Rhammatocerus brasiliensis shows polymorphism of B chromosomes, but the magnitude of B-chromosome occurrence and the factors that may contribute to their dispersion in the species remain unknown thus far. The present study analyzed the occurrence and dispersion of B chromosomes in R. brasiliensis individuals from 21 populations widely distributed in the Brazilian Northeast. The genetic connectivity between 10 populations was verified through analysis of ISSR markers from 200 individuals. Of the 21 populations, 19 presented individuals with one B chromosome, three with two, and one with three B chromosomes. The B chromosome is of medium size and constitutive heterochromatin (CH) located in the pericentromeric region. A variant B chromosome was observed in three populations, similar in size to that of chromosome X, gap and CH, and located in the terminal region. B chromosome frequencies in different populations varied from 0% to 18,8%, mean 8,5%. The wide distribution of the B chromosome is likely a consequence of the positive gene flow among the analyzed populations. B-chromosome occurrence in populations of R. brasiliensis possibly follows the population genetic structure of the species and, owing to the existence of a variant, its origin may not be recent.The grasshopper  B chromosomes, also known as supernumerary chromosomes, are extra elements of the karyotype. These chromosomes do not obey the Mendelian laws of inheritance and do not present homology with the other chromosomes of the A complement  and Eyprepocnemis plorans  of this family are used as models to better understand the occurrence, origin and evolution of the B chromosomes    B chromosomes are dispersed among R. brasiliensis populations according to gene flow existing among them; 2) based on the relatively low frequency of B chromosomes in R. brasiliensis, as verified in different populations by rnambuco , and Bahrnambuco , R. brasR. brasiliensis individuals from 21 populations from the Brazilian Northeast, of which 10 were also analyzed regarding population genetic structure. In addition, the position of the constitutive heterochromatin (CH) was established in the B chromosomes of the species.With this in mind, we analyzed the occurrence, frequency and distribution of B chromosomes in R. brasiliensis (Bruner 1904) were collected from 21 populations in the Brazilian Northeast, of which 13 were located in the state of Pernambuco (PE), one in Sergipe (SE), one in Alagoas (AL), one in Paraíba (PB), one in Ceará (CE), one in Piauí (PI), and three in Bahia (BA) (Specimens of hia (BA) . Of thesThe grasshoppers were collected with the help of an entomological net and transported to the Laboratório de Biodiversidade e Genética de Insetos (LBGI), Instituto de Ciências Biológicas da Universidade de Pernambuco. The collections were permitted by ICMBio/SISBIO under the license number 16278-1.R. brasiliensis were analyzed by conventional staining. First, the classic technique of testicular follicle squashing was performed, consisting in the maceration of the gonads in one drop of 45% acetic acid. Subsequently, the slides were subjected to heat of 65 °C for approximately 3 min for fixation of the chromosome preparation onto the slide, then stained with 5% Giemsa. The C-banding technique was performed according to 2] altered to 2 min, in individuals carrying B chromosomes from the populations of VIT and LC (Pernambuco – PE).The karyotypes of 590 adult male specimens of The occurrence, frequency, and distribution of B chromosomes were analyzed in a total of 1.274 specimens. Of these, 394 were individuals from 10 populations being analyzed for the first time  and 196 were individuals from four populations with samplings being expanded . The data for the remaining specimens were obtained from the literature  was performed to estimate the maximum and minimum number that the frequency of B chromosomes may reach in an analysis with a population size of 1.000.000 specimens.R. brasiliensis specimens was extracted from the leg muscle according to protocol described by Hind III marker (Fermentas Life Sciences).The genomic DNA of 2, 0,2 mM of dNTPs , 1 pmol of primer  and 1 U Taq polymerase (Invitrogen). The reactions were carried out in a thermocycler (Biosystems) as follows: Standard program at 94 °C for 4 min; 35 cycles at 94 °C for 30 s; annealing step (with temperature varying for each primer) for 45 s; extension at 72 °C for 2 min; and final extension at 72 °C for 7 min. The PCR products were visualized in 1,8% agarose gel using a transilluminator (Gel Logic/Carestream MI SE).Polymerase chain reaction (PCR) amplification was performed from a total volume of 10 μL containing 5 ng of DNA, 1X PCR buffer, 5 mM of MgClR. brasiliensis individual, with 33 presenting high numbers of amplified DNA fragments with good resolution. Selection of the most informative primers was accomplished by applying the 33 pre-selected primers to a sample of nine specimens from three populations . Based on the genotyping, the data were grouped into a binary matrix of presence and absence. The analysis of the most informative primers was carried out according to the parameters number of total, monomorphic and polymorphic DNA fragments, and polymorphism indices: Polymorphism Information Content (PIC)  were tested in one nt (PIC)  and Resont (PIC) , Table 1loci were excluded from the genetic analysis.The reproducibility of the ISSR markers was verified through analyses based on two independent PCR amplifications for all seven primers that were applied to all individuals of the studied populations. Non-reproducible E), Wright's fixation index (FST) and molecular variance (AMOVA) were calculated with the software Arlequin 3.5 (ST) were calculated in the software Popgen 1.31 (ST) and inbreeding  differ from zero; ii) model =0, which assumes absence of inbreeding within the populations; iii) model theta=0, based on the absence of population differentiation; and iv) the free model, where the values are chosen randomly, independent of a priori information. The choice of the most suitable model was based on the parameters deviance information criterion (DIC), an analog of the Akaike information criterion (AIC) in the selection via Bayesian models, and Dbar, a measure of the level of adjustment with which the model adapts to the analyzed data .The genetic diversity index (GD), mean expected heterozygosity , medium (M4 – M8) and small pairs (P9 – P11). In addition, 2n = 24, X0 + B was seen in 107 individuals, with the B chromosome being acrocentric and of medium size  identified by Fisher's pairwise exact test, namely: ITA–JP, 0.032; GRA–SUR, 0.031; GRA–ST, 0.012; GRA–OUR, 0.045; GRA–MAC, 0.008; GRA–JP, 0.007; GRA–JN, 0.031; GRA–PIC, 0.045; and BEZ–JP, 0.038  and SAL–PE the lowest (2.32%). The values for total frequency of males with B chromosomes in the populations varied from 0% to 18.8%, with a mean of 8.5% . A signiP, 0.038 .The C-banding procedure revealed positive C bands in the pericentromeric region of all chromosomes of the A complement, as well as in the B chromosomes . An addiE) in the populations was 0.311, with OUR–PE and MAC–AL presenting the highest and lowest values, corresponding to 0.36 and 0.28, respectively . Among the 95 analyzed loci, 71.05% were polymorphic, with the populations OUR–PE and ARA–SE presenting the highest and lowest percentages of polymorphism (90.5% and 13.6%), respectively . In addiectively .ST, AMOVA) and the dispersion capacity of R. brasiliensis  reached 15.32% among the populations and 64.68% within them. Moreover, low genetic differentiation and high gene flow were observed upon analysis of the fixation index, Fnd ST–PE . The ManThe genetic clustering analysis performed with the software Structure presented K=2 as highest value, according to the method described by R. brasiliensis is well represented in Acrididae grasshoppers . Moreover, studies in other grasshopper species such as Eyprepocnemis plorans  with four acrocentric B chromosomes , with two acrocentric B chromosomes  presented variation between 0.285 in MAC–AL and 0.363 OUR–PE. These values are similar to those described for other insects, such as the grasshoppers Pezotettix giornae      and JN–CE (0.290). Moreover, the total expected heterozygosity (HT= 0.298) was greater than the subpopulation heterozygosity (HS= 0.270), indicating that there may be a deficit of heterozygotes.As previously observed in other organisms  . AlthougR. brasiliensis is related to its population genetic structure, given the wide dispersion of the B chromosome verified in the karyotyped individuals, positive gene flow (Nm = 2.3869), and low genetic differentiation FST (0.15) and GST (0.17) among the analyzed populations. Owing to the migratory behavior of R. brasiliensis , constitute the preferred food of grasshoppers , Dichromorpha viridis  and Orphulella pelidna   and Ramburiella hispanica , a positive relationship was also observed between the distribution of plants used as food and the genetic connectivity between populations (The data from molecular variance (AMOVA), which was greater within the populations (84.68%), when associated to Structure data, corroborate the low indices of genetic differentiation, where the presence of a mixture of two genetic profiles is related to migratory habit, wide mobility, and flight capacity of iliensis , 1995. Nr, 1838) . For othulations .R. brasiliensis individuals seem to be capable to overcome these obstacles and maintain a positive gene flow among the analyzed populations. This way, no correlation was observed between the occurrence of B chromosomes and the distribution of R. brasiliensis in different landscapes. For this species, the altitude of the analyzed populations did not seem to influence the frequency of B chromosomes either. This observation differs from that of Although the populations of Pernambuco and other states occupy different altitudes (range 0 to 999 m), as well as potential geographic barriers , R. brasiliensis, the broad distribution of B chromosomes indeed arises from the extant gene flow among the analyzed populations. The migratory habit coupled to the high flying capacity of R. brasiliensis, besides the wide offer of food resources in the sampled localities, are factors that certainly contributed to the genetic connectivity of its populations, and consequently for the broad dispersion of the B chromosome. A variant B chromosome, with CH blocks in the pericentromeric and terminal regions, was detected and is probably currently in the regeneration stage of the cycle. The presence of one such variant B chromosome reinforces the hypothesis of the non-recent origin of the B chromosome, considering that successive generations are necessary for such a chromosome to be nearly neutralized, accumulate modifications and be dispersed among distinct populations.Based on literature evidence that B chromosomes are only transmitted vertically, it is suggested that, for the species


