Grapholita molesta is a cosmopolitan pest of stone and pome fruit species including peach and apple, and historical records indicate that it has invaded North and South America, Europe, Australia and Africa from its putative native range in Asia over the past century.Invasive pest species have large impacts on agricultural crop yields, and understanding their population dynamics is important for ensuring food security. The oriental fruit moth G. molesta. Approximately 15 individuals from each of 26 globally distributed populations were genotyped. A weak but significant global pattern of isolation-by-distance was found, and G. molesta populations were geographically structured on a continental scale. Evidence does not support that G. molesta was introduced to North America from Japan as previously proposed. However, G. molesta was probably introduced from North America to The Azores, South Africa, and Brazil, and from East Asia to Australia. Shared ancestry was inferred between populations from Western Europe and from Brazil, although it remains unresolved whether an introduction occurred from Europe to Brazil, or vice versa. Both genetic diversity and levels of inbreeding were surprisingly high across the range of G. molesta and were not higher or lower overall in introduced areas compared to native areas. There is little evidence for multiple introductions to each continent (except in the case of South America), or for admixture between populations from different origins.We used 13 microsatellite loci, including nine newly developed markers, to characterize global population structure of G. molesta appear to be infrequent, which is surprising given its rapid worldwide expansion over the past century. We suggest that area-wide spread via transport of fruits and other plant materials is a major mechanism of ongoing invasion, and management efforts should therefore target local and regional farming communities and distribution networks.Cross-continental introductions of  Global interest in the ecology of agricultural systems has driven a substantial amount of fundamental research into plant-herbivore interactions, modes of plant (i.e. weed) and invertebrate dispersal and adaptation, and the role of multi-trophic interactions in community dynamics. A growiThe mechanisms that facilitate the establishment and spread of invasive pests are of particular interest to ecologists, evolutionary biologists and policy makers for a number of reasons; agricultural pests are responsible for substantial yield losses in agricultural systems, and there are considerable economic incentives to understand mechanisms of pest invasions. Also, because invasions often occur relatively rapidly, they provide useful case studies to understand the ecological factors (both biotic and abiotic) that mediate population dynamics and the establishment of range limits,5. FinalGrapholita (=Cydia) molesta Busck (Lepidoptera: Tortricidae) is one example of an insect pest species that continues to cause significant economic damage to crops on a global scale[Grapholita molesta is a major pest of stone and pome fruit species mainly belonging to the Rosaceae family, including peach, apple, pear, nectarines, cherries, quince, and persimmons (Ebenaceae)[G. molesta is now distributed throughout temperate regions of Asia, Europe, The Americas, Africa, and Australia.The oriental fruit moth al scale. Grapholenaceae),9. Assumenaceae), G. moleG. molesta has not been accurately traced, although historical records permit the reconstruction of a rough timeline of continental invasions. In Europe, G. molesta was first recorded in Slovenia in 1920[G. molesta specimen recovered from a shipment of Japanese pears[G. molesta is presumed to have dispersed to the neighboring states of Virginia and Maryland, and northward to Ontario, Canada. By the mid-1940s, it had spread from the mid-West to California, Washington state, and Oregon[G. molesta was introduced around 1910, and has since spread to most of the stone and pome fruit growing regions on that continent[G. molesta was first identified in New Zealand in 1976[The global demographic history of  in 1920, in sout in 1920, and has in 1920, howeverse pears. After id Oregon and refeontinent and refeontinent. In othe in 1976, and in  in 1976.G. molesta over regional spatial scales. Population structure of G. molesta in a major fruit-growing region of Northern Italy was relatively low[G. molesta and to develop strategies for its control, little is known about its ongoing global dispersal patterns.A number of regional studies have yielded some information about local factors that contribute to the dispersal and population structuring of vely low, and couvely low and anthvely low. Based ovely low. Anothervely low. In spitG. molesta, in order to retrace invasion routes, and to investigate global population dynamics. We hypothesized that i) patterns of population structure will reflect evidence of multiple introductions via human-mediated dispersal in the invaded range of the species, and ii) patterns of isolation-by-distance will be absent or weak, since international trade routes likely determine patterns of global population structure, and iii) genetic diversity will be reduced in introduced compared to native populations. We also tested whether populations are structured according to host species (predominantly peach and apple).In this study, we used a set of 13 microsatellite loci (including nine newly developed ones) to characterize global population structure of G. molesta larvae (sampled from fruits or shoots) or adult males (captured from pheromone traps) were collected from fruit trees at 26 worldwide locations between April and October 2011  , 2.0 μL dNTPs , 0.5 μL of each forward and reverse primer (2 μM), 0.1 μL HotstarTaq  and 0.5 μL DNA (10–35 ng⁄ μL). Reactions for the remaining nine newly developed loci were carried out in a mix of 5.7 μL ddH2O, 1.0 μL PCR buffer (Qiagen), 1.0 μL dNTPs , 0.2 μL of each forward primer (2 μM), 0.5 μL of each reverse primer (2 μM), 0.5 μL M13 primer , 0.1 μL HotstarTaq  and 0.5 μL DNA (10–35 ng⁄ μL). Locus-specific PCR conditions are reported in Additional fileAll polymerase chain reactions (PCRs) were performed as singleplex reactions. Reactions were performed in a Labcycler thermocycler  in a total reaction volume of 10 μL. The forward primers of the loci eneScan 500 LIZ size standard , and running the products on a laser detection system . DNA sizing and allele definitions were performed using GeneMapper 4.0 software (Applied Biosystems). SSR data are archived at the Dryad repository (http://dx.doi.org/10.5061/dryad.h658g).Genotyping was carried