The southern pike is a freshwater top-predator fish that is endemic to rivers and lakes across Northern and Central Italy and is threatened by population decline and hybridisation with the northern pike. In this study, we focused on a southern pike population that has been recently introduced in an artificial lake, outside the species’ native range, and used genetic data to achieve a two-fold goal: (1) to investigate genetic diversity and (2) assess whether this population entirely consists of pure southern pike or is affected by hybridisation with the northern pike. The following conclusions were drawn from the results: (1) the genetic diversity of the introduced population is as large as that observed in wild populations, and (2) the presence of hybrid individuals is likely, which might be the descendants of old crosses between southern and northern pike. Several introduction events, a large number of founders, and hybridisation itself may explain the high genetic diversity that has been found in the introduced population. The present study may help further understand the genetic drivers of the successful establishment of introduced populations in new habitats. Moreover, the southern pike population from the Alto Flumendosa Lake may also be used as a test case to study how hybridisation evolves in isolated populations. Esox cisalpinus (Teleostei: Esocidae), that has been introduced outside the species’ native range. Using microsatellite markers, this study’s objective was to gather baseline genetic information and assess the presence of hybrids between this species and E. lucius in the introduced population. The resulting estimates of genetic diversity and effective population size are comparable to those observed in the species’ native range. Although different methods yield contrasting and uncertain evidence regarding introgressive hybridization, the presence of late-generation hybrids cannot be completely ruled out. Large numbers of breeders as well as multiple introductions of genetically divergent cohorts and introgressive hybridisation may explain the high genetic diversity of this recently introduced southern pike population. The present study issues a warning that the conservation of southern pike’ introgressive hybridisation between northern and southern pike might be underestimated. The genetic information gathered herein may unravel the origin, number of introduction events, and evolutionary trajectory of the introduced population. This information may help us understand the evolution of introgressive hybridisation in the southern pike’s native areas. Biological invasions are a major threat to the conservation of biodiversity, as invasive species affect native biota through competition, predation, pathogen introduction, habitat alteration, and hybridisation. The present study focuses on a southern pike population,  Biological invasions are now considered to be one of the major threats to biodiversity conservation as they may have wide ecological effects, such as affecting entire community structures ,2. InvasEsox cisalpinus Bianco and Delmastro 2011,  an esocid endemic to Italian freshwater habitats, which has recently been raised to the species rank as it is genetically and morphologically distinct from the northern pike, E. lucius [E. lucius [E. cisalpinus populations are declining due to fishing pressure and habitat deterioration,, with decreases that reached peaks up to 90% in many localities [E. cisalpinus was recognised as a species distinct from E. lucius. Therefore, the use of non-local breeders originating from Central and Northern Europe to produce large numbers of hatchery-reared juveniles has been unintentionally introducing northern pike into Italian freshwater habitats for decades [E. cisalpinus and E. lucius [This is also the case of the southern pike, . lucius ,15. As m. lucius ,17,18. G. lucius . As the . lucius ,19, evencalities ,17. Thercalities . The pic decades . The sym. lucius . Therefo. lucius .E. cisalpinus, was also not native [Salariopsis fluviatilis [The picture is worsened by the use of pike for recreational purposes as they are viewed as high-prize game fish . Indeed,t native . These ot native , which it native , whereast native ,27,28. At native ,30,31, apers. comm.). Therefore, the current population could have been founded by a mixture of several cohorts and wild adult individuals. In this context, the gathering of information on this population is fundamental as genetic data may help address issues regarding its introduction, origin, and genetic makeup that were only partly unravelled by the previous study [E. cisalpinus clade and the overall mtDNA diversity was comparable to that observed in wild southern pike populations from the mainland [Information on the introduction of pike in Sardinia is scant, but it should date back more than a decade to when anglers started to report their presence. Moreover, both captivity-born juveniles and wild adult breeders might have been introduced on several occasions, as catches included both small and large pike , the rudd Scardinius erythrophthalmus , the bleak Alburnus alburnus , the goldfish Carassius auratus , the European perch (Perca fluviatilis), the Italian spined loach (Cobitis bilineata), and the roach Rutilus aula , the latter representing the first observation in Sardinian freshwaters [Salmo trutta  and the rainbow trout Oncorhynchus mykiss  is also reported, as the lake was being actively restocked with both species until a few years ago.Currently, the lake harbours mostly allochthonous fish, such as the tench shwaters . MoreoveE. cisalpinus and E. lucius from four localities  were caught with shore-fishing equipment (fishing rod). Individuals of E. cisalpinus variety from Carignano-La Loggia  and Trasimeno Lake  as well E. lucius from Drava river  were caught by electrofishing , 1 μL of PCR products mixed with 9.90 μL of Formamide and 0.10 μL of GeneScan 500(-250) LIZ size standard  was run on an ABI PRISM 3730xl Genetic Analyser (Applied Biosystems) by an external service . Microsatellite alleles were scored and binned using STRand 2.4.110 analysis software [A set of 16 microsatellite markers was amplified using four multiplex Polymerase Chain reaction (PCR) procedures, which were performed in accordance with the protocols outlined by Gandolfi et al. . Reactiosoftware .https://doi.org/10.5061/dryad.dm97908).Departures from Hardy–Weinberg proportions (HWP) and linkage disequilibrium (LD) were tested as suggested by Waples . First, The microsatellite dataset was corrected for the presence of null alleles, allelic dropout, and other genotyping errors using the Maximum Likelihood approach implemented in MICRODROP 1.01 . The metTwo methods were used to detect outlier loci. BayeScan 2.1  comparesNA), the allelic richness (AR), the expected and the observed heterozygosity (HE and HO), and the inbreeding coefficient (FIS) were computed using diveRsity 1.9.9 [Ne) was estimated using the linkage disequilibrium (LD) method [Within population summary statistics, population size reductions and contemporary effective population sizes were assessed following Cossu et al. . The numty 1.9.9 . The con) method ,45,46 im) method , settingFST estimator θ (1984) [DEST [Global and population pairwise genetic differentiation was estimated using diveRsity to compute the Weir and Cockerham’s θ (1984)  and Jost4) [DEST  indexes;4) [DEST .K = 1–7). For each K, 10 independent runs were performed, each consisting of 100,000 iterations following a burn-in period of equal length. Two methods were used to detect the most likely number of clusters in the data: the ΔK statistics [K, if any, by means of CLUMPAK [Genetic structure was investigated using Bayesian model-based clustering implemented in STRUCTURE 2.3.4 . Only geatistics , which ratistics , which aatistics , which w CLUMPAK . The R p CLUMPAK  was thenIn addition to model-based Bayesian clustering, Discriminant Analysis of Principal Components (DAPC), which does not rely upon a population genetic model , was carTwo alternative methods, which are specifically tailored to detecting hybrids by assigning individuals to a given genotypic class, were used. These methods, implemented in NEWHYBRIDS 1.1 beta  and SNAPE. cisalpinus (TRA) and E. lucius (DRA) could not be the parental populations of any hybrid individual. Therefore, simulations mimicked realistic scenarios of northern pike genomic introgression into southern pike populations that aimed to determine how sampling might affect the assignment of individuals to hybrid classes. In the first scenario (S1), the two reference populations were used to simulate hybridisation in a hatchery between E. lucius and E. cisalpinus; both northern and southern pike from Europe and Trasimeno Lake, respectively, are used for restocking purposes in hatcheries [N = 500 individuals). Gametes were sampled with replacement from a multinomial distribution from the given allele frequencies of reference populations. Then, 50 individuals were randomly sampled to create a population that was a mixture of both pure and hybrid southern pike, which was analysed together with the original reference populations. In the second scenario (S2), the evolution of ideal microsatellite markers was simulated in four populations using EASYPOP 2.01 [Although Gandolfi et al.  showed ttcheries . The funPOP 2.01 . A hierap < 0.05), those showing an excessive number of heterozygotes (3 out 63) did not exceed the number of tests expected by chance based on the cumulative binomial distribution . In contrast, the HWP departures due to a heterozygote