Phragmites australis were used to survey putative native populations of the species in Quebec, and chloroplast DNA (cpDNA) PCR-RFLP analyses identified only one native population, whereas the same analyses revealed that the Kashmir populations are invasive. We compared the native population of P. australis in Quebec (QN), ten populations invasive to Quebec (QE), and five populations invasive in Kashmir, India (KE) using morphometric traits. Using nine cpDNA microsatellite loci, we also compared nine KE populations, ten QE populations, and the QN population. Phenotypic variation was observed among and within populations. Only dry mass of flowers varied across regions. Characterization of morphotypes defined three distinct haplotypes. A bimodal distribution of stem diameter (SD), internode length (IL), leaf length (LL), and leaf width (LW) suggests that a major gene may control growth traits or occurrence of co-selection. High genetic differentiation was observed between populations (RST = 0.353) and haplotypes (RST = 0.133 to 0.418), indicating limited gene flow and probable local adaptation. Principal coordinates analysis and the neighbor-joining phylogenetic tree clearly distinguished the three haplotypes. Among-populations phenotypic difference (SPT) was lower than overall RST for plant height, SD, and fresh and dry mass of flowers and seeds, whereas PST estimates for LL and LW exceeded among-populations RST, suggesting divergent selection, while local adaptation might have occurred in IL, LL, and flower masses. Genetic drift probably influenced among-populations IL differences. The origin of differences in traits influencing competitive success between invasive and native wild populations of alien species is subject of debate. Herbarium-based information sources from 2005 onwards about nativity and distributional range of  The spread of invasive alien species (IAS) has received a great deal of attention, given the adverse ecological and economic effects that they can impose on invaded ecosystems ,2. Why iThe phenotypic differences within and among populations of invasive species in their native and non-native ranges are widely speculated ,12. The PST ;  Trin. ex Steud; Poaceae) is a polyploid perennial grass species with clonal architecture  were amplified using the primers described by Saltonstall [RsaI for the trnLb region and HhaI for the rbcL region following the manufacturer’s protocol . Restriction fragments were electrophoresed in ethidium bromide-stained 3% TAE agarose gels and visualized using UV light. We also searched for matches with a Blast tool provided by NCBI. To confirm the results, PCR products were sequenced on an ABI 16-capillary genetic analyzer 3130XL . Sequences were edited and aligned using BioEdit version 7.0.5 [P. austalis haplotype. Phylogenetic analyses were conducted using MEGA 7 software [To verify whether tonstall  was usedtonstall . PCR proon 7.0.5 . The BLAon 7.0.5  was usedsoftware , and thesoftware .P. australis across regions, we used a set of 9 cpDNA microsatellite loci, viz., PaGT-4, PaGT-8, PaGT-9, PaGT-11, PaGT-12, PaGT-13, PaGT-14, PaGT-16, and PaGT-22, which were developed by Saltonstall , 0.5 mM Fluorochrome , and 1 unit of Taq DNA polymerase (Sigma-Aldrich). Multiplexes PCR employed an annealing temperature of 55 °C and amplified loci PaGT 4 with PaGT 14, and loci PaGT 8 with PaGT 13. Single PCR employed an annealing temperature of 55 °C for loci PaGT 9 and PaGT 12, and 56 °C for loci PaGT 11, PaGT 16, and PaGT 22. Single PCRs were subjected to the same reaction conditions as the multiplexes  Ampijkl is the average value for the dependent variable for the ith region (Quebec or Kashmir), for the jth population nested in the ith region, for the kth haplotype (invasive or native) nested in the jth population and further in the ith region, and for the lth plant nested in the kth P. australis haplotype nested in the jth population further nested in the ith region; μ is the overall mean; τi, βj(k), δ(ij)k and e(ijk)l corresponded, respectively, to region effect , to population effect , to haplotype effect , and to plant effect , the last term being the source of experimental error in the model. Post hoc multiple comparison of means was performed using Duncan Multiple Range Tests. PST values for each measured character were estimated using the following formula [VAP is the among-population variance and VWP the within-population variance.Variance components were estimated in the Statistical Analysis System  with Pro formula ,96:PST =h2) estimate was computed as follows, as P. australis dispersal most often occurs through seeds than stolon and rhizomes [To determine whether putative phenotypic differences are due to adaptive processes or not, an analogous of narrow-sense heritability , the mean number of alleles (A), the mean number of alleles per polymorphic locus (Ap), allelic richness (AR), and observed and expected heterozygosities (HO and HE) for each population [RST; [i the frequency of the allele i in all populations. Genetic similarity (GS) [ij is the number of fragments common to individuals i and j, and (Ni + Nj) is the total number of fragments in both individuals.Given that sion 1.3 . These spulation , togetheon [RST; ). Prior on [RST;  were eston [RST; . To meason [RST; :PIC = 1 ity (GS)  was calcP. australis populations through the software microsatellite analyser (MSA) version 4.05, using the formula of Nei and Li [To assess the phylogenetic relationships between individuals, the distance matrix was computed as genetic distance (GD) among i and Li , as follxy is the number of fragments common to individuals x and y, and (Nx + Ny) is the total number of fragments in both individuals.Where NP. australis using Phylip and Mega 6 software. Relationships between genetic and geographic distance were investigated using Mantel tests in the Isolation By Distance Web Service  of the 20 populations of WS 3.23; ). MantelP. australis exhibit better growth-related traits, especially fresh and dry mass of flowers and seeds. PCoA based on Bruvo distances clearly distinguished invasive and native populations from the sample regions. Highest levels of genetic diversity were found in invasive populations of P. australis in Kashmir Himalayan wetlands. The observed among-population phenotypic differences in the surveyed P. australis are explained by divergent selection, local adaptation, and genetic drift. Understanding the phylogeographic basis of such a diversity gradient remains an open challenge. Cluster and structure analysis delineated all populations of native and invasive P. australis from Quebec and Kashmir into five main groups, shedding some light on their evolutionary relationships. The important level of the genetic diversity reported in the present study should be considered in managing the native and invasive P. australis populations in Quebec and Kashmir. The results provide a first step for further investigations based on sampling from a larger global range, including native European populations of P. australis.In conclusion, it appears that invasive populations of


Nelumbo nucifera Gaertn., a perennial aquatic macrophyte species, has been cultivated in several Asian countries for its economic importance, and medicinal uses. Two distinct ecotypes of the species are recognized based on the geographical location where the genotypes are adapted, i.e., tropical lotus and temperate lotus. The genetic diversity levels and differentiation of the tropical lotus from poorly studied geographic regions still remain unclear. Here, the population genetic diversity and structure of 15 tropical lotus populations sampled from the previous understudied natural distribution ranges, including India, Thailand, and Australia, were assessed using nine polymorphic SSR markers.P = 0.004). Besides, AMOVA analysis revealed a higher variation among populations (59.98%) of the three groups. Overall, the populations used in this study exposed a high level of genetic differentiation (FST = 0.596).The SSR markers used to genotype the 216 individuals yielded 65 alleles. The highest and lowest genetic diversity estimates were found in Thailand and Indian populations, respectively. STRUCTURE analysis revealed three distinct genetic clusters, with relatively low admixtures, supported by PCoA cluster analysis. Low levels of gene flow (mean Nm = 0.346) among the three genetic clusters signified the Mantel test for isolation by distance, revealing the existence of a positive correlation between the genetic and geographic distances  could be explained by limited gene flow and clonal propagation. Conserving the available diversity using various conservation approaches is essential to enable the continued utilization of this economically important crop species. We, therefore, propose that complementary conservation approaches ought to be introduced to conserve tropical lotus, depending on the genetic variations and threat levels in populations.The nine polymorphic microsatellite markers used in our study sufficiently differentiated the fifteen tropical  Nelumbo nucifera Gaertn. (Lotus), a perennial aquatic macrophyte species, belongs to the genus Nelumbo in the family Nelumbonaceae. Cultivation of lotus dates long back in history as an ornamental and vegetable in several Asian countries  evaluate the level of genetic diversity of the tropical lotus populations from the previous poorly studied natural distribution ranges, and (ii) estimate the degree of differentiation and population structure of N. nucifera.Here, we genotyped 15 tropical N. nucifera populations comprising of 216 individuals were sampled from the natural distribution range in Australia, India, and Thailand  for the 15 N. nucifera populations were estimated. GenAlEx version 6.5 , expected heterozygosity (HE), observed heterozygosity (HO), the number of alleles per locus (NA), Shannon’s information index (IS), the number of private alleles (Np), and inbreeding coefficient (FIS).The Cervus version 3.0 program , the analysis of molecular variance (AMOVA) was done in Arlequin version 3.1  (Earl N. nucifera was constructed and displayed in DISTRUCT version 1.1 (Rosenberg To examine the level of genetic variation among r/) Earl , was user/) Earl  while emN. nucifera populations were assessed using the Bottleneck version 1.2.02 program  among the genetic groups (K = 3), in the previous 4Ne generations (Ne = effective population size), was estimated by MIGRATE-n version 3.7.2 program Beerli . A Bayesa Beerli .All SSRs markers observed significant Hardy–Weinberg deviations. The nine microsatellite markers used in the present study yielded high polymorphisms in all populations. For each microsatellite marker, the effective number of alleles (Ne) varied from 1.223 to 1.956 (mean = 1.475). Observed (Ho) and expected heterozygosity (He) estimates ranged from 0.067 to 0.631 and 0.140 to 0.474, respectively    and the number of observed (NA) per population ranged from 1.140 to 2.023 and 1.333 to 2.667, respectively. The heterozygosity levels, observed and expected, ranged from 0.044 to 0.824 and 0.081 to 0.470, respectively. The average expected heterozygosity (HE = 0.358) was higher in Thailand than in both India and Australia. Similarly, Shannon’s information index varied from 0.129 to 0.730. Private alleles were detected in nine of the 15 populations examined. Eleven of the 19 observed private alleles were found in populations sampled from Thailand, and population T4 had the highest count (6) . This observation reflects the presence of high cross-pollination levels among populations. However, only four populations  had positive FIS values, suggesting that there existed inbreeding among the individuals of these populations. Populations T4 and I3 had the highest and lowest genetic diversity (HE), respectively. Overall, the microsatellite markers showed a low genetic variation in N. nucifera populations.Eleven populations showed low levels of coefficient of inbreeding , except for two Australian populations that were assigned together with the Indian populations  and lowest (0.005) genetic distance was found in the populations