out by denaturing 1 μL PCR product in 9 μL formamide  with 0.09 μL GenAlEx v. 6.41[eneland 3.1.4[rlequin 3.01[We identified departures from Hardy–Weinberg equilibrium (HWE) using G v. 6.41. We calcnd 3.1.4,26. We tuin 3.01, accordiuin 3.01.enAlEx using a Euclidean distance metric according to[enAlEx using 999 permutations.Genetic and geographic distances were calculated in Grding to, and misenAlEx, using 999 permutations, and also calculated global (mean) FST value between populations[To better understand the partitioning of regional population genetic structure, we carried out an AMOVA in Gulations. We defitructure version 2.3[tructure assumes HWE and no linkage disequilibrium between loci, the software is robust to deviations from these assumptions, particularly if no spurious populations are observed across multiple runs[K) of K groups and the individual percentages of membership assigned to them according to their molecular multilocus profiles[K (K = 1–10), using a burn-in period and a run length of the Markov Chain Monte Carlo (MCMC) of 30 000 and 100 000 iterations, respectively. Based on a trial run, we found that incorporating higher values of K, and longer burn-ins or MCMC did not appreciably change the results. Five runs were carried out for each value of K. We calculated ΔK according to[istruct[A Bayesian model-based analysis was performed to infer population structure using Ssion 2.3,32. Althple runs. Data werding to. Figure [istruct.eneland 3.1.4[tructure, the software uses a MCMC strategy[K), and assigns individuals to the most appropriate population basing on individual multilocus genotypes. We carried out ten independent MCMC simulations  using the non-spatial approach, during which we allowed K to vary between 1 and 20. Afterwards, the model with the highest mean logarithm of posterior probability was post-processed. Geneland was used to estimate inbreeding coefficients (FIS) of inferred clusters.We also carried out an independent analysis of spatial structure using the R package Gnd 3.1.4,26, whicstrategy to deterigrate-N[We also attempted to estimate the magnitude and direction of historical gene flow between our 26 sample populations using a Bayesian coalescent approach implemented in the software program Migrate-N. HoweverHE), observed heterozygosity (HO), and allelic richness (NA) using GenAlEx. GenAlEx was used to identify private alleles from each continent, and from Japan and the Azores.We calculated expected heterozygosity . Incorporating host species (peach or apple) rather than geographic region as the “regional” factor revealed that host species did not account for any of the variation in the data set, while variation within and between populations accounted for 70% and 30% of the variation, respectively (P < 0.001). A significant pattern of isolation-by-distance was detected (P < 0.001), although this pattern explained a low proportion of the variation in the data set (R2 = 0.111).Global tructure yielded a modal value of ΔK at K = 4  and Spain, or vice versa, which would allow us to infer the direction of gene flow between continents. We found a similar number of private alleles among the Brazilian versus the French/Spanish populations; compared to Brazilian populations, there were 11 alleles that were specific to France (Lafitte-sur-Lot)/Spain, with allele frequencies that varied between 0.033, and 0.310. Similarly, there were 13 alleles that were specific to Brazil compared to France (Lafitte-sur-Lot)/Spain, with allele frequencies that varied between 0.017 and 0.433. The direction of gene flow between Brazil and Europe could therefore not be unambiguously traced using molecular genetic data.In order to further investigate the relationship between the two Brazilian and the two European populations that clustered together based on the Seneland Cluster 6), one population from Spain (Geneland Cluster 4), and the South African population (Geneland Cluster 13).Measures of genetic diversity were high in native as well as in many introduced populations Table . MeasureFIS estimates corrected for null alleles (computed using Geneland), were high across the range of sampled populations, and did not differ appreciably among populations from the introduced versus invaded ranges , which belongs to the same tribe (Grapholitini) as G. molesta[C. pomonella were carried out at scales varying from a few to several hundred kilometers, impairing comparisons between the two species. Nonetheless, low levels of population structure among C. pomonella populations from some regions relative to G. molesta could be the combined result of higher dispersal abilities of the former species compared to the latter[C. pomonella in some countries, which can stimulate flight in this species  to mode. molesta) to high. molesta; Italy[. molesta) among de latter-49 and tiewed by).eneland suggested greater population substructure compared to the analysis from Structure. Evidence indicates that G. molesta was introduced to Australia from mainland Asia. The data also support a North American source of G. molesta in Argentina and Chile, The Azores and South Africa, since historical records indicate that G. molesta was present in North America from the beginning of the 20th century, but arrived much later in the latter three regions. However, a previous study[In the present study, clustering outcomes from two distinct Bayesian approaches were largely congruent, except that the analysis from Gus study suggestetructure), and shared ancestry between Western European and Brazilian populations was also supported by results from Geneland. The two European populations that shared ancestry with populations from Brazil showed little evidence of admixture with other European populations. It is possible that these two populations from Europe were derived from the same source as other European populations, but were isolated from other populations on the same continent, and therefore became differentiated over time. If this is the case, Brazilian populations are likely derived from European origins. However, it is also possible that G. molesta was recently re-introduced to Europe from the South American part of its range. Chile is the primary source of off-season peach and nectarine imports to Europe, and Brazil exports nearly 90% of its apple crop to Europe[C. pomonella individuals with putative South American origin in France, suggesting that South America may be a common source of introduction for important stone and pome fruit pests to Western Europe. Moreover, other invertebrate pests of distantly related plant host species have recently been introduced from South America to Europe, including the