deficit exceeded these expectations (25 out 63 tests). Overall, 12 out of 16 loci departed from the HWP in at least one population, which decreased to 9 after applying FDR correction for multiple testing (14 out 63 tests). HWP departures were only found in E. cisalpinus populations, with Alto Flumendosa Lake (FLU), Trasimeno Lake (TRA), and Carignano-La Loggia (CAR) showing heterozygote deficits at seven, five, and two loci, respectively.All microsatellite loci, except Elu2, were polymorphic at the 5% level across populations, with the number of alleles ranging from 4 to 33 . Among tp < 0.05). After applying FDR correction, the number of locus pairs showing LD decreased to 17; the number of locus pairs at LD per population ranged between one (TRA) and seven (CAR), with only the locus pair EmaB120-Elu19 showing LD in two populations (FLU and TRA). However, as previous studies did not evidence LD between these loci , we deemed it unlikely that LD was due to a truly non-random association.Overall, 78 out of 480 locus pairs showed LD, which exceeded those expected by chance, based on a cumulative binomial distribution ; therefore, the dataset was corrected for allelic dropout and 5 out 16 microsatellite loci were not considered for downstream analyses as they were either monomorphic (Elu2) or showed >12% missing data in at least one population .Overall, rates of allelic dropout  were low at most loci, ranging between 0 and 0.08, except for five loci, namely, B457, B422, Eluc014, and Elu51, which showed rates ranging between 0.15 (B457) and 0.39 (Elu51). A slight correlation between the number of missing data and homozygotes was observed across individuals . The following sub-sections show the results based on the dataset that was corrected for genotyping errors. This dataset, together with the raw data (which were not corrected for genotype errors), input data for simulations, R scripts for running the analyses, and a comparison of outcomes based on both the corrected and uncorrected genetic data are available on figshare repository (Mean and per-locus summary statistics depicting genetic diversity within populations are shown in HE) and observed (HO) heterozygosity were found in the population from Alto Flumendosa Lake (referred to as FLU hereafter). In contrast to the northern pike population, all southern pike populations showed an excessive number of heterozygotes  with a 95% confidence interval (CI95 hereafter) that did not span zero. The southern pike populations also displayed smaller mean allelic richness (AR) than the northern pike population from the Drava river (DRA hereafter), with the smallest value was recorded at TRA (AR = 2.90 ± 0.40). Mean estimates of the contemporary populations’ effective size (Ne) as well as the CI95 were finite  and TRA the smallest . However, after setting S = 20 for all populations, the Ne estimates were very similar across all populations , with heterogeneous genotypic frequencies amongst samples evidenced by Fisher’s exact test of population differentiation . Hereafter, only results based on reported , as thosme trend . The gloFST values and exact G tests after correction for multiple testing (FDR method) evidenced genetic divergence amongst all population pairs , albeit the CI95 slightly overlapped.Both the pairwise on pairs . As expeK and Puechmaille’s statistics clearly supported K = 4 as the best Bayesian-clustering solution (q > 0.90). Both Δsolution . The gensolution  and 96% q < 0.80); among these individuals, only those from CAR showed a small membership (0.06 ≤ q ≤ 0.10) to the cluster in which all northern pike were assigned  were admixed  closely mirrored those obtained by Bayesian clustering, regardless of whether the sampling localities  or K-MeaE. lucius and E. cisalpinus, whereas the remaining DFs showed the genetic structure within the latter. The second DF separated FLU from the mainland populations, whilst the third DF highlighted the differences between CAR and TRA.Since the inferred clusters perfectly matched the sampling localities , only thThe Bayesian algorithm implemented in NEWHYBRIDS did not show consistent results across the five replicate runs . E. cisalpinus), FLU, and CAR were classified as pure with high posterior probabilities (q > 0.9) and assigned to the same genotypic class . In contrast, in 2 out of 5 runs, all individuals from FLU and one from CAR were classified as hybrids and assigned to the F2 genotypic class with q > 0.50; however, 51 out of 58 individuals from FLU were classified as F2 hybrids when q > 0.90 was used as the criterion to assign individuals to a given genotypic class. In contrast, more than half of the individuals from DRA (the reference population for E. lucius) could not be assigned to any genotypic class, even when adopting a more liberal threshold (q > 0.50). In three out of five runs, ten individuals were classified as pure and assigned to the parental population P2 using this threshold. Only ten individuals from DRA (the reference population for E. lucius) were classified as pure and assigned to the other parental category (P2) using the most liberal threshold (q > 0.50) in three out of five runs. Otherwise, they were left unassigned across all runs if more conservative criteria were used (q > 0.75) and even when the most liberal threshold was adopted in two out of five runs.In three out of five runs, nearly all individuals from TRA  from TRA, FLU, and CAR to the parental population P1 under the most liberal threshold . q > 0.75, the fraction of individuals assigned to P1 decreased to 55%, and not even a single individual was assigned when the most conservative threshold was adopted. Overall, 7 out of 106 southern pike were classified as second-generation backcross hybrids: one individual from TRA and six from FLU showed assignment posterior probabilities barely exceeding 50% for this genotypic class. All individuals from DRA were assigned to the other parental population, P2, when q > 0.50 (At q > 0.50 . The fraq > 0.5). SNAPCLUST also successfully assigned most of the individuals from DRA at q > 0.75 . Both methods detected hybrids only when the most liberal threshold was enforced, except for three out four F2 hybrids, which were detected by NEWHYBRIDS even at q > 0.9. Parental individuals were not misclassified as hybrids , whereas 21% of hybrids were assigned to a parental class . SNAPCLUST detected backcrosses slightly more effectively than NEWHYBRIDS .In contrast to the observed data, S1 showed consistent results within and across both methods . OverallThe trends outlined above resembled S2, even though both methods performed slightly better than S1 . All parEsox cisalpinus introduced in Alto Flumendosa Lake are comparable to those of wild populations living in closed freshwater habitats without signs of a founder effect. This population does not entirely consist of pure southern pike, as the presence of late-generation hybrids cannot be completely ruled out. Our results exclude Trasimeno Lake as a potential source population and hence complement those of our previous study [The present study highlights the fact that the genetic diversity and effective population size of the population of us study , which eFIS 95% confidence intervals encompassed zero in three out of four populations  after genotyping errors were corrected , albeit this population has been undergoing supportive breeding since 1970 [Ne estimate, which is twice to five-fold smaller than those obtained over different time periods by Lucentini et al. [S = 20). Indeed, Ne estimators tend to underestimate effective population size when the sample size ratio corresponding to the true effective population size (S/Ne) is too small [S = 20 to estimate Ne  could entirely consist of pure southern pike, even though caution is needed in drawing such a conclusion, as pure individuals might be the descendants of seldom, historical hybridisation events . The oldThe uncertainty with which individuals from the Drava River (DRA) are classified as pure northern pike or left unassigned deserves some consideration. We may speculate that it might be the footprint of the historical introgression of the southern pike genome into the Danubian lineage ,20,69. TE. cisalpinus. Based on the levels of mtDNA variation, Casu et al. [Ne indicates that this population might have been founded by 66 breeders (CI95 = 36–183). Moreover, this is likely a conservative estimate, as it has been estimated by a sample consisting of individuals of different ages; therefore, including individuals with overlapping generations might have introduced a downward bias in Ne estimates [Ne estimates represent the effective population size of the combined wild and hatchery populations [eN estimates obtained in the present study can be compared with those resulting from the introduction of northern pike in north American lakes [eN could have been small over long time periods. Thus, Miller and Kapuscinsky [Compared to the other southern pike populations analysed in the present study, FLU shows higher levels of genetic diversity , whose mu et al.  hypothesstimates ,71. To tstimates ; therefoulations ,72. The s CI95 = 6–183. Moan lakes , which suscinsky  suggesteAlthough more populations should be included to infer the potential origin of FLU, TRA can be excluded as the primary source of breeders for southern pike introduced into Alto Flumendosa Lake; for instance, the lower genetic diversity of the latter is at odds with this scenario. Therefore, we may speculate that the origin of FLU should be searched in hatcheries and/or wild populations of Northern Italy, even