sampled from Australia  (Table P = 0.004)  than within populations 40.02%) of the three countries, supported by high levels of genetic differentiation (F6) Table . In addi6) Table  and geog0.02% of N. nucifera populations revealed that the mutation scale effective population size (θ) for the three genetic clusters were 0.09137, 0.09711 and 0.09759, respectively. Among the populations the computed mutation scaled migration rate (M) ranged from 4.163 (M2- > 1) to 23.631 (M2- > 3). The gene flow was higher from India to Thailand populations (Nm = 0.577). Low gene flow occurred from India to Australia populations (Nm = 0.095). The study also found bidirectional gene flow among the genetic clusters. Overall, low gene flow (mean Nm = 0.394) was obtained among the three genetic clusters of N. nucifera populations. The details of the historical gene flow analysis among populations are shown in Additional file The results of the bottleneck analysis are outlined in Additional file N. nucifera populations sampled from natural distribution ranges in Australia, India, and Thailand. The genetic diversity (HE) values varied from 0.160–0.277; 0.081–0.216 and 0.254–0.470, in Australia, India and Thailand populations, respectively. This highlights the presence of wide genetic variability within each genetic group. The overall findings of this study exhibited low genetic diversity levels (mean HE = 0.245), which is mainly explained by low gene flow levels and clonal propagation. The result of the inbreeding coefficient analysis revealed that A1, A2, I1, and I2 populations had significant positive FIS values, which may be attributed to the high level of matings between closely related individuals. This phenomenon might have contributed to the observed low level of genetic diversity in these populations. The mean expected heterozygosity found in this study is greater than that recorded for the tropical lotus (HE = 0.152) by Liu et al. (E = 0.320) reported for the same ecotype by Yang et al. (Ottelia acuminata (HE = 0.351) by Zhai et al. (Ottelia acuminata var. jingxiensis (HE = 0.441) by Li et al. (E) in this study was found among the populations from Thailand (HE = 0.360), whereas the least genetic diversity was found in Indian populations (HE = 0.156). The higher genetic diversity found in Thailand populations might be related to the inherent broad genetic base of the germplasm or the presence of suitable growing conditions for the species in this country. The highest genetic diversity values (HE = 0.470), was found in T4. From the result, we can suggest that the higher genetic diversity level revealed in this population might have been accumulated during a long evolutionary history of the population. The highest genetic diversity and private alleles , were found in T4 , generally greater than the current study PIC. Overall, the present results portrayed that the SSR markers used are handy for genetic diversity studies in the N. nucifera germplasm.The mean PIC value (0.593) detected in this study indicates that the markers are effective for population genetic studies of u et al. , which ou et al.  on N. luN. nucifera contributed to low genetic diversity. Positive FIS values observed in four N. nucifera populations  in the present study is lower than the previous findings reported for N. nucifera  and the low gene flow (0.346) found in this study contributed to the observed genetic structure, supported by significant IBD patterns in the study area . Zhang et al.  have already been affected by fragmentation.Gene flow may have a significant impact on the genetic differentiation of the local populations Storfer . It playi et al.  also repE) within crop species plays a critical role in crop improvement programs , and to those that have exhibited bottlenecks. We recommend that complementary conservation approaches should be effected to maintain endangered and the declining populations of tropical lotus.Further studies using additional samples from all the species distribution areas and more markers should be conducted to gain more insights into the population genetic structure of Additional file 1: Table S1. Polymorphism information of the nine SSRs markers used in the present study.Additional file 2: Table S2. Pairwise fixation index (FST) values between populations of tropical N. nucifera , and Nei genetic distance .Additional file 3: Table S3. Bottleneck analysis in 15 tropical N. nucifera populations.Additional file 4: Table S4. Migrate-n results of historical gene flow among the three genetic groups.


Aedes aegypti is among the best‐studied mosquitoes due to its critical role as a vector of human pathogens and ease of laboratory rearing. Until now, this species was thought to have originated in continental Africa, and subsequently colonized much of the world following the establishment of global trade routes. However, populations of this mosquito on the islands in the southwestern Indian Ocean (SWIO), where the species occurs with its nearest relatives referred to as the Aegypti Group, have received little study. We re‐evaluated the evolutionary history of Ae. aegypti and these relatives, using three data sets: nucleotide sequence data, 18,489 SNPs and 12 microsatellites. We found that: (a) the Aegypti Group diverged 16 MYA (95% HPD: 7–28 MYA) from its nearest African/Asian ancestor; (b) SWIO populations of Ae. aegypti are basal to continental African populations; (c) after diverging 7 MYA (95% HPD: 4–15 MYA) from its nearest formally described relative (Ae. mascarensis), Ae. aegypti moved to continental Africa less than 85,000 years ago, where it recently  split into two recognized subspecies Ae. aegypti formosus and a human commensal, Ae. aegypti aegypti; (d) the Madagascar samples form a clade more distant from all other Ae. aegypti than the named species Ae. mascarensis, implying that Madagascar may harbour a new cryptic species; and (e) there is evidence of introgression between Ae. mascarensis and Ae. aegypti on Réunion, and between the two subspecies elsewhere in the SWIO, a likely consequence of recent introductions of domestic Ae. aegypti aegypti from Asia. The vast majority of work has been on these two subspecies that we here refer to as Aedes aegypti sensu stricto (s.s.), to distinguish them from the newly studied populations from the islands in the southwestern Indian Ocean, which we will refer to here as Aedes aegypti sensu lato (s.l.). We note that while the clear‐cut distinction of the subspecies designations is questionable in some instances  on the islands east of Africa have received comparatively little attention  following the manufacturer's recommendations and including the optional RNase A step, and stored at −20°C until further use. Aedes pia is a morphologically distinct member of the subgenus Stegomyia and is basal to the other two members of the Aegypti species group: Aedes aegypti and Aedes mascarensis , and we used previously published sequence data from Soghigian et al.  for which fossil calibrations are known. We used nucleotide sequence data for estimating the evolutionary relationships of Stegomyia, rather than SNP data detailed elsewhere, as the SNP chip data captured from samples more distant from Ae. aegypti s.s. is limited  and because fossil calibrations are not available for the Aegypti Group but are for Aedes and other Culicidae . The alignment derived from Soghigian et al. , generated following sequencing protocols detailed more thoroughly in Soghigian et al. , and a group for "island" populations of Ae. aegypti originating from Europa, Madagascar, Mayotte and Réunion.For fine‐scale population structure and admixture analysis, we generated a SNP data set with populations of us Table . For popAe. pia, we overrode default software settings on allowable missing data for a given sample ; and second, we utilized the OTV Caller plugin for Axiom Analysis Suite to exclude genotype calls from SNPs that might contain off‐target variants, such as those that could occur due to insertions, deletions or mutations at probe sites. Finally, we exported all polymorphic SNPs for downstream analyses.Genotype calls were made using Affymetrix Axiom Analysis Suite 3.1 (Life Technologies–Thermo Fisher Scientific), for both newly genotyped samples and previously studied populations. This integrated software suite calls genotypes from probe florescence values, excluding probes (and thus SNPs) whose fluorescence deviates from expectations, as might result from probe misbinding. For probe quality control and SNP calling, we used default software settings, save for two exceptions: first, in order to include SNPs called from Ae. aegypti  and Africa. Microsatellite genotyping was performed as described previously . We used a nested analysis of molecular variance (AMOVA) from the package poppr , and also visualized alternative K values based on biologically reasonable ancestral clusters due to similarity in K values past K = 4. We also assessed whether the removal of the genetic cluster associated with Aedes pia would provide qualitatively different results from the inclusion of this outgroup species. Additionally, we performed principal component analysis to evaluate genetic structure of populations and species as implemented in the R package adegenet , which was consistent with previous results in Soghigian et al. , for example relative missing data  and population clustering results.We evaluated the evolutionary relationships among populations of n et al.  that sug2.7Z scores were indicative of admixture between two parent populations to produce the third sibling population. p‐values were calculated from these Z scores with false discovery rate correction for multiple comparisons with the R function p.adjust  could bias estimates of allelic variation between populations, as Treemix uses allele frequency estimates to infer admixture through the F3 test. Using the R function dplyr, we resampled, without replacement, each Ae. aegypti population to four individuals. For each resampling, we conducted F3 tests as implemented in Treemix as we describe above. We resampled our populations 100 times, ran Treemix on each resampled data set and then summarized the resampled mean estimates of the F3 statistic with parametric 95% confidence intervals. This summary method, rather than p‐values, was chosen due to the large number of three‐population comparisons  during resampling.To ensure that specific characteristics of our data set in terms of missing data and uneven sampling did not bias our admixture analysis, we estimated two additional F3 statistics per population triplet and compared them with our complete data set. First, to evaluate whether missing data influenced our Treemix results, we refiltered our data set as above, but with the flag –geno 0 in Plink, thus requiring a SNP position be present in all samples to be included. We then used this data set in Treemix, as above, to estimate F3 statistics and 2.8Ae. aegypti s.s. and their relatives on Europa. Particularly, we were interested in those events that occurred more recently in evolutionary history and for which nuclear and mitochondrial genes did not resolve the divergence times or directionality. Furthermore, Aedes aegypti SNP chip only assays two alleles at each locus and was designed to deliberately emphasize population‐level differences among Ae. aegypti aegypti, possibly neglecting informative variation in African Ae. aegypti. Based on the admixture detected in other island populations (see below), we focused this analysis on comparisons between the