pine-infesting woodwasp Sirex noctilio, which was introduced from Chile to Switzerland[Unexpectedly, Brazilian populations clustered together with two European populations  pattern of isolation-by-distance, suggesting that regional introductions occur in a relatively stepwise manner. Also, we found little evidence for substantial admixture between populations sampled from different continents, which, combined with the clustering outcomes, implies that multiple cross-continental introductions are unlikely. We note however, that sampling from Australia and Africa was limited to one population each, and we cannot make definitive continent-wide conclusions for these regions based on these sample sizes.G. molesta disperses primarily through the movement of fruit, bins and plant material between orchards[G. molesta likely accounts for the high levels of inbreeding observed in our study, since close relatives are probably geographically constrained to the same or neighboring orchards. Combined, these findings suggest that improved management of this invasive pest at regional and community levels (e.g. by area-wide pest management) and at the national level (e.g. by quarantine regulations) may be efficient strategies to limit its ongoing spread.It has earlier been suggested that orchards,21. The orchards. Flightsorchards,47, and orchards. The pooG. molesta populations are structured according to host plant species. Although G. molesta has been shown to perform better on peach, its primary host, compared to apple[We did not find any evidence that to apple, serial to apple. Such seG. molesta in most non-native areas, as is suggested to occur in other invasive species. Levels of genetic diversity were reported from the non-native range of 14 species of invasive insects[Ceratitis rosa, Drosophila pseudoobscura, Polistes dominulus, and Rhagoletis completa) exhibited HE values above 0.3. In our study, HE exceeded 0.5 in most populations from both the native and invaded range. High levels of genetic diversity observed in this study could be a result of high microsatellite mutation rates in lepidopterans[Estimates of genetic diversity among different geographic regions were high overall. A six times higher number of private alleles among Asian populations compared to populations from other continents provides, for the first time, convincing evidence that Asia is indeed the native range of this species, as is frequently suggested in the literature. We found that genetic diversity levels are surprisingly high across most of the invaded range, except for a few populations . It is therefore unlikely that founder effects impair the adaptive potential of  insects, and of opterans, and/or G. molesta populations, and may contribute to the ability of G. molesta to adapt to pest management regimes. For example, there is considerable genetic variation within G. molesta populations with regard to olfactory response of female moths to their host plants[G. molesta has developed insecticide resistance in at least some parts of its invaded range[High levels of genetic diversity may account for high levels of heritable phenotypic variation within t plants. Similared range,60.G. molesta, with the possible exception of a reintroduction to Europe from South America, or two independent introductions to South America. This is likely a result of the poor natural dispersal abilities of G. molesta, combined with stringent international trade regulations that aim to prevent its movement across international borders. Although bottlenecks have occurred in a subset of introduced populations, we argue that, overall, reductions in genetic diversity will not prevent G. molesta from adapting to new control measures or changes in climatic conditions, and we therefore suggest that improved community-based control measures, in addition to more stringent national and intracontinental regulations regarding the regional transport of plant material, will be most effective for containing its ongoing spread.Unlike many other invasive pests (reviewed by), which The authors declare that they have no competing interests.SD and DM conceived of and designed the study. HK carried out the molecular genetic work, analyzed the data, and drafted the manuscript. All authors read and approved the final manuscript.Appendix 1. Locus specific PCR conditions. Description: Detailed description of PCR conditions.Click here for fileAppendix 2. Pairwise FST values. Description: Pairwise FST values from 14 clusters derived from analysis in GENELAND.Click here for file


Frankliniella occidentalis (Pergande), is an invasive species and the most economically important pest within the insect order Thysanoptera. F. occidentalis, which is endemic to North America, was initially detected in Kunming in southwestern China in 2000 and since then it has rapidly invaded several other localities in China where it has greatly damaged greenhouse vegetables and ornamental crops. Controlling this invasive pest in China requires an understanding of its genetic makeup and migration patterns. Using the mitochondrial COI gene and 10 microsatellites, eight of which were newly isolated and are highly polymorphic, we investigated the genetic structure and the routes of range expansion of 14 F. occidentalis populations in China. Both the mitochondrial and microsatellite data revealed that the genetic diversity of F. occidentalis of the Chinese populations is lower than that in its native range. Two previously reported cryptic species (or ecotypes) were found in the study. The divergence in the mitochondrial COI of two Chinese cryptic species (or ecotypes) was about 3.3% but they cannot be distinguished by nuclear markers. Hybridization might produce such substantial mitochondrial-nuclear discordance. Furthermore, we found low genetic differentiation  among all the populations and strong evidence for gene flow, especially from the three southwestern populations  to the other Chinese populations. The directional gene flow was further supported by the higher genetic diversity of these three southwestern populations. Thus, quarantine and management of F. occidentalis should focus on preventing it from spreading from the putative source populations to other parts of China.The western flower thrips,  Among the different ecological, genetic, and evolutionary features that determine whether an invasion will succeed or fail, genetic characteristics have received increasing attention Frankliniella occidentalis (Pergande), is an invasive species and the most economically important pest within the insect order Thysanoptera, which includes more than 5500 described species F. occidentalis causes