though the genetic divergence from CAR (Po drainage basin) is as large as that between FLU and TRA . Indeed,The baseline genetic information gathered herein may help assess the origin, introduction events, and the potential spread of the southern pike population that has been recently introduced to Alto Flumendosa Lake. Integrating these genetic data with those of wild and hatchery populations from Italy may unravel the origin of the invading population. The population from Alto Flumendosa Lake is likely a mixture of pure and hybrid southern pike that are introgressed by the northern pike’s genome. Uncertainty with respect to the assignment of the individuals to pure or hybrid genotype classes, and thus in the estimation of their proportions in the introduced population, may be due to the limited capacity to detect late-generation hybrids . Thus, aE. cisalpinus was first described [Overall, the present study highlights, for the first time, that the introgression of northern into the southern pike genome might be underestimated, albeit caution is needed when drawing such a conclusion. Indeed, this result stems from only two simulated scenarios, yet they are scenarios representing realistic cases that may occur in the wild . More tescribed ,26, may The present study aims to assess the genetic makeup and the occurrence of hybrid individuals in a southern pike population that has been introduced in Alto Flumendosa Lake, Sardinia, outside its native range and expand on the results of a previous study . This poOverall, genetic information might help unravel the origin, number of introduction events, and evolutionary trajectory of an alien fish population in a new habitat ,74. In p


Aquarana catesbeiana, in Brazil on the basis of 324 samples collected from feral and captive groups at 38 sites in seven of the nine states where feral populations occur. We genotyped all samples using previously developed, highly polymorphic microsatellite loci and performed a discriminant analysis of principal components together with Jost’s D index to quantify pairwise differentiation between populations. We then amplified 1,047 base pairs of the mitochondrial cytochrome b (cytb) gene from the most divergent samples from each genetic population and calculated their pairwise differences. Both the microsatellite and cytb data indicated that bullfrogs comprise two populations. Population grouping 1 is widespread and possesses two cytb haplotypes. Population grouping 2 is restricted to only one state and possesses only one of the haplotypes from Population grouping 1. We show that there were two imports of bullfrogs to Brazil and that there is low genetic exchange between population groupings. Also, we find that there is no genetic divergence among feral and captive populations suggesting continuous releases. The limited genetic variability present in the country is associated to the small number of introductions and founders. Feral bullfrogs are highly associated to leaks from farms, and control measures should focus on preventing escapes using other resources than genetics, as feral and captive populations do not differ.Non-native species are a major problem affecting numerous biomes around the globe. Information on their population genetics is crucial for understanding their invasion history and dynamics. We evaluated the population structure of the non-native American bullfrog,  Precautionary policies to prevent new introductions and dissemination of already introduced populations are essential6, as are efforts to control and eliminate established non-native species8. The effort and cost of controlling invasive species can be prohibitive, especially for species that are distributed over large areas and are continually reintroduced9, like the north American bullfrog (hereafter bullfrog), Aquarana catesbeiana10, in Brazil.The introduction of non-native species as a result of human actions is one of the major causes of wildlife threat and extinction around the world12 and consuming a broad diversity of animal taxa, including small vertebrates18. Egg clutches comprise as many as 20,000 eggs and frogs have a fast growth rate, reaching sexual maturity within 1 year of metamorphosis19.Native to eastern north America, bullfrogs are, relative to most anurans, large and voracious predators, exceeding 150 mm in adult body length21 and have found that bullfrogs can have a significant impact on native fauna22: Kupferberg23 reported larvae competition among native species in consequence of phytoplankton change in pools invaded by bullfrogs. The voracity and generalist diet of introduced bullfrogs have led to concerns about possible decrease of native species’ populations24. Similarly, given that bullfrogs are resistant to chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) infection27, one of the primary causes of precipitous amphibian population declines, the occurrence of Bd in a large proportion of bullfrogs in Brazilian frog farms31 suggests that feral bullfrogs might be an important disease vector. For example, bullfrog presence has been found to be a positive predictor of both Bd prevalence and Bd load in the north American frog Rana boylii32. In Brazil, the most direct evidence of impacts on native frogs is provided by experimental studies that provided evidence of a change in the vocal behavior of native males, that increased their vocal frequency in response to bullfrog vocalizations34, although this represents niche overlap and does not necessarily impact species diversity. A recent report indicates that bullfrogs in Brazil seem to have little influence on native amphibian population dynamics35, but a study focused on its known and potential impacts indicates that the presence of bullfrogs can cause changes in activity and habitat use preferences of native species36.Although the specific responses of native fauna to introduced bullfrogs remain poorly understood, many studies have examined the bullfrog invasion around the world38. There are over 150 known breeding facilities in the country, that produce around 400 tons/year, moving around 1.9 million USD38. According to published accounts, bullfrogs were first imported to Brazil to produce meat for human consumption in 1935 by a Canadian technician named Tom Cyrril Harrison who brought either 300 individuals39 or pairs40 from an unknown locality to a Brazilian government breeding facility in the city of Rio de Janeiro42. Hundreds of tadpoles were subsequently sent to new breeding facilities around the country in an agricultural program encouraged by federal and state governments42. The state-based characteristics of this governmental program facilitated the appearance of feral bullfrog populations in different political regions of the country, as today feral bullfrogs are widespread in the south and southeast regions of Brazil, with additional localities in the central-west, northeast, and north41. Escape and release of bullfrogs from farms are spatially correlated with feral bullfrog populations45, being the most likely cause considering the disconnected distribution of these populations in Brazil, although the dispersive potential of bullfrogs is not well documented. Although advances in technology and increased market demand have enabled bullfrog farming to expand greatly over the last 10–15 years46, many of the earlier frog farms failed in the 1990s38, resulting in massive releases of bullfrogs when businesses closed . In the 1970s, an additional 20 pairs of adult bullfrogs were imported to Brazil28 from the University of Michigan to São José do Rio Preto in São Paulo state by Luiz Dino Vizotto . Little information about these individuals is available, and the outcome of this introduction is not confirmed nor discussed in the literature. The same is true for small-scale occasional introductions supported by individual breeders, without any official registers, such as a supposed bullfrog batch introduced from Mexico in the early 2000’s38.Although there is very limited knowledge about the possible impacts of bullfrogs over native species in Brazil, this species is widespread in the south and southeast regions of the country, with limited distribution in the northeast and north38. If only one event was successful, followed by strong selective pressures of breeding facilities, the genetic diversity of bullfrog populations in Brazil should be quite limited. The limited information about introduction history of the bullfrog in Brazil raises the possibility of different genetic scenarios. At least one population was successfully introduced in the country, and other lineages could have also contributed genetically to the introduced populations.If all the introduction events were successful, different lineages in the country might be going through an introgressive hybridization process due to the common practice among farmers of purchasing breeding stock from multiple farms in different states with the goal of increasing the genetic diversity of the frogs bred in their facilities 48, and can be helpful to develop management and control programs50. Efforts to understand the genetic structure of introduced bullfrog populations have been undertaken in several regions, such as Europe51, China43 and western USA52. Unlike the invasion history in Brazil, Europe had several events of introduction from different origins51, and the western region of the USA is continually flooded with individuals from the native range52. China also had different events of introduction, although little genetic diversity was found43. These examples can serve as a baseline for comparison