Europa population, and populations from East and West Africa. Allele numbers, percentage of missing data, percentage of polymorphic loci, average observed (Ho) and expected (uHe) heterozygosities, and Fis values were estimated in GenAlEx , East Africa (EA) and Europa. The data sets were from: (1) La Lope Forest (Gabon—WA) and Lunyo (Uganda—EA); (2) Ouagadougou (Burkina Faso—WA) and Kisumu (Kenya—EA); and (3–5) independent combinations of randomly drawn individuals from WA  and EA populations . We tested three scenarios . As such, we performed divergence time estimation on the combined nuclear and mitochondrial nucleotide data set. The topology recovered from our divergence time estimate was similar to the likelihood analyses; thus, we report only the results from the divergence time analysis here  and African Stegomyia 39 MYA (95% HPD: 21–64 MYA). Our results support the monophyly of the Aegypti Group, and its affinity to other Afrotropic Stegomyia , with the Ae. aegypti SNP chip . An analysis of molecular variation on our SNP data found significant genetic differentiation among subspecies Aaf and Aaa, island populations and species in the Aegypti Group . Since the results of our primary SNP data set achieved qualitatively the same conclusion to that of the other two data sets investigated, and because of the increased power of the larger sample size in terms of SNPs and number of individuals per population, we report the specific results from the primary data set here . Tests of admixture ; Réunion, with evidence of admixture between Ae. mascarensis and a population of Aaa, with Ae. mascarensis + Jeddah again assigned the lowest Z score ; and Mombasa, the likely result of admixture between a population of Aaf and a population of Aaa, with Johannesburg + Jeddah assigned the lowest Z score . Both the filtered‐for‐completeness data set and the resampled subset of four Ae. aegypti individuals per population found the same likely admixed populations and same originating parent populations having the lowest F3 statistics as in the primary analysis. However, the F3 statistics estimated were lower, concordant with the reduced power from fewer SNPs and/or fewer individuals per population from which to estimate allele frequencies.Due to the aforementioned analyses indicating evidence of admixture in the southwestern Indian Ocean, we utilized F3 tests implemented in Treemix to formally test for potential admixture, with three separate analyses: the primary full SNP data set , a data set with no missing data derived from this data set  and a data set where populations of e Figure  found stAlthough Jeddah was a common contributor in the lowest F3 scenario across all analyses, all Asian populations tested were suggested as putative ancestors of these admixed populations Tables  and S19.Ae. aegypti s.s. in our maximum‐likelihood analysis, diverged prior to continental Aedes aegypti diversifying within Africa, rather than representing a re‐introduction from the continent. We used representative populations from East and West Africa to explicitly model whether populations in Europa diverged prior to, or after, the divergence of East and West African populations of Aedes aegypti on continental Africa  less than 85,000 years ago  to confirm that the population of Europa, sister to 44.1Ae. aegypti have occurred. But to fully understand the history of this mosquito, we need to consider its relationship to other members of the subgenus Stegomyia and, in particular, other members of the Aegypti Group. Our analysis  is less than 20 MY  is less than 9 MY  indicates that the common ancestor of the Aegypti Group was most likely found throughout the islands in the southwestern Indian Ocean.Our analyses on the divergence times of species in the Aegypti Group on Indian Ocean islands Figure  correspo Michon,  and MaurAe. aegypti s.s. entered Africa less than 85,000 years ago, as the divergence of Europa and continental African populations was younger than 85,000 years across all five ABC analyses  and Ae. aegypti formosus (Aaf). This has been speculated to have begun when human settlements developed in sub‐Saharan Africa and water was stored during the dry season. The full separation of subspecies occurred ~500 years when European slave trade brought Ae. aegypti s.s. to the New World beginning in the 16th century  indicate these populations have been on Madagascar for >7 million years.We find strong evidence that  Figures  and 3. A4.3Ae. mascarensis or Ae. aegypti from Sri Lanka) we have underestimated or missed potential admixture events, as the relative F3 statistics inferred from our analysis of resampled populations were slightly lower than those from the full SNP data set. However, our resampling provided qualitatively the same results as our full data set. This indicates that small sample sizes can detect admixture that is consistent with observations from larger samples of the same population, at least given the relatively large number of SNPs in our study.On the islands in the southwestern Indian Ocean, we find evidence for recent introgression among members of the Aegypti Group. These results indicate a component of the admixture is Aaa from Asia, rather than from New World Aaa or from Aaf. In our analysis, Aaa from Saudi Arabia was most often identified as the Aaa parental population in admixture analyses, but we note that the inference of ancestral population origins with Treemix is limited to the populations used in the analysis, and thus, future sampling may yield different results; additionally, all Asian populations we tested had similar strength of support as Saudi Arabia to be the origin of the parental Aaa involved in introgression in these island populations Tables . This inAe. aegypti on Réunion exhibit a particular scutal morph that appears intermediate between Ae. aegypti and Ae. mascarensis . Other Ae. aegypti from Réunion—differentiated from Aaf in scaling patterns—have been reported to be primarily a sylvatic mosquito  to Ae. aegypti s.s. on continental Africa. Moreover, we find strong evidence of a cryptic genetic lineage on Madagascar. It is also clear, however, that while our limited sampling in this region of the world has revealed important insights, more intense sampling and analyses of collections are likely to reveal a more complete picture of the evolutionary history of the Aegypti Group.JS, AGS and JRP designed research. JS, AGS, VR and GLG performed research. AGS, VR, GLG, ABF and JRP contributed samples. JS and AGS analysed data. JS, AGS, VR, GLG, ABF and JRP wrote the manuscript.Supplementary MaterialClick here for additional data file.


Aedes aegypti in continental North America with some of the newest to illuminate the range of genetic diversity and structure that can be found within the invasive range of this important disease vector. Aedes aegypti populations in Florida have probably persisted since the 1600‐1700s, while populations in southern California derive from new invasions that occurred in the last 10 years. For this comparison, we genotyped 1,193 individuals from 28 sites at 12 highly variable microsatellites and a subset of these individuals at 23,961 single nucleotide polymorphisms (SNPs). This is the largest sample analyzed for genetic structure for either region, and it doubles the number of southern California populations previously analyzed. As predicted, the older populations (Florida) showed fewer indicators of recent founder effect and bottlenecks; in particular, these populations have dramatically higher genetic diversity and lower genetic structure. Geographic distance and driving distance were not good predictors of genetic distance in either region, especially southern California. Additionally, southern California had higher levels of genetic differentiation than any comparably sized documented region throughout the worldwide distribution of the species. Although population age and demographic history are likely driving these differences, differences in climate and transportation practices could also play a role.The genetic diversity and structure of invasive species are affected by the time since invasion, but it is not well understood how. We compare likely the oldest populations of  Aedes aegypti, the primary vector of yellow fever, dengue, Zika, and chikungunya.Invasive species are a threat to conservation, human health, and the economy. We present a case study showing that even within an invasive species' range, its genetic diversity and structure can vary dramatically depending on the age of the populations. Specifically, we compare ‘old’ and ‘new’ populations of the invasive mosquito  They are the second most common threat associated with species that have gone extinct since the 1500s  using Axiom_aegypti, a high‐throughput genotyping chip that has 50,000 probes . Microsatellite data for eight of the 15 southern California populations and ten of the 16 Florida populations, as well as SNP data for six of the Florida populations, are being published here for the first time (Table 2.3O), expected heterozygosity (HE), and the inbreeding coefficient (FIS) were calculated for each population using Genepop, and allelic richness was estimated by rarefaction (N = 30) using the software HPRARE v. 1.0  for microsatellites with Genepop v. 1.1.4. and tested for significance using an exact conditional contingency‐table test with the following parameters: 10,000 dememorizations, 500 batches, and 10,000 iterations per batch ) and geographic and driving distances using a Mantel test with 9,999 permutations. Driving distance was calculated by finding the fastest driving routes between pairs of sites using Google Maps.We calculated pairwise genetic differentiation .To explore genetic structure, we conducted twenty independent runs of STRUCTURE v. 2.3.4 for 33.1p‐values for the other 15 loci. Similarly, 59 out of 1,976 (3.0%) locus pairs showed significant evidence of being in linkage disequilibrium after a Bonferroni correction, and there were not enough data to determine the p‐values for 136 locus pairs. These numbers are similar to what we would expect by chance, and there was no microsatellite or microsatellite pair that was consistently problematic. As such, we assumed that these are independent, neutral loci, and all 12 microsatellites were included in the analyses.Considering each population separately, there were 372 microsatellite loci (12 microsatellites × 31 populations) we tested for deviations from the Hardy–Weinberg equilibrium and 2,112 microsatellite locus pairs we tested for linkage disequilibrium. Four out of 357 (1.1%) microsatellite loci across populations were out of Hardy–Weinberg equilibrium after a Bonferroni correction, and there were insufficient data to determine the t test, p < 10–5). There was no statistically significant difference in the inbreeding coefficient between the two regions on average (p > .05). Four Florida sites and eight southern California sites had negative FIS values, indicating less relatedness than expected under random mating  were significantly higher in Florida than in southern California Table   calculated with microsatellites were significantly greater than zero (p < .05), except for Palm Beach County 2013 and 2018 (p = .22) (Dryad doi:10.5061/dryad.pnvx0k6jn). The FST values (mean ± SD) among Florida sites (0.033 ± 0.018) were significantly lower than the FST values among southern California sites (0.23 ± 0.10) . Since three of the sites are represented twice in the dataset, we repeated the analysis after excluding the older