enormous damage by directly feeding on greenhouse vegetable and ornamental crops and by transmitting plant-pathogenic tospoviruses F. occidentalis is endemic to North America in an area west of the Rocky Mountains from Mexico to Alaska F. occidentalis has rapidly invaded most countries in the world and now exists on every continent but Antarctica The western flower thrips, F. occidentalis was first discovered in flowers from Myanmar at the Kunming International Floral Festival of China in 2000 F. occidentalis, it is first necessary to know its genetic diversity, population structure and pathways of range expansion in China.In China, F. occidentalis have been hampered by a lack of polymorphic molecular markers. Presently, only 6 polymorphic microsatellites are known Genetic tools, such as mitochondrial DNA (mtDNA) and microsatellites, can reveal the origins of newly established populations, their genetic makeup and their routes of migration F. occidentalis and used them and the mitochondrial COI gene to characterize the genetic structure of 14 introduced populations of F. occidentalis in China. The primary goals of this study were to (1) investigate the genetic diversity and differentiation of introduced populations across China and (2) identify the potential avenues of dispersal and determine whether their range expansion in China is the result of a bridgehead effect.In this study, we isolated new polymorphic microsatellites markers for F. occidentalis female adults were sampled representative of 14 sites in China during May 2009 to August 2010  No specific permissions were required for these collections because the thrips are pests on common crops; (b) The location is not privately-owned in any way; (c) The field studies did not involve endangered or protected species.Total genomic DNA was extracted by homogenizing a single female adult in a 50 µl mixture of STE buffer  in a 1.5 ml Eppendorf tube. The mixture was incubated with 2 µl proteinase K (10 mg/ml) at 37°C for 30 min, followed by 5 min at 95°C. The samples were centrifuged briefly, and used immediately or stored at −20°C for the PCR reactions.2; Fermentas), 0.2 mM dNTPs (Takara), 1 µl of DNA (concentration not estimated) and 0.4 µM each of the oligonucleotide primers. The thermal profile used an initial denaturation step of 95°C for 3 min followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 45 s. A 7 min final extension at 72°C was added at the end of cycle to increase copy number. Negative controls were included in both DNA isolation and PCR reactions.All the individuals sampled were sequenced with primers C1-J-1751 and C1-N-2329 to yield a 571-bp fragment of the mitochondrial cytochrome c oxidase subunitIgene (COI) 5′-TACCGTGAGGGAAAGTTGAAA-3′) and 28sR4076 (5′-AGACTCCTTGGTCCGTGTTT-3′) We also amplified a 456-bp fragment of the D2 domain of 28S (28SD2) nuclear ribosomal DNA from a sub-set of samples possessing the five different mtDNA haplotypes found in this study 5, (AC)6(TC)5 or (AC)6(AG)5 were used in this study, details of genomic library construction and microsatellite isolation was described by Lian et al. 2; Fermentas), 0.2 mM dNTPs (Takara), 1 µl of DNA (concentration not estimated), 0.3 µM forward primer, 0.3 µM fluorescent (FAM or HEX) labeled new ssr primer ((TC)6(AC)5, (AC)6(TC)5 or (AC)6(AG)5). The thermal profile used an initial denaturation step of 95°C for 3 min followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 45 s and a final 10 min extension at 72°C. Two additional microsatellites  were selected and PCR conditions were described previously by Brunner & Frey Samples were genotyped at ten microsatellite loci developed for Nh), nucleotide diversity (π) and haplotype diversity (Hd) were calculated using the program DNASP v5 Sequences were assembled with CodonCode Aligner 3.6.1  and manually edited before creating consensus sequences. The resulting consensus sequences of all individuals were aligned using Clustal X 2.0.11 NA), observed heterozygosity (HO), unbiased expected heterozygosity (uHE), mean number of alleles (Na), and number of private alleles (NP) were assessed using GenAlEx 6.41 AR) was calculated with FSTAT 2.9.3.2 HS). We used the one-side group comparisons in FSTAT with 1000 permutations to test for significant differences in allelic richness and gene diversity between the two groups inferred by population-pairwise FST analysis (see below).The population genetic diversity indices such as total alleles per locus  Asymmetric pairwise measures of recent gene flow were estimated using two different genetic approaches: (i) assignment tests and detection of first generation migrants for each population implemented in Geneclass 2 F. occidentalis individuals from 14 populations across China revealed five haplotypes  ranged between 0.172 and 0.656 with the lowest in HRB  from 506 plotypes  and formCoexistence of two different forms HO ranged from 0.085 to 1.000  based on all 10 loci was strongly correlated with the AR value based on 6 loci without the 4 problematic loci . Similar patterns were observed for the unbiased expected heterozygosity . Thus, all 10 loci were used in the subsequent analysis. The genetic diversity indices uHE and AR were not statistically significant across all 14 populations . The average allelic richness (AR) ranged from 3.776 to 5.191 alleles in QTX and DL localities, respectively. uHE ranged from 0.621 to 0.770 in QTX and CC, respectively  in southwestern China exhibited a statistically significant higher allelic richness , gene diversity  and a lower FST  than the other group which includes the other populations. The same results were obtained when the two small populations  were excluded from the comparative analysis.The allelic richness  indicating a low but significant population differentiation in China. When considering all the populations, 70 of the 91 pairwise comparisons tested were significantly different from zero  appeared homogenous, with no significant differentiation following the use of Bonferroni correction, suggesting the occurrence of extensive gene flow among these three populations. Additionally, JQ seems to be an isolated population since it was significantly differentiated from the other populations (mean FST = 0.079)  .F. occidentalis revealed that K = 2 was the best fit of the data for the 14 populations in China  migrants, of which 20 migrated from the three southwestern populations to almost all of the other populations . The majority of putative migrant genotypes (110/135) were restricted to southwestern China and the migration events were highly directional, from the Southwest to other parts of China. GeneClass also identified 