in new genetic research, and give indications about the invasiveness of a non-native population.Information on introduced bullfrog population genetics is crucial to understand invasion history, structure and dynamics of gene pool exchange between populations52, we expect introduced bullfrog population in Brazil to be less diverse than other non-native populations previously studied in different regions of the globe, as fewer introduction events were reported in Brazil in relation to other areas. Considering that more unreported introduction events could have happened, our main goal in this study was to assess the genetic structure of Brazilian bullfrog populations and possible gene flow between them. To achieve our goal, we focused on four main questions: (1) Are captive and feral populations genetically different? (2) Does bullfrog genetic structure corroborate the hypothesis that there were two or more introduction events in Brazil? (3) Is there gene flow between populations? (4) Do Brazilian bullfrog populations present lower genetic diversity than non-native populations from other regions?Because multiple introduction events can create introduced populations that present more diverse gene pools53. Farm animals were not sacrificed for this research. Tissues were stored in 99% ethanol and kept at − 20 °C until DNA extraction, which we performed with the DNeasy Blood and Tissue extraction kit  following the manufacture’s guidelines.We obtained 324 bullfrog skin, liver, and/or muscle tissue samples, of which 128 were purchased from 11 farms and 194 were collected from feral specimens or sampled from museum or private collections Fig. . Specime54. Multiplex PCR was performed on a Veriti™ thermal cycler (Applied Biosystems) with a thermal profile consisting of 95 °C for 7 min followed by 10 cycles of 95 °C for 30 s, touchdown from 62 to 57 °C for 45 s, and 72 °C for 30 s, followed by 30 cycles of 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s, and final extension at 72 °C for 7 min. The reaction mix, with a total volume of 10 µl, contained (1.0 µl) buffer, (0.5 µl 2 mM) dNTPs (0.5 µl) fluorescent dye ,  of mixed forward and reverse primers, (0.125 µl) Taq polymerase, (3.375 µl) distilled deionized water, and (3.0 µl) template DNA. Later, we diluted the PCR products to a proportion of 1:4 and submitted them to sequencing by a third party, not changing service provider to avoid bias due to calibration differences. We scored results using Gene Marker v. 2.6.3 (SoftGenetics) and tested for the presence of null alleles, allele dropout, and stuttering using Microchecker55. We also genotyped 91 samples (24.1%) a second time to evaluate the percentage of homozygotes that were actually null alleles. We included control samples in each procedure.We amplified seven nuclear microsatellite loci using the library developed by Austin et al.40, we anticipated two possible scenarios: (1) extremely low genetic divergence and diversity among bullfrog populations in Brazil. This could have been caused by limited genetic diversity in the founding event of Brazilian populations, together with strong selective pressure in breeding facilities; 2. Increased genetic diversity caused by founding populations that were prevenient from different native populations. Considering that each scenario would require different analytical methods, we chose for assessing the genetic structure using two different analytical toolkits. A flowchart with the analytical decision-making is available in Fig. Given that published sources report a total of 340 or 640 bullfrogs being imported to Brazil .For scenario 1, we performed a discriminant analysis of principal components (DAPC)58 to quantify pairwise genetic differentiation among state populations, which was calculated with the mmod R package59. Next, we tested the significance of differentiation between pairs of populations using the DEMEtics R package60 with 10,000 permutations. We applied the Benjamini–Hochberg Procedure61 to control for false discovery rates (FDR) and avoid type I error62 using the p.adjust function from stats package63. We used the gstudio package64 to calculate the following diversity indices for each genetic population: expected (He) and observed (Ho) heterozygosity, number of alleles (A) per locus, effective number of alleles (Ae) per locus, and size-corrected Wright's inbreeding coefficient (Fis). Through a permutation procedure with 100 batches of 1,000 iterations, we checked for Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) in each state population by performing a probability test in Genepop ver. 3.465. To avoid type I error, P values were corrected following the Benjamini–Hochberg Procedure.We used Jost’s D index57. We estimated admixture using STRUCTURE software66. This analysis was performed using nine runs for every K (from K = 1 to K = 9) using state populations in the locprior model and a burn-in period of 10.000 steps and 20.000 Markov chain Monte Carlo (MCMC) repetitions. The best fitted K number of populations was calculated using STRUCTURE HARVESTER67, and interpreted through the Evanno method68.Considering scenario two, we performed a principal coordinate analysis (PCoA) to visualize genetic differentiation using the R package Adegenet69 to perform these analyses.We also calculated the differentiation among and within state populations and feral and captive populations through an analysis of molecular variance (AMOVA). We used Arlequin softwareb gene (cytb). We used a combination of the primers MVZ15L70 and cyt-bAR-H71 and a thermal profile for polymerase chain reaction that consisted of 95 °C for 10 min, followed by 45 cycles at 95 °C for 30 s, 50 °C for 40 s, and 72 °C for 40 s, with a final extension step at 72 °C for 5 min. The reaction mix, with a total volume of 25 µl, contained (0.15 µl) Go Taq G2 Flexi DNA Polymerase (Promega corporation), (2.5 µl) Go Taq flexi Buffer,  dNTPs,  MgCl2, 1.0 µl of each primer (10 pM), (15.35 µl) distilled deionized water, and (2.0 µl) template DNA. PCR amplification products were cleaned using Agencourt AMPure XP DNA Purification and Cleanup kit , and they were sequenced by a third-party using fluorescent-dye labelled terminators  with an ABI 3730XL . Sequences are available on GenBank (MT668579 and MT668580).On the basis of the microsatellite results, we selected the 18 most divergent samples from each genetic population  with the G-INS-I strategy. We trimmed the sequence alignment to a length of 937 bp. We used Arlequin 3.569 for most genetic analysis. We calculated the haplotype diversity (Hd) and nucleotide diversity (π) indices and report values ± SD for each genetic population. We also calculated the pairwise differences between the introduced populations using the θST index and evaluated its significance by performing 10,000 permutations. Using the p.adjust function from stats package63, we performed the Benjamini–Hochberg Procedure61 to control for FDR.We used Geneious ver. 10.2.3The presence of null alleles was indicated for all loci, whereas allele dropout and stuttering measures were not significant. Genotyping error was estimated to be less than 2%. The state-based total-data DAPC Fig.  showed eP = 0.001). Most pairwise comparisons indicated significant LD  that differ from each other in 14 base pairs (π = 0.005324 ± 0.003); Haplotype A was more frequent than Haplotype B, occurring in 80% of specimens (Hd = 0.3556 ± 0.159). Population grouping 2 contains Haplotype B exclusively. The population differentiation test between the two population groupings was significant after FDR correction .40. The introduction history of this species resulted in high rates of homozygosity, high inbreeding coefficient values, low allelic richness, and consequent overestimations of LD and HW disequilibrium. These characteristics are predictable, as they are associated with the founder effect and inbreeding in these populations following their importation to Brazil and subsequent intensive reproduction and presumed artificial selection. Populations with low genetic variation, as expected in cases of non-native populations with few founders or populations that have undergone major reductions73, present special conditions that require appropriate methods. As such, we used DAPC to analyze genetic structure because it makes no assumptions about HWE, LD, or gene flow, like more commonly used analyses such as STRUCTURE66.All bullfrogs in Brazil descend from a relatively small number of founders introduced at most 85 years ago, in at least two different events75, the common practice of discarding loci inferred to possess null alleles [e.g.78 is contraindicated in cases such as the introduced bullfrog in Brazil. Specifically, Microchecker indicates the presence of null alleles on the basis of excess homozygotes being evenly distributed across homozygote classes55, which is precisely the situation expected to occur in recent invasions subjected to high levels of inbreeding [e.g.,9. The indication of null alleles in all genes reinforces this point, as all genes underwent the same biological process and none of them are in HWE. Nevertheless, the double genotyping of almost a quarter of all microsatellite data showed that the presence of null alleles is not significant. Thus, we included all loci in our analyses.Similarly, although null alleles are known to inflate measures of genetic differentiation and create false homozygotesConsidering that other non-reported introductions could have happened, increasing the gene pool and contradicting our first methodological toolkit assumptions, we reanalyzed