site from each pair, and the difference persisted (p < 10–32). FST values for pairs between Florida and southern California (0.18 ± 0.057) were significantly lower than pairs within southern California (p < 10–4). Similarly, all pairwise FST values calculated with SNPs were significantly greater than zero. The FST values (mean ± SD) among Florida sites (0.038 ± 0.013) were significantly lower than the FST values among southern California sites .All pairwise FST and genetic distance  using microsatellites. According to a Mantel test, there was a negative relationship between linearized FST and geographic distance within southern California , and no significant relationship between linearized FST and driving distance . However, a plot of geographic distance versus genetic distance suggests a “U” shape rather than a negative relationship , although the relationship appears negative in a plot of geographic versus genetic distance .There was a slight and marginal correlation between linearized p Figure . Using SK levels, Montclair clusters with Mission Viejo CA (e.g. K = 3) or as its own group (e.g. K = 5) (not shown). Within Florida, Bayesian clustering analysis identified one group . For southern California microsatellites, PCA showed differentiation among some populations, especially Santa Ana 2015, Garden Grove, and Mission Viejo . However, in southern California, Santa Ana 2015 and Santa Ana 2017 cluster separately using STRUCTURE . Additionally, the mean pairwise FST values within southern California sites collected in 2014 and 2015 is 0.30 ± 0.12, which is significantly higher than the mean pairwise FST values within sites collected in 2015 and 2016 (0.18 ± 0.065) .In terms of temporal stability, there was no indication of genetic difference between the two years for Palm Beach County or Key West using STRUCTURE, PCA, or DAPC. The pairwise E Figure  and k‐meg Figure , and the4Aedes aegypti mosquito provides a clear example of this phenomenon. This species evolved from a forest‐dwelling mosquito in Africa to an anthrophilic disease vector found on every continent but Antarctica. It is the primary vector for numerous debilitating and costly diseases; the global burden dengue alone is immense with around 100 million new cases each year  create environmental, economic, and health problems around the globe, and the FST , and more southern California populations showed evidence of inbreeding  is higher than mean FST values between Africa and other continents (0.11–0.14).Contrary to expectations, Florida had no relationship between driving distance and genetic distance and only a marginal increase in genetic distance with geographic distance Figure . Even moAe. aegypti, and indeed its allelic richness is lower than populations sampled in Africa , more work and demographic history inference is needed, and we are currently analyzing these regions further in the context of North America more broadly.Ae. aegypti. Indeed the temporal instability of Santa Ana CA may indicate the population decreased to low levels in the winter, and perhaps even went locally extinct, before getting reseeded the following year. Differences in transportation networks, perhaps combined with differences in climate and seasonal events, could also affect the genetic connectivity among the populations.Although we believe time since invasion is the most important factor driving the differences between Florida and southern California, the differences in climate between the regions could also have an effect. Florida is more tropical: It has more precipitation, higher humidity, a smaller daily temperature range, warmer winters, and wetter summers Figure . It is rFST among the 2016–2017 sites, perhaps indicating a diminishing of the most extreme effects of bottleneck.This work builds on previous studies from Florida and southern California by increasing the number of individuals and sites sampled, as well as the number of genetic markers. Our work supports previous findings of minimal structure  or the regions (Florida is larger and has different transportation patterns than Réunion Island)  and old (Réunion Island) populations of a similar species, Ae. aegypti has experienced during its global expansion. The unusual genetic patterns in southern California compared to other regions around the world make it especially intriguing for further study.We provide this case study to illustrate that even within its invasive range, the population genetics and structure of an IAS can vary dramatically. As such, a “one size fits all” control measure may not be appropriate for controlling an invasive species; rather the control methods should be tailored to the region in question and may need to be adjusted over time. Moreover, the marked differences between the two regions considered here evoke the diversities of invasion histories and novel environment The authors have no competing interests to declare. However, we would like to include this disclaimer due to an author affiliation: The findings and conclusions in this publication are those of the authors and should not be construed to represent any official USDA or US Government determination or policy.Evlyn Pless: Conceptualization ; Data curation (lead); Formal analysis (lead); Investigation (lead); Methodology (lead); Project administration (supporting); Visualization (lead); Writing‐original draft (lead). Kristen A. Hopperstad: Resources ; Writing‐review & editing . Nicholas Ledesma: Resources ; Writing‐review & editing . Daniel Dixon: Resources ; Writing‐review & editing . Jennifer A. Henke: Resources ; Writing‐review & editing . Jeffrey Powell: Conceptualization ; Funding acquisition (lead); Project administration (lead); Supervision (lead).https://doi.org/10.5061/dryad.83bk3j9p7, SNP dataset (unfiltered and filtered): https://doi.org/10.5061/dryad.8gtht76m8.This article has earned an Open Data Badge for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at Microsatellite dataset:


Bemisia tabaci (Gennadius), is an important invasive agricultural pest worldwide, causing severe damage to numerous crops by feeding or transmitting plant viruses. In this study, we monitored the dynamics of two invasive whitefly cryptic species, Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED), in Jiangsu, China, from 2005–2016. We found that B. tabaci MED quickly established and asserted dominance over MEAM1, resulting in their population displacement in Jiangsu in only three years (from 2005 to 2008). We further investigated the possible mechanisms underlying the successful invasion and competitive displacement from a genetic perspective. Based on sequencing of mitochondrial gene sequences from large numbers of whitefly samples, multiple invasion events of MED were validated by our genetic analyses. MED invaded Jiangsu starting from multiple introduction sites with secondary and/or subsequent invasive events. This may favor their invasion and displacement of MEAM1. This study advances our understanding of the mechanisms that enabled the successful invasion of MED. Despite the severe ecological damage and economic loss caused by invasive species, the factors contributing to successful invasion or displacement remain elusive. The whitefly,  Biological invasions are of great concern as they have a pronounced impact on the native ecosystem, biodiversity, and the economy. Displacement events are intimately linked to invasion biology and continue to increase at alarming rates . HoweverBemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is a cryptic species complex containing at least 40 morphologically indistinguishable species [Arabidopsis thaliana. The whitefly,  species ,3,4,5. A species . MEAM1 e species . In the  species  also det species . Shortly species ,11,12,13 species . A few s species ,16, inse species , host pl species ,19,20,21 species , and eve species ,24. For  species  found th species  identifi species ,27. For  species  found thIn Jiangsu province, MEAM1 was initially found in 2001, whereas MED was first detected in 2005, and since, they have spread over most of Jiangsu’s regions in only a few years . In thisB. tabaci samples were collected from greenhouses and cultivated fields across six municipal level administrative units  in Jiangsu Province from 2005 to 2016 (from August to October)  . Collect2 (25 mmol/L), 2 µL dNTPs (10 mmol/L), 2 µL forward and reverse primers (20 µmol/L each), and 2 µL of template DNA. The PCR reaction program was initialized at 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min, with a final extension for 5 min at 72 °C. Amplification products were purified and sequenced by IGE Biotechnology Co., Ltd. .Total DNA was extracted from individual whiteflies following the protocol of Luo et al. . The speB. tabaci sample was blasted against NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the subspecies/biotype. The sequences in this study were deposited in GenBank (accession numbers: KF142153–KF142157). Each sequence was translated into amino acids to detect whether there was a frameshift mutation or nonsense codon to exclude the pseudogenes. The protein sequences could also be found in GenBank. The haplotype diversity (H) and nucleotide diversity (π) were calculated using DnaSP v. 5.0 [F-statistic (FST) values were calculated using Arlequin v. 3.5 [Trialeurodes vaporariorum, was used as an outgroup. Tree data were visualized and edited using FigTree v. 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). Tajima’s D and Fu’s Fs tests for all populations (pooled) were used to test for neutrality using Arlequin v. 3.5 [Sequence fragments of the mtDNA CO1 gene (555 bp) were assembled using ContigExpress to obtain a consensus sequence and then aligned using the Clustal X 1.83 program . Each seP v. 5.0 . The medP v. 5.0  and usedP v. 5.0  was usedn v. 3.5  to estimn v. 3.5  with 3,0n v. 3.5 . The gren v. 3.5 . Harpendn v. 3.5  implemenn v. 3.5 . The conn v. 3.5 .B. tabaci mtCOI sequences showed that among the total collected whiteflies, 21.7% were identified as MEAM1 (invasive), 77.4% as MED (invasive) and 0.9% as Asia II 3 (indigenous). Both MEAM1 and MED were widely distributed across Jiangsu, while Asia II 3 was rarely detected . Based on alignment of mtCO1sequences, we found that only one haplotype (named HapB1) was identified from MEAM1 individuals; however, four haplotypes  could be defined from MED populations. Of these haplotypes, HapQ3 was the most widely distributed haplotype and was shared among all the populations; HapQ1 was present in all populations except the Wuxi population; HapQ2 and HapQ4 were only detected with low numbers in the Yangzhou and Nantong populations, respectively. Taken together, all populations had at least two haplotypes, except Wuxi. In reference to the time period, HapQ3 was first detected in 2005 from all the populations, except Lianyungang and Suqian; only MEAM1 individuals were found in these two populations in 2005 and 2006; HapQ1 was first detected from Yancheng and Nantong in 2005; HapQ2 and HapQ4 were first detected from the Yangzhou and Nantong populations in 2007 and 2006, respectively. The temporal and spatial distribution of each haplotype can be viewed in The six MED populations showed different levels of genetic variation based on the genetic indexes of H and π. Although the overall level of genetic diversity between MED populations was low, Nantong and Yancheng had higher haplotype diversity (0.352 and 0.285) in comparison to the other four populations. In addition, Nantong had the highest nucleotide diversity, whereas