cine GCs B, group STAT3 knockout could impair the fertility with a significant reduction in litters and litter sizes [STAT3 knockout had no effect on fertility [C/EBPβ knockout also resulted in the subfertility with a reduction of corpus luteums in 70% [STAT3 and C/EBPβ had exhibited an essential role in folliculogenesis and female reproduction. Previous studies recommended that C/EBPβ might display a cis-regulatory role in mammalian GCs [STAT3 in humans, mice, and pigs (STAT3 had significantly increased or decreased along with the overexpression or depression of C/EBPβ (C/EBPβ was likely to play a positive regulatory role for STAT3 in porcine GCs.It is widely known that the regular cell apoptosis and proliferation of GCs supported the development of follicles in mammals ,6. In mier sizes , but oocertility . Moreoves in 70% . These rlian GCs ,32. We pand pigs , and furf C/EBPβ . This obC/EBPβ binding sites were predicted at the promoter of STAT3 (STAT3 might be a direct target of C/EBPβ. Based on the dual luciferase reporter system, the deletion of −1397/−1387 binding sites resulted in a significant reduction of the relative luciferase activity. This observation was in accordance with results shown in C/EBPβ was further confirmed to bind at −1397/−1387 of STAT3 by ChIP (C/EBPβ directly targeted at STAT3 to positively regulate the expression of STAT3 in porcine GCs.In pigs, four putative of STAT3 A, sugges by ChIP E. These STAT3 in GCs may be related to ovarian leptin resistance and decreased fertilization in polycystic ovarian syndrome women [STAT3 is required to regulate the expression of luteinizting hormone receptor mRNA during the cell differentiation of GCs in mice [STAT3 appears to be stronger in the ovulatory follicles as compared to the small follicles and dominant follicles, and these results mean that STAT3 in GCs may be associated with the follicular growth [STAT3 got involved in the functions of GCs and follicular development. Most importantly, in human GCs, promoting ovarian GCs apoptosis and affecting the steroidogenesis could suppress the activation of STAT3 [STAT3 could enhance the early apoptosis rate of mouse GCs [STAT3 might induce the apoptosis of mammalian GCs. In the present study, we found that STAT3 inhibited cell apoptosis and promotes cell proliferation in porcine GCs (STAT3 on cell apoptosis and cell proliferation in mammals. Previous studies have found that lower levels of phosphorylated me women . STAT3 i in mice . In bovir growth , and phor growth . Furtherr growth . These oof STAT3 , and redouse GCs . These rcine GCs , and theC/EBPβ is essential for ovulation and luteinization [C/EBPβ double-mutant could change expressions of Prlr, Abcb1b1, Plxnd1 and Cyp19a1 in mouse GCs, and moreover, putative C/EBPβ binding sites have been identified in the promoters of these genes [C/EBPβ knockdown could alter Star, Cyp11a1, and Hsd3b2 expressions in granulosa tumour-derived KGN cells [Runx2 mRNA levels in periovulatory GC of mice and thus might impact cell differentiation of GCs to become luteal cells [C/EBPβ was an important transcript factor in mammalian GCs. Moreover, by binding at the promoter, C/EBPβ was likely to be essentially required for Star, which was a vital accessory protein required for biosynthesis of steroid hormones from cholesterol, in response to hormones in rat [C/EBPβ inhibited cell apoptosis and promoted cell proliferation in porcine GCs (C/EBPβ enhanced the antiapoptotic and pro-proliferation effects of STAT3 in porcine GCs (STAT3 was a target of C/EBPβ in mammalian GCs. Collectively, in porcine ovarian GCs, C/EBPβ targeted at −1397/−1387 region to promote the expression of STAT3, and moreover, C/EBPβ might enhance the antiapoptotic and pro-proliferative effects of STAT3. These present works could provide usefully biological information for further studies on the molecular mechanism of C/EBPβ and STAT3 in ovarian follicular development.nization ,40. Prevse genes . C/EBPβ GN cells  and resual cells . These os in rat , mouse [s in rat , human [s in rat , and pors in rat  GCs. In cine GCs . We alsocine GCs . These r


Asellus aquaticus, using a genome‐wide SNP approach (Genotyping‐by‐sequencing). It was hypothesized that genetic diversity in A. aquaticus would be positively correlated with dose rate. A. aquaticus was collected from six lakes in Belarus and the Ukraine ranging in dose rate from 0.064 to 27.1 µGy/hr. Genotyping‐by‐sequencing analysis was performed on 74 individuals. A significant relationship between geographical distance and genetic differentiation confirmed the Isolation‐by‐Distance model. Conversely, no significant relationship between dose rate and genetic differentiation suggested no effect of the contamination gradient on genetic differentiation between populations. No significant relationship between five measures of genetic diversity and dose rate was recorded, suggesting that radiation exposure has not significantly influenced genetic diversity in A. aquaticus at Chernobyl. This is the first study to adopt a genome‐wide SNP approach to assess the impacts of environmental radiation exposure on biota. These findings are fundamental to understanding the long‐term success of aquatic populations in contaminated environments at Chernobyl and Fukushima.Analysis of genetic diversity represents a fundamental component of ecological risk assessments in contaminated environments. Many studies have assessed the genetic implications of chronic radiation exposure at Chernobyl, generally recording an elevated genetic diversity and mutation rate in rodents, plants, and