our dataset with a more traditional approach. This included PCoA and STRUCTURE, to visualize genetic differentiation and admixture, respectively; and AMOVA, to teste differentiation among populations.28. Specifically, although we observed that samples from most areas overlap extensively by occupying the same multivariate space in the DAPC (Population grouping 1), samples from Minas Gerais did not (Population grouping 2). Similarly, the best number of Ks indicated for the STRUCTURE analysis was three. Although there was a difference in the interpreted number of populations between the two used approaches, it could have happened due to biases in both of them. The DAPC approach is subjected to a certain amount of subjectiveness, as different populations are interpreted by their distribution in the multivariate space. In this case, the population from Parana, for instance, has a distribution that does not completely overlap with the rest of samples named here as Population grouping 1. This could be the third K represented in the STRUCTURE approach. At the same time, as mentioned before, the assumptions about HWE, LD, or gene flow in the STRUCTURE analysis could also be responsible for inflating the real number of Ks.Both methodological toolkits presented similar results, with low differentiation among population groupings. Our state-based analysis of microsatellite data corroborates published accounts that there have been only two importations of bullfrogs to Brazil, a well-known importation of 300–600 individuals in 1935 and a lesser-known importation of 20 pairs in the 1970s44, breeding facilities effectively maintain bullfrog populations across the country in an infinite island model. This process was documented in a recent work32, where authors investigated the historical and present bullfrog trade among Brazilian states. In their work, the states of São Paulo, Rio de Janeiro and Minas Gerais seem to be central to this market. They did not contrast the trade of living bullfrogs from the meat trade, what might explain why Minas Gerais still has differentiated genetic traits even though it is deeply entangled in the commercial net.The results of the cytb analysis corroborate the DAPC results and further suggest that either founders of Population grouping 1 contained both mtDNA haplotypes, or some degree of introgression of the haplotype from Minas Gerais (Population grouping 2) into Population grouping 1 has occurred but not the other way around. This might be correlated with the decrease of frog farmers in the state of Minas Gerais, previously known as one of the biggest producers in the country. Farmers from other states, like São Paulo where some haplotypes from population grouping 2 were found, made efforts to enhance the genetic diversity of their breeding population by importing animals from other states while breeders from Minas Gerais closed or reduced their facilities, preventing Population grouping 2 from receiving migrants from Population grouping 1. We therefore conclude that populations from the same genetic units are probably exchanging migrants, since there is no signal of genetic differentiation between them, what agrees with the information that farmers keep exchanging animals. This practice is unlikely to be effective, given the low genetic variation of bullfrogs in Brazil, and might also have contributed to preventing the genetic differentiation of introduced populations. That is, by exchanging "migrants" with each other and replenishing feral populations with constant leaksThe lack of differentiation among populations prevents the use of genetic resources to diagnose and control leaks from breeding facilities. As shown in both DAPC and AMOVA, captive and feral specimens do not differentiate. This result can be explained not only by biological processes, like the low number of founders and intense inbreeding following importation, but also by the common practice among farm managers of purchasing breeding stock from multiple farms in different states with the goal of increasing the genetic diversity among the frogs bred in their facilities.43—where also only two haplotypes were found among over 500 samples—and in some degree in Europe (5 haplotypes among nearly 400 samples from 8 countries)47, although European countries have more populations and more genetic diversity, due to the relatively high number of introduction events that happened in the continent (at least 25)51. The process of transforming introduced populations into a single population due to exchange of migrants may obscure population and invasion genetics inference, as the genetic signal is lost making it nearly impossible to clarify the history of events that followed the introductions. This also brings consequences to management efforts as important information like the origin and the dynamics of the introduced populations are let largely unanswered.The practices of bullfrog farmers in Brazil tend to generate an undifferentiated population in the country over time. The same process seems to have occurred in other countries where populations were analyzed with the same genetic markers used in this study. Similar patterns were observed in China52. Although the genetic diversity of bullfrogs in Brazil seems to be low, especially when compared to populations in its native range (42 haplotypes were found in the United States79), farming does not seem to suffer any impacts.The number of introduction events seems to be directly related to the degree of genetic diversity in the region, and the bullfrog populations in Brazil exhibit the lowest number of mtDNA haplotypes of all studied non-native populations of this species examined so far35. Conversely, we observed a small percentage of anomalous animals when visiting farms, and farmers did not report any negative effects in their business. Research focused on other invasive amphibian species, such as the cane toad (Rhinella marina + R. horribilis species complex), show that reduction in genetic diversity after introduction does not seem to affect ecologically relevant traits80. This matter should be accounted when evaluating the potential of bullfrog invasiveness. Feral bullfrogs are highly associated to leaks from farms, and control measures should focus on preventing escapes using other resources than genetics, as feral and captive populations do not differ. The limited genetic variability present in the country is associated to the small number of introductions and founders.The exceptionally reduced genetic variation found in the population from Minas Gerais might explain the observation of morphological anomalies reported by Ferrante et al.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Plutella xylostella (L.), the most widely distributed Lepidopteran pest that developed resistance to almost all current insecticides, migrates heterogeneously across space and time. To investigate its real-time genetic pattern and dynamics, we executed four samplings over two consecutive years across Southern China and Southeast Asia, and constructed population network based on contemporary gene flow. Across 48 populations, genetic structure analysis identified two differentiated insect swarms, of which the one with higher genetic variation was replaced by the other over time. We further inferred gene flow by estimation of kinship relationship and constructed migration network in each sampling time. Interestingly, we found mean migration distance at around 1,000 km. Such distance might have contributed to the formation of step-stone migration and migration circuit over large geographical scale. Probing network clustering across sampling times, we found a dynamic P. xylostella metapopulation with more active migration in spring than in winter, and identified a consistent pattern that some regions are sources  while several others are sinks  over 2 years. Rapid turnover of insect swarms and highly dynamic metapopulation highlight the importance of temporal sampling and network analysis in investigation of source-sink relationships and thus effective pest management of P. xylostella, and other highly dispersive insect pests.Genetic makeup of insect pest is informative for source-sink dynamics, spreading of insecticide resistant genes, and effective management. However, collecting samples from field populations without considering temporal resolution and calculating parameters related to historical gene flow may not capture contemporary genetic pattern and metapopulation dynamics of highly dispersive pests.  