23 individuals as potentially first-generation , our results shed light on the dynamic aspects of the invasion process soon after the introduction.This study constitutes the first attempt to understand the pattern of genetic variability in F. occidentalis in China are lower than those reported in the native regions based on mitochondrial and microsatellite DNA markers Nh = 5, Hd = 0.549) were much lower than those reported in the 12 native populations in the United States  in the 14 invasive populations were lower than those in the United States (uHE = 0.855 and HS = 0.853) Nh and uHE) for F. occidentalis in China was comparable to those reported for populations of fire ant Solenopsis invicta in newly invaded areas in China, Australia, New Zealand and the Caribbean  and/or null alleles. Brunner & Frey also found a significant deviation from HWE of F. occidentalis within two different habitats in its native area FIS<0.276 with a mean of 0.188), since a female thrips could survive long enough to mate with her own haploid male progeny because of their special haplodiploid sex determination, potentially long adult lifespan and rapid immature development rate Coccotrypes dactyliperda whose principle mating strategy was inbreeding with an average inbreeding coefficient of FIS = 0.27 within populations in California Kladothrips xiphius, K. arotrum, K. antennatus) were also highly inbred  which is comparable to the differentiation level of an invasive fruit fly pest B. invadens across its introduced range in Africa  from the source populations in southwestern China. This is especially the case for DH, which is separated by desert and xeric grasslands from other desert-edge localities where more host plants could survive. The main means of spreading of F. occidentalis is the movement of floricultural and horticultural products Analysis of Np = 5) seems to be an isolated population with significant and much higher FST compared with other populations. Its isolation is also supported by the Bayesian analysis. The isolation of JQ might be the result of genetic drift, natural selection or a separate introduction event(s). Multiple introductions seem unlikely given JQ has only two mitochondrial haplotypes which also exist in most populations across China. The presence of a greater number of nuclear private alleles in JQ is probably due to the low sample number in the founder populations and high allele number of microsatellites.Furthermore, JQ with more private alleles  Bemisia tabaci also has several biotypes with different degrees of invasive success F. occidentalisOur mtDNA results (but not the microsatellites) confirmed the presence of two previously described forms of F. occidentalis cryptic species in China arises from other factors, such as natural selection, change of habitat. Alternatively, if these two forms are different ecotypes, it was possible for hybridization to occur in China where they were sympatric, because they are not completely reproductive isolated In China, these two forms were about 3.3% divergent in terms of their mtDNA, but they cannot be distinguished by microsatellite markers. Moreover, the 28SD2 sequences of these two cryptic species in China were identical to each other and corresponded to the WFTG/HD which reported in its native range F. occidentalis neighboring populations BS, DL and KM are genetically similar and form a single population and differ little from several other populations with which they share co-ancestry. Furthermore, these three populations have an asymmetric migration towards other populations and have a slighter higher microsatellite genetic diversity than do other populations. Consistent with the nuclear microsatellite variation, these three populations have a higher mitochondrial DNA diversity. Together, these analyses provide compelling evidence that the invasion started in southwestern China , where F. occidentalis was initially found. This hypothesis is supported by the fact that KM is a center for floriculture production and transportation and a major centre for international imports of plants in China F. occidentalis. This appears to be another case of the Bridgehead effect Solenopsis invicta, which is native to South America, was inadvertently introduced into the southern United States, where it formed the bridgehead populations that act as a source of the California populations and other populations in China, Australia, New Zealand and the Caribbean Diabrotica virgifera virgifera, which is native to Mexico, established a bridgehead population in the United States for its later invasion of Europe Harmonia axyridis also followed the establishment of a bridgehead population in eastern North America The F. occidentalis in China as revealed in this study can help to improve management strategies. Strict trade quarantines and local eradication should be imposed to prevent further introduction and spread of the putative three source populations of F. occidentalis because of the high rates of gene flow from the three southwestern populations to the rest of the populations in China. Preventing contaminated plants from the putative source populations from being transported and population suppression or local eradication are crucial to eradicating this pest in other areas. To eradicate F. occidentalis from an area, it is necessary to reduce the population by various means, such as by using insecticides and chemical attractants, to a level below the Allee threshold or to apply biological controls to increase the Allee threshold New knowledge on the genetic diversity and population structure of Table S1Frankliniella occidentalis populations in China.Genetic diversity at ten microsatellite loci in 14 (DOC)Click here for additional data file.Table S2Basic indices calculated using COI gene and ten microsatellites based only on WFTG individuals and haplotype distribution in Chinese populations.(DOC)Click here for additional data file.Table S3STF matrix obtained using 10 microsatellite loci. All WFTL individuals were omitted from the respective population and were treated as a single population.Pairwise (DOC)Click here for additional data file.Table S4Results of assignment test and detection of first generation migrants (F0) based on WFTG individuals, with source populations list by column and recipient populations by row. Populations with sample size of ≤10 individuals were not included.(DOC)Click here for additional data file.Table S5Within-population tests for heterozygosity excess P-values.(DOC)Click here for additional data file.Table S6Results of AMOVA test on mitochondrial and microsatellite markers.(DOC)Click here for additional data file.