the genetic diversity of Lianyungang and Wuxi were much lower .FCT values from the SAMOVA analyses suggested three was the optimal number of population groups . The six populations were clustered into the three groups as follows: (1) Lianyungang and Wuxi, (2) Suqian, Yancheng and Yangzhou, and (3) Nantong . Likewise, the genetic differentiation between Suqian, Yancheng, and Yangzhou was also low. However, the genetic differentiation between Suqian and Wuxi was the highest (FST = 0.13861) (We further investigated the population genetic structure of MED populations. Monitoring of  Nantong . Interes0.13861) .D and Fu’s Fs statistics. The values of Tajima’s D and Fu’s Fs for all samples were negative , but not significant (p > 0.05); however, a sudden expansion model with a small and insignificant Harpending’s Raggedness (HR) index failed to be rejected , which indicated a relatively good fit to a model of demographic expansion [To uncover the demographic history of MED in Jiangsu, neutrality tests were conducted using Tajima’s xpansion  or a recxpansion ,44. FurtWe further explored the possible source of MED individuals in Jiangsu based on phylogenetic construction. Based on the constructed phylogenetic tree , four haB. tabaci species, MEAM1 and MED, were introduced to numerous countries or regions, resulting in large yield loss among different crops. In China, MEAM1 appeared to displace the indigenous whiteflies since it invaded China in the mid-1990s [The ongoing dispersal of exotic species or the rearrangement of species’ geographical distribution is one of the most striking biological outcomes of global changes ,45. In tid-1990s . Interesid-1990s ,47. In tid-1990s . In a shid-1990s ,27. Impoid-1990s . TherefoArctotheca populifolia, adapted to new environments with very little neutral genetic diversity [Eichhornia crassipes) and sweet briar (Rosa rubiginosa) are also successful invaders with low neutral genetic diversity [We first evaluated the genetic variation of a large number of whitefly individuals. As expected, MED populations had a higher level of genetic variation than MEAM1. In particular, only one mitochondrial haplotype could be detected from MEAM1 individuals, whereas four haplotypes of MED were defined; therefore, the overall MED level of haplotype diversity and of nucleotide diversity were much higher than MEAM1. Previous studies had investigated the genetic diversity of MED populations in China ,48,49,50iversity . In addiiversity ,53.Furthermore, it is well known that multiple invasion events by a species have been proposed as a means through which the loss of genetic diversity is overcome ,54. In sFirst, we noted that the initial percentages of MED in whitefly populations of the mid-southern part were much higher than those in the northern regions (Suqian and Lianyungang). This potentially indicated that MED could have invaded Jiangsu starting from the mid-southern region. Indeed, MED could not be found from Lianyungang and Suqian until 2007. Furthermore, based on the index of genetic diversity and the number of haplotypes, we could easily find that MED individuals in Yancheng and Nantong had the highest level of genetic diversity. Because “ancestral” populations typically possess higher levels of genetic diversity ,58,59, wSecond, due to the high population densities of whiteflies and frequent agricultural trading in Jiangsu, secondary and/or subsequent invasions of MED to Jiangsu are possible. Previous studies demonstrated that MED initially invaded and established populations in the Yangtze River Valley and eastern coastal areas ,13,48, aTo some extent, the Yangtze River probably acted as a physical barrier for MED, because all populations had the haplotype HapQ1 except Wuxi, which is the only population located in the southern part of the Yangtze River. We suspect that Wuxi could be one of the introduction sites. Furthermore, the reason why MED in Wuxi did not successfully displace MEAM1 could be because MED had only one haplotype and expressed the lowest level of genetic diversity. This may support our idea concerning the contribution of genetic traits in successful invasion and displacement.Finally, we utilized phylogenetic construction to explore the possible source for MED individuals in Jiangsu. The MED individuals can be separated into two subclades, MED(Q)1 and MED(Q)2, from the Mediterranean . In our B. tabaci MED in Jiangsu. Specifically, MED first invaded Jiangsu separately starting from either Nantong or Yancheng in 2005, then formed bridgehead populations, and proceeded to invade other regions in Jiangsu. In addition, from 2005 to 2007, there were probably secondary and/or subsequent invasive events into Yangzhou and Nantong (In summary, we monitored the invasion of MED populations along with the rapid displacement of the MEAM1 populations in Jiangsu over a 12-year period. We gave evidence that the multiple invasions of MED occurred in Jiangsu based on our monitoring and genetic data. Importantly, the multiple introductions probably enhanced the ability of MED populations to occupy Jiangsu and dominate over MEAM1 by accelerating its geographic expansion. Finally, we inferred the invasive routes of  Nantong b. The co


Cyrtomium falcatum is a homosporous fern species native to eastern Asia. Two subspecies belonging to a sexual diploid race of C. falcatum are recognized: subsp. littorale and subsp. australe. Subspecies littorale shows intermediate selfing rates, while subsp. australe is an obligate outcrosser. We aimed to evaluate the process of mating system evolution and divergence for the two subspecies using restriction site associated DNA sequencing (RAD-seq). The results showed that subsp. littorale had lower genetic diversity and stronger genetic drift than subsp. australe. Fluctuations in the effective population size over time were evaluated by extended Bayesian skyline plot and Stairway plot analyses, both of which revealed a severe population bottleneck about 20,000 years ago in subsp. littorale. This bottleneck and the subsequent range expansion after the LGM appear to have played an important role in the divergence of the two subspecies and the evolution of selfing in subsp. littorale. These results shed new light on the relationship between mating system evolution and past demographic change in fern species.Evolution of mating systems has become one of the most important research areas in evolutionary biology.  If repeated selfing is effective at purging genetic load, predominant selfing may constitute a stable state when inbreeding depression levels are low4. Theoretical studies predict that a population bottleneck favors selfing because it can reduce inbreeding depression3. Empirical studies also suggest a close relationship between bottlenecks and selfing. For example, in Capsella (Brassicaceae), the self-compatible C. rubella separated from the self-incompatible C. grandiflora in the last glacial period, and this speciation was associated with a major population bottleneck in C. rubella5. The selfing species C. rubella then expanded its distribution into new northern habitats that may have emerged after the Last Glacial Maximum (LGM)6. Since selfing would confer an advantage when filling such new habitats, the environmental shifts that occurred during the last glacial period and as it ended are considered to have favored the evolution of selfing. However, the general patterns of mating system evolution and their relationship to the demographic history of species have not yet been closely examined.The evolution of mating systems has long been one of the most important areas of study in evolutionary biology. Transition from outcrossing to selfing is a common evolutionary pathway in angiospermsCyrtomium falcatum (L.f.) C.Presl. Since self-incompatibility systems such as those based on S-loci are not observed in ferns or other seedless land plants, their mating system would be expected to be determined simply by the balance between inbreeding depression and the advantages of selfing, both of which could be affected by demographic history. The life cycle of homosporous ferns differs from that of seed plants as they have free-living gametophytes that potentially bear both male and female gametangia. The gametophytes are capable of three types of mating: gametophytic selfing (selfing within a gametophyte), sporophytic selfing (crossing between two gametophytes of the same sporophyte) and sporophytic outcrossing (crossing between two gametophytes from separate sporophytes)7. Among land plants, gametophytic selfing is unique to homosporous ferns and lycophytes and monoecious bryophytes, and it is considered to be an extreme form of inbreeding because it results in a zygote that is homozygous at every gene locus. Sporophytic selfing is equivalent to selfing in seed plants. These two types of selfing are responsible for inbreeding in homosporous ferns. Elucidating the evolutionary patterns and process of selfing in homosporous ferns and comparing them to those in seed plants is crucial for understanding the fundamental processes of mating system evolution in vascular plants.To deepen our understanding of the relationship between selfing evolution and demographic history (e.g. the occurrence of population bottlenecks), in this study we focused on a homosporous fern species, Cyrtomium falcatum is a homosporous fern in the family Dryopteridaceae, and is native to Japan, Korea, South China, Taiwan, and Indochina. Two cytotypes have been identified: sexual diploid and apogamous triploid8. Matsumoto8 recognized two forms of the sexual diploid cytotype from the apogamous triploid, C. falcatum subsp. falcatum. These diploid cytotypes were recently formally described as two subspecies: C. falcatum subsp. littorale S.Matsumoto ex S.Matsumoto et Ebihara and C. falcatum subsp. australe S.Matsumoto ex S.Matsumoto et Ebihara9. Cyrtomium falcatum subsp littorale is distinguished by its smaller plant size, fewer numbers of pinnae, and the indusium does not have a black spot, cf. C. falcatum subsp. australe. Subspecies littorale is distributed in the north-eastern part of Japan and laboratory experiments have demonstrated variation in sexual expression (gametangium formation) in gametophytes among its populations8. Sporophytes referred to as the separate type (S-type) produce gametophytes bearing antheridia and archegonia that are separated both spatially and temporally. Conversely, sporophytes of the mixed type (M-type) produce gametophytes that bear antheridia and archegonia simultaneously in a mixed manner8. Variation in the sexual expression of gametophytes can profoundly affect the rate of gametophyte selfing in natural populations10. Our recent study using microsatellites showed that levels of inbreeding (FIS) and gene diversity (h) differed significantly between S-type  and M-type  populations, suggesting higher selfing rates and lower genetic diversities in M-type populations11. Subspecies australe is distributed in the southern part of Japan and exhibited little selfing ability when its gametophytes were cultured in isolation8. Only S-type sexual expression has been observed to date in subsp. australe8. Subspecies littorale may be a lineage derived from subsp. australe because the former subspecies has specialized ecological and morphological characters, including saxicolous habitat, early maturation, progenesis, white indusium without a central black spot, and considerable levels of selfing. The variation in the mating systems of the derived subspecies and the predominance