Locusta migratoria    and Plutlostella . Howeverlostella  and formlostella . AnalysiNm refers to historical gene flow and thus inadequate for contemporary population dynamics. In addition, Nm calculated by FST need several assumptions that real populations are likely violating and do not provide accurate estimation of migration  directions . Most poigration . To bettigration .For inseigration , pedigreigration . Furtherigration  can leadigration  could fuPlutella xylostella  has developed resistance to almost all insecticides, causing enormous damage to global cruciferous vegetable and oil crop industries  are recorded by radar . Among tgionally . Widely gionally . In thisP. xylostella populations in four Asian countries including China, Myanmar, the Philippines, and Vietnam  following the instruction manual. The extracted DNA of each individual was tested based on OD 260/280 ratio measured by UV-1600 Spectrophotometer. All the individuals were with high DNA concentration and quality, and suitable for microsatellite  genotyping.A total of 15 published SSR markers were selected for genotyping . The PCRNa), effective population size (Ne), Shannon index (I), observed heterozygosity (Ho), expected heterozygosity (He), and allelic richness (Ar). In addition, we treated populations of the same sampling time interval as a metapopulation and estimated genetic variation in each metapopulation by using number of alleles and number of private alleles based on the rarefaction method in ADZE  across the sampled populations from a metapopulation .Further genetic structure analysis found that the populations with higher genetic variation clustered together (cluster1), while the other populations formed a second cluster . We doubThe parentage analysis based on Colony identified the best configuration of offspring and parental individuals in each sampling time interval and was used to infer contemporary gene flow between source-sink populations. We found similar migration events among populations identified by markers using different error rates. This supports the accuracy of kinship assignment analysis . We thusThe population network was constructed using the presence of gene flow between two populations as edge, with the arrow representing the direction and the size of node indicated the degree centrality . In time 1, FJ1, FL1, GD1, GX1, GZ1, HN1, and JX1 were with high degree centrality and receiving immigrants from other populations , while FWe further summarized the geographical distance of each population pair with gene flow. A total of 115 population pairs with contemporary gene flow in the overall 249 pairs were identified . The datWe further used Newman’s modularity clustering in igraph to construct the migration network. To distinguish from the former network, we used betweenness as undirected and weighted edges. Overall, we found a highly differentiated pattern of clustering across sampling time intervals, and clustering analysis found samples from spring (time 1 and time 3) have lower clustering number compared with that in winter (time 2 and time 4) . At timede novo mutations) or as source population that could mediated gene flow into the other populations. In time 1, highest degree centrality was found in Fujian (FJ1) and Guizhou (GZ1). Across four clusters of time 2, Guangxi (GX2) was the population with the highest node degree , For many insect pests, resources and habitats are ephemeral across time and space  due to fP. xylostella in overwintering regions. This could be explained by low level of autonomous control over migration direction . To be specific, less clusters in spring (time 1 and time 3) compared with that in winter of the same year (time 2 and time 4) were observed. This pattern is likely due to more active windborne migration in spring that contributed to the linkage across regional populations  should be conducted for a comprehensive understanding of migration at certain geographical distances and its contribution to regional metapopulation dynamics.Based on modularity clustering analysis, we found a differentiated clustering pattern across different sampling times, which supported a dynamic metapopulation of irection  and the irection . We founirection . The religration  that forulations . More aculations  as well Nilaparvata lugens (Stål) (Sogatella furcifera (Spodoptera frugiperda (Nilaparvata lugens (Stål)  as well as Indo-China Peninsula  were always the source for populations in our four samplings. This pattern has also been identified in a recent analysis based on genome-wide SNPs , where ps (Stål) ; Sogatelurcifera ; Spodoptugiperda ] and gens (Stål) ]. The yes (Stål) , but alshai gulf . Neverthhai gulf .www.mdpi.com/journal/insects/special_issues/population_genetics, e.g., Nm, that represent historical gene flow are unsuitable for partially migrating populations of agricultural insects (Current insect ecology has witnessed a boom in population genetics/genomics studies of agricultural pests [ insects . Pedigre insects .P. xylostella populations crossing the Bohai Strait during the spring compared with those in winter. This temporal differentiation of insecticide resistance strains is likely due to variation in insecticide pressure between Southern and Northern China (Investigation of contemporary gene flow is important especially for insecticide resistant pest monitoring. For example, rn China . Furtherrn China  could beP. xylostella across its overwintering region in Southern China and Southeast Asia. Our results depicted a rapid population turnover and dynamic metapopulation of this highly dispersive pest and highlighted the importance of temporal sampling and population network analysis in understanding contemporary genetic pattern and regional source-sink dynamics of agricultural insect pest. These results are expected to shed light on monitoring of the real-time dynamics of insecticide resistance and identify regional key populations towards more effective and sustainable management in the long term. More broadly, methods of this study could also be applied to other highly dispersive insect pests under modern agricultural regime.In this research, we investigated the temporal variation in genetic makeup of


Neosalanxtaihuensis sampled from 11 populations in the six typical river basins of China. Using the COI gene sequencing technology, the N. taihuensis population’s genetic difference within and between river basins was investigated. Significant levels of genetic subdivision were detected among populations within basins rather than between basins. Population history dynamics showed that N. taihuensis populations experienced a population expansion during the glacial period in the late Pleistocene. These results suggest that different populations should be considered as different management units to achieve effective conservation and management purposes.In the current study, we estimate the genetic diversity of the salangid Neosalanx taihuensis  is a commercially important economical fish endemic to China and restricted to large freshwater systems with a wide-ranging distribution. This fish species has continuous distribution ranges and a long-introduced aquaculture history in Chinese basins. However, the research on its population genetic differentiation within and between basins is very limited. In this regard, 197 individuals were sampled from 11 populations in the Nenjiang River Basin (A1–A4), Songhua River Basin (B1), Yellow River Basin (C1–C2), Yangtze River Basin (D1), Lanchang River Basin (E1–E2) and Huaihe River Basin (F1). Based on the COI sequence, the N.taihuensis population’s genetic difference within and between river basins was investigated. The haplotypes and their frequency distributions were strongly skewed, with most haplotypes (n = 13) represented only in single samples each and thus restricted to a single population. The most common haplotype  was found in all individuals. The analysis of molecular variance (AMOVA) revealed a random pattern in the distribution of genetic diversity, which is inconsistent with contemporary hydrological structure. The mismatch between the distribution and neutrality tests supported the evidence of a population expansion, which occurred during the late Pleistocene (0.041–0.051 million years ago). Significant levels of genetic subdivision were detected among populations within basins rather than between the six basins. Population history dynamics showed that N. taihuensis experienced an expansion during the glacial period in the late Pleistocene. Therefore, different populations should be considered as different management units to achieve effective conservation and management purposes. These results have great significance for the evaluation and exploitation of the germplasm resources of N. taihuensis.The salangid  The genetic structure of a population is dependent on the interaction of the species biology and the environment in which it resides. Populations from different basins often show significant genetic differences resulting from isolation, while a basin population demonstrates no or low levels of genetic difference . There iNeosalanx taihuensis  is a usually one-year-lived freshwater fish species from the Salangidae family, which is distributed mainly in the Yangtze River and affiliated lakes and which has a continuous distribution in Chinese river basins [N. taihuensis has therefore been introduced to numerous lakes and reservoirs in more than 20 Chinese provinces since the 1980s [N. taihuensis has been studied for more than 100 years, but most of the studies have focused on taxonomy [N. taihuensis.The salangid, he 1980s ,8. The Lhe 1980s . Both bahe 1980s . The Yelhe 1980s . The Nenhe 1980s . N. taihtaxonomy , biologytaxonomy  and moletaxonomy . HoweverN. taihuensis, a status analysis of its genetic diversity is necessary.Molecular markers are useful tools for the assessment of genetic diversity and population structure, in addition to the various influential factors. It is well known that nuclear and mitochondrial (mtDNA) DNA markers are extensively used to evaluate genetic diversity and population structure in freshwater and marine fish species. MtDNA allows for the study of the geographic structure and historical demography of a species, because it is maternally inherited without recombination and has a relatively fast mutation rate . It is cCulter alburnus from Chinese freshwater lakes [The evolution speeds of mitochondrial cytochrome oxidase I (COI) is suitable for the detection of population differences and have been widely studied . It is oer lakes . In thisN. taihuensis were collected from 11 populations in the NjRB (A1–A4), ShRB (B1), YeRB (C1–C2), YzRB (D1), LcRB (E1–E2) and HhRB (F1) from November to December in 2019  following the Kit protocol. The COI fragment was amplified using COIF: 5′-CCTTTGGCAGGAAACTTGGC-3′ and COIR: 5′-TGGTAAAGAATGGGGTCGCC-3′ as primers in a 50 μL reaction volume mixture containing 25 μL Premix Taq , 1.0 μL each of 10 μM primer, 3.0 μL total genomic DNA approximately 100 ng as template and 18–22 μL of sterile distilled water. PCR was performed according to the reaction method as follows: 98 °C for 5 s, 30 cycles at 98 °C for 10 s, annealing at 56 °C for 30 s and a final extension at 72 °C for 2 min. Purified PCR products were directly sequenced using primers COIF and COIR from both ends in a semiautomated DNA analyzer . To avoid the errors in amplification and sequencing, all singletons among polymorphism sites were verified by additional amplification and sequencing.Total DNA was extracted from the fish tailfins using E.Z.N.A.Protosalanx hyalocranius (accession number MK523756.1) and genetic distances were generated for UPGMA phylogenetic tree reconstruction using models of substitution by MEGA 9.0 . Genetic differentiation was estimated by calculating pairwise distances between populations [N.taihuensis was estimated to be one year [COI sequences were edited and aligned using Clustal X 1.81 software. Fish diversity was evaluated by determining COI haplotype diversity (Hd) and nucleotide diversity (π) . These tulations . Pairwisulations . The relulations . Furtherulations ,29. The ulations . The timone year . We usedone year  and the one year .All the COI sequences of mtDNA regions were homologous in length and aligned using cluster X. A total of 197 COI sequences with an aligned length of 647 bp were generated from six river basins. Eleven polymorphic sites and thirteen haplotypes were detected among all samples. The average nucleotide composition for all individuals was highest for G (33.32%), followed by A (26.04%), T (21.43%), and C (19.22%), The G/C contents (52.54%) were significantly higher than the A/T contents (47.47%), showing a strong G/C bias, respectively .In this study, 13 different haplotypes were identified in the 197 samples . The nump < 0.05). The highest degree of genetic differentiation occurred between the TH and XLD populations (Fst = 0.5387), and the lowest degree of genetic differentiation occurred between the LH and LHP populations  followed by H1 (0.2995), H2 (0.1929) and H3 (0.0964), which indicated H4 was the most probable ancestral haplotype, as it was shared with five river basins. In fact, a high frequency of shared haplotypes  was found among all basins, which produced a pattern of population relationships associated with the ancestry of the haplotypes found within each population rather than with the hydrological network .Protosalanx hyalocranius served as a separate branch of the out group. The results of the NJ tree basically corresponded to the results of the haplotype network tree, but there was no significant population pattern, and haplotypes were cross-distributed among populations.The NJ (neighbor-joining) tree was based on the COI marker, and the same samples gave similar results . The NJ ’s Fs value and Tajima’s D test of the COI gene had negative values, but the statistical test was not significant , indicating that the population deviated from neutral evolution. The mismatch distribution showed a unimodal pattern  occurring during the Late Pleistocene.Neutral test analysis showed that Fu pattern . Both thN. taihuensis may have experienced a rapid expansion of the population after the bottleneck effect cause [N. taihuensis population. Contrary to Gaoyou Lake  and Luoma Lake (0.361 ± 0.103 and 0.00062 ± 0.00062) [Genetic studies are of great importance, since they can improve our understanding of genetic diversity and evolution, and therefore provide constructive guidance for the conservation and management of genetic resources, as well as the development of breeding programs ,35. Genect cause . Similar0.00062) .N. taihuensis populations in China. The higher haplotype and lower nucleotide results are basically consistent with previous studies [N. taihuensis [Changes in fish genetic diversity are affected by many factors, including geographic isolation, human activities, habitat differences and mitochondrial evolution rates . The ext studies . The resihuensis ,43.N. taihuensis as a usually one-year-lived “R-strategy” creature, a high fecundity and abundant resources are important biological competition strategies. In addition, combined with its inherent biological characteristics, N. taihuensis is a cold-water fish, and the suitable water temperature is low [N. taihuensis.The genetic diversity and population structure of the same species in different geographical locations are often related to the living environment . The Yane is low . The late is low . The LcRN. taihuensis. An AMOVA showed that the genetic variation of N. taihuensis mainly came from within the population (90.37%). The degree of differentiation was at a moderate level (0.05 ≤ Fst = 0.09 < 0.25), and among different basins, the genetic differentiation was the largest (Fst = 0.5387) between the TH and XLD populations. Within the same basin, the genetic differentiation (Fst = 0.2146) between the HSH and LHP populations was the highest, and the maximum variation rate between different basins was higher than that within the same basin. It may be that geographic isolation affects the genetic differentiation of species, resulting in different genetic diversity of species, but this study found that no obvious geographical pattern was formed, and the genetic differences between groups were low [In this study, the COI gene sequence was used to analyze genetic differentiation among the populations of were low .From the perspective of phylogenetic evolution, the analysis of the UPGMA tree based on genetic distance showed that their close relationship and geographic proximity were basically consistent. This study took population samples from different waters in the north and south, and the UPGMA tree was roughly divided into three branches ; the NjRB, Songhua River and XLD population in the northern water system formed Clade 2; the southern water system YzRB, LcRB and HhRB formed Clade 3. Due to the inherent biological characteristics of Clade 1, most samples were in relatively closed water bodies for a long time, and the gene exchange with other groups was limited, forming a distance isolation model organism and resulting in a long genetic distance. The haplotype network map constructed from the 13 haplotypes was radically distributed around the main haplotype, and different groups intersected with each other, which basically corresponded to the results of the NJ phylogenetic tree. There was no significant group pattern.p values can be considered as showing historical signs of population expansion [N. taihuensis may have experienced population expansion. N. taihuensis originated in the middle of the Tertiary and experienced alternating Quaternary and interglacial climate during its evolution [N. taihuensis population at 0.041–0.051 million years, both earlier than the last major glaciation of the Quaternary glacial period, about 18,000 years ago. It is speculated that the last large glacial period had a great impact on the N. taihuensis population, during which N. taihuensis lived in a shelter. With the end of the last glacial period, the warming caused the sea level rising, and the N. taihuensis population in Taihu Lake spread out [In general, patterns of genetic diversity in species are closely related to their evolutionary history. Population historical evolution is usually detected in two ways: one is to observe the nucleotide unpaired distribution map based on the infinite-site model; the other is neutrality tests ,46. In txpansion ,47,48. Mread out . During N. taihuensis have been attracting significant attention in China. It is worth noticing that the population size of N. taihuensis and its fishing yield have decreased rapidly in recent years [N. taihuensis, so it is urgent to make an effective plan to protect this economically and ecological important species. Furthermore, as a one-year-lived species, all populations should be managed and conserved separately [N. taihuensis’s nature reservoir.As one of the most important commercial species in the salangid family, the fish nt years . Overfisnt years . Consequnt years ,53. At tparately . MoreoveN. taihuensis populations, further indicating that the population had an overall genetic diversity The low level of genetic diversity may lead to a reduction of species fertility and an increase of harmful recessive factors, which result in species degradation. As a native fish species, N. taihuensis should be paid enough attention to maintain its genetic diversity. The YzRB served as the main origin for this species, and it should be protected more. As a usually one-year-lived “R-strategy” creature, the N. taihuensis is more sensitive to habitat changes, so habitat protection and water pollution control should be given priority. In a later stage, more nuclear marker methods can be analyzed to explore the genetic diversity, in order to provide more basic information for the germplasm resources of N. taihuensis.The results showed a high haplotype diversity and a low nucleotide diversity between the