An integral part to understanding the biology of an invasive species is determining its origin, particularly in pest species. As one of the oldest known invasive species, the goals of this study were to evaluate the evidence of a westward expansion of Hessian fly into North America, from a potential singular introduction event, and the population genetic structure of current populations. Levels of genetic diversity and population structure in the Hessian fly were compared across North America, Europe, North Africa, Western Asia, and New Zealand. Furthermore, Old World populations were evaluated as possible sources of introduction. We tested diversity and population structure by examining 18 microsatellite loci with coverage across all four Hessian fly chromosomes. Neither genetic diversity nor population genetic structure provided evidence of a westward movement from a single introduction in North America. Introduced populations in North America did not show identity or assignment to any Old World population, likely indicating a multiple introduction scenario with subsequent gene flow between populations. Diversity and selection were assessed on a chromosomal level, with no differences in diversity or selection between chromosomes or between native and introduced populations. Invasive species, particularly insects, can have damaging consequences to natural environments and agro-ecosystems Mayetiola destructor (Say), is an introduced wheat pest, where it is one of the oldest known invasive species First reported in North America in 1779, the Hessian fly, Hessian fly is a galling midge that is dependant on its host for survival. The primary host is wheat, although other closely related grass species are suitable when wheat is not available Despite the long time span since the introduction of Hessian fly into North America, we wanted to test if there is clear evidence of westward movement of Hessian fly colonization to support the rich historical accounts of a single introduction. Additionally, we wanted to test if we could identify a clear source population for either North America or New Zealand from currently sampled populations, as these locations represent two separate invasions.It has been suggested that parts of West Asia may be the origin of Hessian fly, along with its host plant, and Hessian fly colonized Europe and North Africa 100 years prior to colonizing North America 2 (Promega), 5 µM dNTPs (Promega), 0.75 U of Taq DNA polymerase (Promega), 1.0 µl of reverse non-fluorescent primer (5 µM) and 0.5 µl of a fluorescently labeled forward primer (10 µM), labeled with one of three Beckman-Coulter dyes , and 6–25 ng template DNA. Cycling conditions were 95°C for 4 min, 6 cycles of 95°C for 1 min, 50°C for 1 min, 72°C for 1 min, 31 cycles of 95°C for 30 sec, 50°C for 30 sec, 72°C for 55 sec, and a final extension of 72°C for 30 min performed in a MJ Research DNA Engine Dyad thermal cycler . After amplification, products were pool-plexed, which is the combining of loci for genotyping. The pool-plexes included 2 groups of nine loci, adding 2 µl of blue, 3 µl of green, and 12 µl of yellow/black dyed products. Genotyping mixes were made from 1 µl aliquot of pool-plexes, 0.5 µl of a 600 bp size standard (Beckman-Coulter), and 40 µl SLS buffer (Beckman-Coulter). Genotyping was then performed using a Beckman-Coulter CEQ8000, per manufacturer’s instructions, and sized with CEQ8000 software. Genotypes were checked for errors using Micro-CheckerThirty collections of Hessian fly were obtained from nineteen states within the United States, one Canadian Province, and six non-North American countries . SamplesreatereeNA enAlEx v6.2 IS), calculated in Fstat v2.9.3 For all applicable software, Cottlenecket al. ottleneck simulations for heterozygosity excess. A mode-shift test was also run to look for any distortion in the distribution of allele frequencies different than the typical L-shaped distribution, indicating a bottleneck M) M>0.68, and should approach 1.Three methods were used to search for a bottleneck to test if any signal of a bottleneck remained, either as a result of initial introduction or range expansion westward. First, a search for a recent population bottleneck was performed in Bess v2.3.1 ess has been shown to perform equally well, if not better, in discerning the number of populations as compared to other commonly used clustering software, especially if geographic admixture is moderate to low or if differentiation was weak ess, 100 independent runs for each K were performed with 50,000 sweeps and a burn-in of 5,000 sweeps, and allowing for admixture. Samples were run in three groups : entire dataset (1 to 16), only North American samples (1 to 16), and only Old World samples (1 to 11). The Deviance Information Criterion (DIC) was used to compare model fits (those with a lower DIC fit better) by taking an average across the 100 independent iterations and plotting against K. Much like the plateau of log-likelihood values in Structurelumpp version 1.1.2 istruct was used to visualize the results To assess population genetic structure, we implemented a Bayesian clustering algorithm, which uses an explicitly spatial prior distribution, as utilized in Tstat v2.9.3 Diversity based on Nei’s unbiased genetic diversity was calculated for each locus in FST. In order to identify if there are any loci experiencing different selection pressures between North America and the Old World, these simulations were carried out with 1) the whole data set, 2) North American populations, and 3) the Old World populations, excluding New Zealand from simulations other than with the whole data set. Simulations were performed using the Fdist method ositanositan was run for 50,000 simulations with both the infinite alleles model (IAM) and the stepwise mutation model (SMM), using both options for neutral and forced mean FST, for each of the three groups tested.Selection was also assessed for each locus by identifying loci with excessive or reduced FM values all greater than 0.68, and all values around 1, indicating equilibrium , very close to a neutral slope of 1.0, indicating among all loci, diversity in North America does not drastically differ from that of the Old World (2 = 0.6604), A2 = 1.0088 (R2 = 0.4384), X1 and X2 combined = 0.9292 (R2 = −1.733) (Little variation was found in the assessment of diversity between North America and the Old World . Comparild World . Additio −1.733) . TherefoThe only loci identified as candidates for selection were Hf119 and Hf174. Both loci were identified as being under balancing selection under the IAM only. Under the SMM, no loci were identified as being under any selection. This result was the same for the North American populations (both in K7 and K8), as well as the Old World populations. Both loci under selection were located on chromosome A1; however, Hf119 is located on the short arm and Hf174 on the long arm All Old World collections consisted of single, independent populations, with the exception of the Israeli collection containing two distinct populations. Finding two populations within the Israeli collection is very interesting as these flies were all collected within the same field, yet have a very distinct population genetic structure. This result may not be entirely surprising as Israel is closely located to the putative origin of wheat and Hessian fly, and many species of wild wheat natively occur within the country None of the introduced populations from North America or New Zealand clustered together with any of the Old World populations . This rehttp://webarchives.cdlib.org/sw15d8pg7m/http:/ers.usda.gov/Briefing/Wheat/maps.htm), as suggested by Black et al. In North America, seven populations were found across a majority of the wheat-growing region, with more Hessian fly collections sampled than in