birds inhabiting contaminated areas. Only limited studies have considered genetic diversity in aquatic biota at Chernobyl, despite the large number of freshwater systems where elevated dose rates will persist for many years. Consequently, the present study aimed to assess the effects of chronic radiation exposure on genetic diversity in the freshwater crustacean,  This study utilized a genotyping by sequencing method to provide a novel assessment of the impacts of chronic radiation exposure at Chernobyl on a freshwater crustacean. No significant impact of over 30 years of radiation exposure on genetic variation was recorded. These findings are fundamental to understanding the long‐term success of aquatic populations in contaminated environments at Chernobyl and Fukushima. The authors recorded increased genetic diversity in G. affinis individuals inhabiting two contaminated ponds  as opposed to individuals from two reference populations. However, one of the aforementioned sites is also heavily contaminated with a suite of other genotoxicants  is a simple, cost‐effective reduced representation method for assessing a vast number of SNPs across the genome  eight reference animals and plants (RAPs), meaning these organisms will be used to support the evolving system for environmental radioprotection  was assessed following Giska et al., , sorted lakeside and immediately preserved in 96% Ethanol. A total of 455 A. aquaticus individuals were collected across all sites during the sampling period. Numbers of A. aquaticus individuals sequenced are shown in Section Field sampling was conducted in May‐June of 2016 at six lakes in Belarus and Ukraine . Sampling dates and environmental conditions are shown in Table 2.3A. aquaticus is presented in Fuller et al.,  and average activity concentrations in water at various depths measured in 2003. Accurate dose conversion coefficients (DCCs) for A. aquaticus were generated within the ERICA tool (Version 1.2) by user‐inputted geometry  and used for subsequent dose assessments using ERICA. Total dose rates for A. aquaticus at each of the six lakes are shown in Table A full description of the methods used to calculate doses to  et al., . Brieflyntium in 986 and a2.4A. aquaticus individuals using a QIAGEN DNeasy Blood & Tissue Kit (QIAGEN) following some amendments to the manufacturer protocol to maximize DNA yield. Individuals were first homogenized in 180 µl of Buffer ATL. Following addition of 40 µl Proteinase K, samples were incubated at 56°C for 16 hr to allow for lysis to occur. After lysis, samples were centrifuged at 20,000 g for 5 min and the supernatant transferred to a new microcentrifuge tube. This step was performed to remove the nonlysed, largely chitinous tissue as this may impact the quality of DNA for downstream applications in A. aquaticus  were performed on 10% of DNA samples to assess suitability for genomic library construction. Digests were performed according to manufacturer protocol and visualized on a 1% agarose gel.DNA was extracted from a total of 74 2.5A full description of GBS methods is provided in Elshire et al., . Briefly2.6Bioinformatics analysis was conducted by collaborators at the Genomic Diversity Facility using the UNEAK  are shown in Table Fst values >0.15 are generally considered to indicate significant differentiation , the two closest sites geographically. A highly significant relationship between geographical distance in kilometers and genetic differentiation was recorded , confirming the Isolation‐by‐distance hypothesis.Genetic differentiation values . This indicates that dose rate did not significantly influence genetic differentiation among populations at Chernobyl. However, geographical distance was found to be a driver of genetic differentiation. A principal component analysis (PCA) plot of genotypes based on genome‐wide filtered SNP data is shown in Figure Conversely, no significant effect of the gradient in dose rate (Euclidean distance) on genetic differentiation was recorded when accounting for geographical distance , the most commonly used measure of gene diversity . Nucleotide diversity (π), a measure of polymorphism within the population, ranged from 0.0016 at Svatoye to 0.0026 at Gorova. No significant relationship between nucleotide diversity and dose rate was recorded .Calculated genetic diversity statistics for each population are shown in Table ity Nei, , ranged Ho) or the within population inbreeding coefficient, F, was recorded . Coupled with the lack of a relationship between genetic differentiation and dose rate, this provides evidence that genetic patterns in A. aquaticus at Chernobyl were not influenced by radiation dose rate. Tajima's D statistic was <0 for all populations .Similarly, no significant relationship between dose rate and observed heterozygosity , which is fundamental to structuring A. aquaticus populations given known limitations on dispersal capacity  and the confirmation of IBD suggests that gene flow and homogenization of populations is likely not responsible for the lack of a relationship between genetic variables and dose rate. This suggests that current dose rates received by A. aquaticus at Chernobyl (maximum of 27.1 µGy/hr) are either insufficient to cause a high rate of mutations and subsequent elevated genetic diversity or that effects may have occurred previously but populations have recovered. These findings contradict the majority of studies which record an increase in genetic diversity in a range of nonhuman organisms at Chernobyl , and N.F., J.T.S, and A.T.F.: wrote the manuscript.