of outcrossing in its ancestral subspecies constitute an evolutionary pattern that makes C. falcatum a useful model for studying mating system evolution in homosporous ferns.12 was performed to acquire a large amount of genetic data for high resolution population genetic analysis in order to evaluate phylogenetic relationships among S-type and M-type populations of C. falcatum subsp. littorale and S-type populations of subsp. australe. RAD-seq data also enabled us to estimate the demographic history of subsp. littorale and subsp. australe. In non-model organisms that are lacking extensive molecular marker development, RAD-seq is a valuable tool for molecular population genetics. We addressed two questions by means of these analyses: (1) Are S-type populations in subsp. littorale more closely related to subsp. australe than M-type ones? (2) Are there any associations between demographic events, lineage divergence and the evolution of selfing? By answering these questions, we aimed to clarify the process of mating system evolution in the two subspecies of diploid sexual C. falcatum.In this work, restriction-site associated DNA Sequencing (RAD-seq)Cyrtomium falcatum were collected from 21 populations in Japan  were in that of C. falcatum subsp. australe9. Of the subsp. littorale populations, Pop6 and Pop8 are S-type and the rest are M-type11. Populations 2, 3, 6, 8 and 11 are the same as those examined in previous microsatellite analyses11; they were referred to as ESAN1, ESAN2, IZU2, SADO and SAND, respectively, in the previous study11. Most of the samples of these populations were collected during the previous work11, but new samples of Pop6 were collected during this study. The remaining samples were collected from natural populations or obtained from plants cultivated in Tsukuba Botanical Gardens, which were originally collected as described by Matsumoto (2003)8. In addition to the sporophyte samples, twelve haploid gametophyte samples were used. The gametophytes were grown from spores of a hybrid between subsp. littorale and australe (A1-55 × A2-2), which was made through artificial crossing by Matsumoto8. The sequence data from these haploid samples were used to check and remove contigs containing paralogous sequences. All of the 95 samples referred to above were subjected to RAD-seq. Details of the sources of the samples are shown in Table Eighty-four samples of 13. To detect genetic structure and population demographic events, we extracted genome wide SNP from samples using Double-digest RAD-seq (ddRAD-seq)14. The DNA library was prepared with two restriction enzymes, EcoRI and BglII as described by Sakaguchi et al.15. Fifty-one bp single-end sequencing was conducted in one lane of the Illumina HiSeq2500  by Macrogen . Finally, 9.2 gigabases with 181 million reads were obtained.Total DNA was extracted from silica-dried sporophyte leaves or from fresh gametophytes using the HEPES/CTAB methodC. falcatum 16 to exclude chloroplast sequences from the data. For this purpose, we used the BWA17 ‘aln’ and ‘samse’ functions with default settings. We extracted chloroplast SNPs using the ‘mpileup’ function of samtools 1.3.118. After mapping, we extracted unmapped single end reads with samtools 1.3.1 ‘view’ function. These unmapped sequences were considered to contain no chloroplast sequences. After excluding chloroplast reads, we converted bam files to fastq files using bamtofastq in Hydra version 0.5.319. Next, we created contigs using pyRAD20. Low quality reads  were discarded. At the within-sample clustering stage, the minimum coverage (number of reads per sample), maximum number of heterozygous sites, and allowed number of alleles were set to 5, 5, and 2 respectively. The maximum proportion of shared heterozygote sites allowed was less than 40%. Variant Call Format (VCF) files were used for the following analysis, which included only SNPs generated by pyRAD. As the first data filtering step, we removed loci with > 20% missing data using vcftools21 because missing data can bias data analysis. We also removed individuals with > 30% missing data to avoid the adverse effects of low-quality DNA data. Next, we removed loci with minor allele frequencies (< 3%) from the non-chloroplast vcf file. Roesti et al. 22 suggested that minor alleles can be uninformative and lead to bias in genome scanning (e.g. detection of outlier loci), which is important when evaluating both selection and neutral genetic structure23. A minor allele frequency threshold of 3% has been widely used in recent NGS-based studies26. We also filtered out chloroplast loci with > 20% missing data in the same way. To estimate genetic structure based on chloroplast data, we used Splits tree427 to construct a neighbor net with the chloroplast data.The raw fastq files were ascribed to individuals based on barcode sequences. These individual fastq files were mapped onto the chloroplast genome sequence of 21, we conducted outlier loci detection. Although population-based outlier loci detection methods have been used extensively, a recent study22 showed that historical or on-going demographic events  can lead to bias in these population-based approaches. Moreover, admixture and a historical bottleneck were observed in C. falcatum populations in our previous study11. We therefore used an individual-based principal component analysis (PCA) approach to detect outlier loci. This approach was implemented using the R package pcadapt28 which robustly detects outlier loci under several population demographic patterns. A principal component number of K = 7 was initially employed based on the trend of the scree plot. However, the number of outlier loci was too large under these conditions. Because the slope of the scree plot began leveling out at K = 3, we instead set K to 3. We also excluded non-neutral loci estimated by pcadapt using vcftools. The remaining loci, which were considered to be neutral, were used for population genetic analysis.To evaluate genetic structure and demography based on neutral genetic information and to avoid bias due to outlier lociFST values at each SNP locus were calculated using hierfstat29. To estimate the effect of differences in mating system, we also used hierfstat to compute the mean inbreeding coefficient, FIS, for populations of more than 4 individuals. According to Hedrick30, the FIS value reflects the relative contribution of selfing sensu-lato (both gametophytic and sporophytic selfing) to outcrossing in each population. To validate the geographical pattern of inbreeding coefficients, we also tested the correlation between FIS and latitude for subsp. littorale and australe populations. We estimated effective population size by linkage disequilibrium using NeEstimator 2.0131.The average gene diversity and 32. To carry out the PCoA, pairwise genetic distances33 between individuals were calculated with the “interpolating missing” option to correct for any bias due to missing data. We also conducted a model-based cluster analysis using STRUCTURE34. Numbers of clusters (K) between 1 and 25 were evaluated under the correlated allele frequencies model35 by running 10,000 burn-in Markov Chain Monte Carlo (MCMC) repetitions and 10,000 subsequent repetitions. We used the CLUMPAK server36 to evaluate multimodality37 among runs for each K and to generate barplots for the different values of K. The probability of the data (Ln P(D)) and ΔK38 were summarized using STRUCTUREHARVESTER39. We estimated a pattern of population bifurcation and events of mixing between populations by using TreeMix 1.1240. TreeMix infers the points at which admixtures happened in a population tree under the assumed number of admixture events. We assumed 1–5 admixture events, and the best tree was defined by likelihood41.To evaluate genetic structure among individuals, we performed principal coordinate analysis (PCoA) using GenAlEx 6.542 and a Stairway plot43. We first generated an EBSP using BEAST 2.4.444 for the two subspecies, excluding the OGAS populations. We used a modified version of the method introduced by Trucchi et al.45, who successfully detected temporal changes in the size of king penguin populations and showed that highly polymorphic loci identified by RAD-seq can reflect general trends that also apply to other kinds of loci. To obtain all sequences with more than four polymorphic sites, following Trucchi et al.45, we sampled one sequence per individual randomly. We used a substitution rate of 0.75 × 10–8 per site per year based on estimates for Arabidopsis thaliana46. The substitution rate parameter for sequences with four polymorphic sites was set to 1.0, and was increased in proportion to the number of polymorphic sites (i.e. it was set to 1.25 for sequences with five polymorphic sites and 1.5 for those with six). We ran 250,000,000 MCMC chains and collected a trace and EBSP log after every 1000 chains. Tracer 1.647 was used to verify that the Estimated Sample Size was greater than 100 for all parameters. We calculated Tajima’s D48 for these loci using DnaSP 649 to evaluate the general trend in the demographic pattern.Population demography in terms of temporal changes in population size was evaluated by generating an Extended Bayesian Skyline Plot (EBSP)43 is a newly developed method for inferring temporal changes in population size by using site frequency spectrum (SFS) data to test demography. To estimate SFS, we used a sequence from the individual in Pop 17 as the ancestral sequence because the TreeMix results indicated that this population was at the base of the population tree. We mapped sequence data for all individuals onto this sequence using BWA17 and used samtools 1.818 to convert SAM files to BAM files and sort the BAM files. We estimated the site frequency spectrum with ANGSD50, using the following options: dosaf 1 (multi sample genotype likelihood estimation with assumed Hardy–Weinberg equilibrium), GL: 1 (genotype likelihood estimation of the samtools model), and RealSFS using the maximum number of iterations (100). We used the same mutation rate as for EBSP and assumed a generation time of three years in the demography estimation for the Stairway plot.Stairway plot v2The non-chloroplast dataset contained 9350 loci before filtering. After excluding low frequency minor alleles and missing data, 2636 loci remained. Pcadapt detected 355 loci estimated to be non-neutral among these loci. Thus, 2281 loci were used for population genetic analyses. For chloroplast DNA (cpDNA), 191 SNPs were extracted and 180 remained after excluding missing data. Although 10 haplotypes were detected, most of the individuals were fixed for haplotype 1 Table  and the littorale. Subspecies australe had higher gene diversity than all the subsp. littorale populations except for Pop8 (SADO). SADO had the highest gene diversity of the subsp. littorale populations, but subsequent analyses suggested that this population belongs to subsp. australe . Taking this result into account, all subsp. australe populations had higher gene diversity than subsp. littorale . At K = 2, the clustering mostly supported taxonomical classification of subsp. littorale and australe; however, all of the SADO and two of the SAND individuals were included in the australe cluster or showed an admixture-like pattern. The OGAS and KASA populations also showed population admixture at K = 2, K = 3, and K = 4. However, at K = 5 the OGAS1—3 populations formed a new cluster. At K = 3, several subsp. australe populations appeared as admixtures of multiple clusters. At K = 4, NOMO and TSUN