any prior study. All collections fit into pseudo-regional populations, with the exception of FlSC and HoMS, which stood independently as their own population. With respect to the populations in the southeastern US, overall similar results were found previously We found similar genetic diversity between all Hessian fly populations, regardless of their origin; however, when observing those populations that are centrally located in North America, such as Central and SouthEast, we observed a slight decrease in diversity and allelic richness in the populations as the distance from the coast decreased. This could be due to the fact that these centrally located populations were made up of more collections; however, it could also be an indication that genetic mixing commonly occurs in these areas. Unfortunately, this pattern of slightly higher diversity in the centrally located populations, and decreasing moving out, neither expressly lends itself to providing evidence of a westward expansion, nor does it refute the possibility. Private alleles were found in the most westward populations , either ositan. The lack of differences in diversity on a chromosomal basis between the North American and Old World groups is supported by the findings that both the North American and Old World populations have the same two loci under selection. This indicates, that despite over 200 years of separation and the high probability of differing selection pressures in the field IS values, and therefore contribute to these populations not maintaining Hardy-Weinberg equilibrium. Inbreeding, however, may exist in these populations, and the population structure could be an artifact of common descent, but a higher resolution of loci would be required to test this hypothesis.As a test to identify if there had been any increasing and/or differing selection pressures in North America compared with our Old World samples, we compared genetic diversity at each locus and for each chromosome . This coThe minimal evidence of a westward movement, along with the lack of differences in diversity and private alleles between North America and the Old World is consistent with evidence of multiple introductions In this study we did not find overwhelming support of a westward expansion, although this result adds to uncovering the invasion history of one of North America’s oldest invasive insects. We did observe that after >200 years, a significant proportion of population admixture still remains in North America, compared to Old World counterparts. It will be interesting, however, to further explore if the population structure in North America is a result of an association between populations and wheat class, or some other factor, adding to the development of hypotheses on the biology of this invasive insect. Furthermore, our data adds to the possibility of a multiple invasion scenario, and due to the methods employed here, provides a baseline for the quantity of data necessary to reconstruct the history further. As the signal of invasion has weakened over the last >200 years, future investigations of Hessian fly may need to be conducted to examine this invasion scenario on a genomic level to further tease apart this dramatic history.


Passer domesticus) in Brazil varies with range expansion, (ii) to determine how genetic diversity and differentiation in Brazil compares to ancestral European populations; and (iii) to determine whether selection or genetic drift has been more influential on phenotypic divergence. We used six microsatellite markers to genotype six populations from Brazil and four populations from Europe. We found slightly reduced levels of genetic diversity in Brazilian compared to native European populations. However, among introduced populations of Brazil, we found no association between genetic diversity and time since introduction. Moreover, overall genetic differentiation among introduced populations was low indicating that the expansion took place from large populations in which genetic drift effects would likely have been weak. We found significant phenotypic divergence among sites in Brazil. Given the absence of a spatial genetic pattern, divergent selection and not genetic drift seems to be the main force behind most of the phenotypic divergence encountered. Unravelling whether microevolution , phenotypic plasticity, or both mediated phenotypic divergence is challenging and will require experimental work .Introduced species are interesting systems for the study of contemporary evolution in new environments because of their spatial and temporal scales. For this study we had three aims: (i) to determine how genetic diversity and genetic differentiation of introduced populations of the house sparrow ( If STP is significantly larger or smaller than STF, then it is possible that the geographic variation in phenotypic traits were more likely shaped by selection rather than governed by genetic drift STP is a rough estimate of STQ, the latter measures variation in quantitative traits by partitioning the variance related to additive genes between and within populations STQ can be challenging because it requires the rearing of several populations in common garden conditions. Therefore, phenotypic measures have been used as a surrogate, but one should be cautious to the possible caveats associated with the use of STPTo better understand rapid evolution and how invasive species adjust to novel environments, population genetic studies should be combined with analyses of spatial phenotypic differentiation Passer domesticus) to address three main questions. First, we compared genetic diversity and population structure between populations in the introduced range in Brazil and the native range in Europe. We expected populations from Brazil to be less diverse than the European populations. Second, we analyzed genetic differentiation among populations in Brazil to understand how the expansion process in Brazil occurred. For example, if house sparrows in Brazil underwent sequential founder events during its expansion, one would expect a pattern of isolation by distance and populations in the expansion front to present reduced genetic diversity. Our third goal was to compare spatial phenotypic differentiation (STP) with genetic differentiation (STF). This method would allow us to evaluate whether phenotypic differences between populations were shaped by selection or genetic drift. In principle, if STP equals STF, differentiation of morphological traits (assumed to be governed by additive genetic variation) is probably the result of genetic drift. However, if STP is larger than STF it means that quantitative traits have diversified more than neutral genetic loci, which could be evidence of directional selection. Alternatively, if STP is significantly smaller than STF, quantitative traits probably diversified less than neutral genetic loci, suggesting that these traits have been under the influence of stabilizing selection In this study we used the house sparrow . In Brazil approval by an ethical committee of the university is required only for captive animals used in experiments. All other types of work with animals are regulated by IBAMA - Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis, which substitutes the ethic committee in approving and evaluating all aspects of projects that involve capture and handling of animals, taking morphological measurements, blood sampling and other procedures. Permits were obtained from IBAMA  for the purpose of this study and MR Lima’s PhD Project and house sparrows were captured [with mist nets] at university campi and private homes , with appropriate permissions of institutions and land owners. After being measured and sampled for blood and feathers, all birds were immediately released. Field methods were carried out so that handling