Rhanterium eppaposum Oliv. (Arfaj), a perennial forage shrub, have depleted due to unethical human interventions and climate change in Kuwait. Therefore, there is an urgent need to conserve this native plant through the assessment of its genetic diversity and population structure. Genotyping by sequencing (GBS) has recently emerged as a powerful tool for the molecular diversity analysis of higher plants without prior knowledge of their genome. This study represents the first effort in using GBS to discover genome-wide single nucleotide polymorphisms (SNPs) of local Rhanterium plants to assess the genetic diversity present in landraces collected from six different locations in Kuwait. The study generated a novel set of 11,231 single nucleotide polymorphisms (SNPs) and indels (insertions and deletions) in 98 genotypes of Rhanterium. The analysis of molecular variance (AMOVA) revealed ~1.5% variation residing among the six populations, ~5% among the individuals within the population and 93% variation present within the populations . Bayesian and UPGMA analyses identified two admixed clusters of the tested samples; however, the principal coordinates analysis returned the complete population as a single group. Mantel’s test returned a very weak correlation coefficient of r2 = 0.101 (p = 0.00) between the geographic and genetic distance. These findings are useful for the native species to formulate conservation strategies for its sustainable management and desert rehabilitation.Natural populations of  Land degradation is an issue of global concern and poses a serious threat to flora biodiversity, soil fertility and food security in arid lands . AnthropKuwait harbors an important genetic resource that contains a valuable gene pool of drought and salt tolerance. Nonetheless, Kuwaiti foliage has been severely degraded, and significant changes in vegetation communities have been observed since 1974 . FurtherRhanterium epapposum, Oliv. (Asteraceae), locally known as “Arfaj”, is the national plant of Kuwait [Rhanterium, thereby reducing it from 30.6% to 2.1% [f Kuwait  and growf Kuwait . The dipf Kuwait . The plaf Kuwait . Camels f Kuwait . Human if Kuwait  have aff to 2.1% ,15. Prev to 2.1% ,17. It iTechnological advancement has brought about a paradigm shift in the way of assessing genetic diversity. The conventional morphological markers have been replaced by the use of first-generation molecular markers such as simple sequence repeats (SSRs), amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPDs) ,19,20. DRhanterium eppaposum in Kuwait, the Gulf Cooperation Council (GCC) and other tropical countries [R. eppaposum populations to estimate genetic diversity and to identify genetic structures within the wild landraces growing in Kuwait. The findings provide a basis to formulate strategies for its conservation and rehabilitation.There are very few published studies on the genetic diversity and population structure of ountries ,14,15,25R. eppaposum through the technique of GBS.To the best of our knowledge, in this study, SNP markers were generated and used for the first time in the non-model species of For the 99 samples, sequencing of the GBS library yielded 40 million raw reads with an average base quality of Q 30 . The seqThese variants were distributed on 44 scaffolds of 50 GB each . MaximumA cut-off of 0.05 for the allowed level of variants for molecular diversity analysis further filtered the usable number of SNPs at each location. Maximum numbers were 8841 at Al Kabd, followed by 8838 at Al Salmi, 8583 at Om Qaser, 8547 at Al Maqwa, 8078 at SANR and 7536 at Mina Abdulla. Among these, approximately 26% were polymorphic at Al Salmi and Al Kabd, 21 % at SANR and Mina Abdulla, 16.5% at Om Qaser and 14% at Al Maqwa .Rhanterium populations. In addition to this, the inbreeding coefficient (FS) ranged from 0.129 to 2.83 (mean −1.095 p = 0.00).There were more transition-type SNPs (28.40%) than transversion-type (19.5%), and the ratio of Ts/Tv was 1.5 (2351/1618). Substitutions were higher than the transitions and transversions. The observed theta (pi) was higher than the expected theta (S) at all the locations yielding a negative Tajima’s D (D < 0). From this, we interpret that there is an abundance of rare alleles in all the ST of 0.02, FIT of 0.06 and a FIS of 0.05 at a confidence interval of 95% was obtained. The values of fixation indices were in agreement with molecular diversity indices  was at a minimum (0.004) between Al Salmi and Al Kabd and a maximum (0.04) between Om Qaser and Mina Abdulla. Almost comparable FST of 0.032 and 0.033 were observed between SANR and Mina Abdulla and between Al Maqwa and Mina Abdulla  from the short-read aligned BAM files (Phred + 33) into a variant call format . Using tHaloxylon salicornicum growing in similar environments also depicted higher locus missing rates [In this study, the number of polymorphic SNPs in Al Kabd populations was the highest. This confirmed our previous predictions based on RAPD, SRAP and ISSR markers ,13,14. Hng rates .Rhanterium populations naturally grow under stressed conditions; consequently, methylation events are expected to be high. The voluminous transitions and exceedingly repetitive sequences can be considered an evolutionary footprint of methylation events [H. salicornicum populations [Acacia pachyceras growing in Kuwait [The average transition/transversion (Ts/Tv) ratio was found to be 1.5. The mutation of methylcytosine to uracil and eventually to thymine might result in transition abundance within a species . In Eukan events ,41,42. An events  and wildulations . A highln Kuwait .Rhanterium population in Kuwait. This was supported by the inbreeding coefficients, which indicate a higher number of observed heterozygotes within the populations [The negative Tajima’s D values are encouraging and suggestive of excessive low-frequency polymorphisms or rare alleles . This coulations .Rhanterium collected all over Kuwait is likely to originate from a common ancestor. The minor genetic differences are believed to be due to recombination events occurring during sexual reproduction [Rhanterium landraces are genetically similar, justifying the need to extend the diversity of future rehabilitation projects by integrating alternative genotypes from neighboring countries such as Saudi Arabia, UAE, Oman, Qatar, etc. In a similar study conducted with ISSR markers [Haloxylon also demonstrated a weak population structure in Kuwait [Rhanterium populations, which agrees with studies on highly outcrossing species [Rhanterium [The findings from a population structure analysis suggest that the large, genetically homogenous group of 98 genotypes of oduction . These f markers , low genn Kuwait . Our ana species . Overallanterium .2. The topography is flat with slightly uneven desert dunes. Frequent sandstorms and winds (Toz) are quite common. These features facilitate the easy dissemination of seeds to otherwise far areas [There was no clear congruence between genetic structure (clusters) and geographic origin in our analyses, i.e., no grouping was based on spatial origin. This was confirmed through the results of Mantel’s correlation test. No isolation by distance pattern was observed. Regular exchange of pollens, critters and reproductive material taking place between these spatially apart but outcrossing species is the only explanation that can be given for this in the present circumstances. Kuwait is a country with a small land area of 17,000 kmar areas . The comar areas . These rar areas .Rhanterium community much in Kuwait. The phenotypic characterization of these populations, however, needs to be undertaken.We speculated differences in the population structure owing to the various soil types in which these populations are growing. The soil types of OmQaser, SANR, KABD, Al Maqwa, Mina Abdulla and Al Salmi were calcigypsids, haplocalcids, torripsaments, acquisalids and Petrogypsids, respectively . It appeRhanterium landraces were used in this study. They were collected from the areas of Al Kabd (n = 21), Sabah Al Ahmed Nature Reserve , Om Qaser (n = 8), Al Maqwa (n = 8), Al Salmi (n = 24) and Mina Abdulla (n = 19). The GPS coordinates of each specimen were recorded and used to mark the locations on Google Maps , as per the manufacturer’s instructions, from young leaves [®BR dsDNA Assay .A set of 99 Kuwaiti gle Maps . The GPSg leaves . All theThe GBS was performed at the University of Minnesota Genomic Centre (UMGC), USA. Genomic DNA was digested using the restriction enzymes PstI + BtgI , and barcoded adapters were ligated to each DNA sample using T4 ligase . Dual indexed SBG libraries for R. eppaposum were pooled and loaded across 4 lanes of a 150 bp single read sequencing run on a NextSeq 550  instrument with a coverage of 10x generating ~40 M raw reads.In-house-developed Gopher-pipelines by the University of Minnesota Informatics Institute in collaboration with the University of Minnesota Genomics Center and the Research Informatics Solutions (RIS) group at the University of Minnesota Supercomputing Institute were used for sequence alignment and variant calling . Per-basThe analysis of molecular variance (AMOVA) was used to partition the genetic variation into inter- and intra-gene pool diversities using Arlequin V3.5 software . For theRhanterium in Kuwait. The variation residing among these populations was found to be 1.5%. Further analysis grouped the assayed samples into three genetic clusters with admixed populations. These results can enhance genotype selection for increased genetic variation and for desert rehabilitation. The findings in this study can also aid in the application of GBS in the characterization of non-model plants with complex genomes.With the application of GBS, it has been possible to generate 10,568 SNP markers for diversity analysis of