appeared as pure clusters; this outcome was observed up to K = 25.In the STRUCTURE analysis of all populations, the probability of the data (LnP(D)) increased progressively with each K until it reached a plateau at = 6 Fig. . The higaustrale as the ancestral form on the basis of gene diversity, so we presumed that subsp. australe would have a lower drift parameter. This was the case when the migration number m was set to 3. All subsp. littorale populations other than the SADO population constituted one group that departed from subsp. australe  near the western limit of the subspecies’ distribution  populations in subsp. littorale were placed in two different positions of the tree: SADO was in subsp. australe, and IZU was in a branch of subsp. littorale. The populations from the Ogasawara Islands were divided into two groups: OGAS1 had a low drift parameter, while OGAS2 and 3 formed a group sister to subsp. littorale  and then increased from 22,000 years BP onwards  and -0.025 (SD: 1.080), respectively.An EBSP analysis was performed using 82 loci for rds Fig. b. The efize Fig. a. The melittorale; the effective population size fell from about 7000 years BP and reached its minimum at 40,000 years BP  and simultaneously experienced population shrinkage in the last glacial period, and that this population became a founder of subsp. littorale. In other words, the postulated population bottleneck event may be closely associated with the divergence between subsp. littorale and australe. Both the EBSP and Stairway plot analyses suggest that the effective population sizes for the two species were similar before the bottleneck event, which supports the second scenario.The two subspecies showed different demographic patterns and levels of genetic diversity. Subspecies littorale would have helped population size recovery after the population bottleneck. Both the EBSP and Stairway plot methods indicated a rapid increase in population size of subsp. littorale after the population bottleneck, which could be explained by range expansion to the north. Gametophytic selfing has been considered advantageous for reproductive assurance during rapid range expansion because it enables single-spore colonization of distant regions53. After the last glacial period, habitats suitable for subsp. littorale would have been empty in the northern area. M-type sexual expression and relatively high levels of selfing  for the subsp. littorale populations to estimate their levels of selfing and obtained F values ranging from 0.22 to 0.7911. The IZU population was known to have S-type sexual expression8 and had the lowest F value (0.22). The present study using genome-wide SNP data yielded lower F values, ranging from 0.053 to 0.427 for the subsp. littorale populations  for estimating allele frequencies, had nearly the same F value (0.233) as that in our previous study11. TreeMix analysis . There are two possible explanations for the non-concordance between sexual expression and tree topology. First, after the evolution of M-type sexual expression favoring selfing in the common ancestor of subsp. littorale populations, S-type sexual expression may have evolved newly in the IZU population or been acquired through introgression from subsp. australe. Second, M-type sexual expression may have emerged later on each of the littorale branches via parallel evolution or gene flow from the population in which M-type evolved first. In this view, IZU is a unique population that has maintained ancestral S-type sexual expression. Although IZU has S-type sexual expression like subsp. australe, the population shows mixed-mating (FIS = 0.233), unlike the obligate outcrossing mating system shown by subsp. australe populations (Table littorale was reported in two distant locations: Sadogashima Island and Izu peninsula8. Because the present study excluded the SADO population on Sadogashima Island from subsp. littorale, only the populations around the IZU population are known to have the S-type sexual expression. Although IZU shows the lowest level of selfing among subsp. littorale populations, it retains a mixed mating system. Therefore, the putative transition from M-type to S-type in IZU is not an unequivocal example of recovery of outcrossing from selfing, which has not been observed in seed plants54. However, it can safely be described as an interesting example in which an evolutionary change that enhanced outcrossing emerged in mixed-mating plants.In our previous study using microsatellite markers, we calculated inbreeding coefficients (11, the evolution of selfing via transmission advantage operates only under a low genetic load and predominant selfing is expected3. Therefore, the existence of mixed-mating even in the M-type populations suggests that reproductive assurance, rather than transmission advantage, is the main factor affecting the evolution of selfing in this species. If this is the case, populations of subsp. littorale are expected to harbor sufficient genetic load to avoid the positive feedback process of selfing evolution driven by transmission advantage3. If genetic diversity and inbreeding depression increased in selfing populations that have been stable for a long time as a result of migration, it would be possible for them to change their sexual expression toward enhancing outcrossing, especially in populations that were not completely selfing.The mating system of ferns seems to be determined by the balance between inbreeding depression and the advantages of selfing. Additionally, it appears that changes in the sexual expression pattern, timing, and the position of the reproductive organs on gametophytes can alter the ease of gametophytic selfing. As discussed previouslyC. falcatum subsp. littorale underwent a population bottleneck during the last glacial period, which appears to have induced the divergence of C. falcatum subsp. littorale and australe. The evolution of selfing may have been favored by reproductive assurance during rapid range expansion after the last glacial period. This is the first study to address the relationship between mating system evolution and range expansion after a glacial period based on demographic inferences, and it sheds new light on mating system evolution and ecological dynamics in both ferns and other plant species.This study successfully showed that Supplementary Information.


Harmonia axyridis, a biological control agent and an invasive alien species. Here, equipped with a type IIB endonuclease restriction site-associated (2b-RAD) DNA sequencing approach, we identified 13,766 and 13,929 single nucleotide polymorphisms (SNPs), respectively, among native and invasive H. axyridis populations. With this information we simulated populations using a randomly selected 3000 SNPs and a subset of individuals. From this simulation we finally determined that six individuals is the minimum sample size required for the accurate estimation of intra- and inter-population genetic diversity within and across H. axyridis populations. Our findings provide an empirical advantage for population genomic studies of H. axyridis in particular and suggest useful tactics for similar studies on multicellular organisms in general.Finding optimal sample sizes is critical for the accurate estimation of genetic diversity of large invasive populations. Based on previous studies, we hypothesized that a minimal sample size of 3–8 individuals is sufficient to dissect the population architecture of the harlequin lady beetle,  Using optimal sample sizes to accurately estimate genetic diversity of large natural populations is an imperative issue in the analysis of evolutionary processes . BecauseGiven these disadvantages of microsatellites marker in genetic analysis, a growing number of researches have recommended to use the genomic data over microsatellites for genetic study ,13,14. FFst in a plant species can be accurately estimated from six to eight individual plants  or x = 100 . At the same time, the ΔK line chart showed a peak at x = 30  individuals appears to have little impact on the mean Ae, Ho and uHe when 3000 SNPs were selected for n = 4  and for n = 15  ] and for n = 15 was 0.0795 [95% CI ]. Simultaneously, the ΔK line chart showed a clear peak at n = 6  with 3000 SNPs was sufficient to recover the genetic diversity found in PLKK populations  ] . Also, aear peak . For theat n = 6 . For Ae 0.0852)] . The ΔK parately .Fst when we set the number of replicates to x = 60; the number of individuals (n) were fixed to three and the number of SNPs were fixed to 3000. For instance, the mean values of Fst for x = 60 were 0.0398 [95% CI ] and for x = 100 were 0.0347 [95% CI ] (n ≥ 6) did not decrease the sample size needed to recover the genetic differentiation of individual populations of H. axyridis based on 60 replicates ] and for n = 15 were 0.0394 [95% CI ]. Similarly, the ΔK line chart showed a peak at n = 6 ] . At the 0.0408)] . Furtherplicates . The meaat n = 6 .H. axyridis, which confirmed our hypothesis that a minimal sample size of 3–8 individuals is sufficient to estimate genetic diversity within and across native and invasive populations. The next step in our studies will be to sample global populations of H. axyridis to investigate the genetic diversity. To our knowledge, only one published study has examined a reliable number of individuals to address invasive population genomics using SNP markers [In this study, we conducted a rigorous empirical determination of the optimal sample size in an invasive ladybeetle,  markers , while s markers ,39,40,41Sample size is an important study design factor that influences population genomic studies ,23,42,43B. tabaci, in which 3–4 individuals were required to recover within-population genetic diversity parameters (n = 20) than that in previous study (n = 10) [n = 25–30 or 30 individuals) from each population to identify the optimal sample size [H. axyridis to estimate genetic parameters [Our results indicate that, in general, even small sample sizes are likely to be sufficient, which is similar to previous studies ,23,42,43rameters . The exaple size , and 35 tudy n = 0 [n = 25tudy n = 0 [n = 25Fst, when a large number of polymorphic SNPs are employed. This minimum sample size in H. axyridis is larger than two individuals for an Amazonian plant species, Amphirrhox longifolia (Violaceae) and three to four individuals for B. tabcai based on empirical analysis [n = 4–6 to measure Fst metrics when using a large number of SNPs (>1000) [Fst (>1000 or ≥1500 SNPs) [Additionally, our results showed that sample sizes of six individuals per population, provide accurate estimates of analysis ,23 (Tablof SNPs >000 [Fst 00 SNPs) , while a00 SNPs) . Thus, w00 SNPs) .H. axyridis to conduct a sample size optimization analysis. The SNP data support the presence of two highly divergent lineages of H. axyridis populations. Lineage 1 is distributed in China and Lineage 2 generally in Poland, which suggests a long-standing barrier to gene flow between these geographic regions  and invasive (European) populations of  regions . Our resn ≥ 6) has little impact on the estimates of genetic diversity within H. axyridis populations. Accurate estimates of Fst can also be easily obtained at a small simple size . The findings demonstrated that SNP markers can accurately estimate the genetic diversity of H. axyridis populations, even when small numbers of individuals are sampled, which provides a starting point for future genome-wide population studies.Our results showed that a sample size greater than six individuals (