time and potential suffering of animals were minimized. House sparrows are considered exotic birds in Brazil, and in the IUCN Red List the species has a Least Concern category. Data on European populations were obtained from a previous study Two hundred house sparrows were released in 1905 in Rio de Janeiro, Brazil 2 plus dNTPs and a PCR buffer especially developed for multiplex PCR), 1 pmol of each primer (forward primers were labelled with either 6-Fam or HEX) made up to 10 µl with ddH20. For PCR conditions see Genomic DNA was extracted from blood samples using a standard protocol with overnight digestion with proteinase K and subsequent phenol-chloroform extraction and alcohol precipitation PCR products of Pdoµ1, Pdoµ6 and Pdo8 were multiplexed and diluted 1∶100, while Pdoµ3, Pdoµ4 and Pdo9 were multiplexed and diluted 1∶50. These multiplex combinations were chosen so that products had different dye labels and differed in range sizes. Labelled size standard MM1000 was mixed with multiplexed PCR products and electrophoresis was conducted in a capillary ABI3730XL sequencer (Applied Biosystems). Resulting data were analysed with GeneMapper 3.0 (Applied Biosystems) for fragment size determination.et al.et al.et al.Genotyping of house sparrow populations from Europe was done in ABI 377  and Hardy-Weinberg equilibrium (HWE) using FSTAT version 2.9.3 ISF. We used non-parametric tests (Wilcox tests) to test for any differences in the genetic diversity estimators of the introduced and native house sparrow populations. We also compared these genetic diversity estimators with data from the literature of other house sparrow populations for studies that has used similar procedures to calculate these estimators To compare genetic diversity between the native range (Europe) and the introduced range (Brazil), we calculated allelic richness (Ar) and private allelic richness (Par) for each population using HP-Rare To test whether house sparrow populations in Brazil had experienced a recent bottleneck, as might be expected if the expansion process occurred via sequential founder effects or because of very small initial population size at the time of release, we used BOTTLENECK version 1.2.02 STF values, which were estimated according to Weir and Cockerham STF was estimated globally and between all pairs of introduced populations. Significance of global STF was evaluated by permutation of genotypes among samples and calculating 95% Confidence Intervals (C.I.) by bootstrapping over loci (number of permutations was set at 1000). Pairwise STF was tested to determine whether it was significantly different from zero by randomizing the genotypes, and a Bonferroni correction was used to control for type-I errors. We also calculated estD defined by Jost STF calculations when using highly polymorphic markers such as microsatellites. estD varies from zero, when there is no genetic differentiation between populations, to one when populations are completely differentiated, and was calculated using the web-based resource SMOGD estD across loci. Moreover, we also used STRSTF, however, it uses variance in allele size (number of repeat units) between populations, because mutations in microsatellites involve the addition or subtraction of a small number of repeat units. We used STR because it is less sensitive to rare alleles than STF.Genetic differentiation among the introduced populations of Brazil was determined by STF, while for esDt and STR we used the library “vegan” STF and estD (as above) for the European populations. These calculations were repeated separately for the Brazilian and European populations because only three loci matched between the studies.We tested for isolation by distance, which is the correlation between geographical distance (using log transformation) and the degree of genetic differentiation, using a Mantel test in Arlequin version 3.5.1.2. Left tarsus, beak height, beak width and beak length of Brazilian sparrows were measured with a digital calliper (0.01 mm) and left wing, tail and body length were measured with a ruler (0.1 cm). Additionally, 770 feathers were plucked randomly from dorsal and breast areas  and three measurements, which consisted of 50 sequential spectra each, were taken from each sample at three random points by lifting the black block that contained the probe to ensure that a different part of the feather was being measured each time. Individual color was characterized by averaging the three spectra, which were interpolated to a step of 1 nm between 300 and 700 nm. We calculated brightness as the area under the spectra curve  and UV-Chroma as the proportion of UV reflectance between 300 and 400 nm.STP) was used to infer the role of genetic drift and natural selection on the different morphological traits of house sparrow populations of Brazil by comparing it with STF. STP is similar to the STQ index, which measures quantitative trait differentiation, however, STP is influenced by environmental, non-additive genetic effects and by the interaction between the environment and genotype . Further, the morphometric traits being considered in this study are known to have substantial additive genetic basis STP−STF comparison can provide initial insights into the evolutionary process that has occurred during the expansion of the house sparrow in Brazil before further inquiries can be made. STP was estimated as:Phenotypic divergence (2h is the heritability (the proportion of the phenotypic variance attributed to additive genetic effects). The scalar c represents the proportion of the total variance that is claimed to occur because of additive genetic effects across the populations. If parameters c and 2h are known for the populations being studied, then STP equals STQc in the wild is very challenging and 2h is population specific 2c/h ratio is critical to how well STP approximates STQ, one can use a sensitivity analysis, which varies this ratio, to infer the robustness of the approximation of STQ by STP2c/h = 1 , that is the proportion of phenotypic variance due to additive genetic effects is the same for both within and between population variance. If STP exceeds STF at this point it will also do so at any point where c>2hSTP exceeds STF when c<2h . The reason is that natural populations are probably under genotype-environmental interactions and/or divergent environmental effects and a low value of 2c/h assumes a larger role of environmental effects in driving between population variance than within population variance . Therefore, the lower the critical 2c/h ratio is (2c/h<1) when STP exceeds STF, the more likely it is that the trait is being shaped by selection where STP were obtained using analysis of variance where body length was entered as a covariate. STP 95% C.I. were calculated, by considering STP to be normally distributed and using critical values of t, to test whether they overlapped with global STF value ±95% C.I. and thus whether STP values were different from STF. The critical 2c/h ratio was obtained by graphically exploring STP and its 95% C.I. as a function of 2c/h and by looking at the approximate value of 2c/h where the lower 95% C.I. of STP meets the upper 95% C.I. of STF2c/h<0.1 means that in order for genetic drift to explain phenotypic divergence, the required additive genetic effect across populations would need to be less than 10% of the additive genetic effect within population. Therefore, this would be a very robust inference that the traits are under selection and not genetic drift Variance components for estimating We also conducted multivariate analysis of variance (MANOVA) to test if populationś centroids of trait means were significantly different from each other. MANOVA assumptions were checked before analysis, and three males and two females were not included in the analysis because they were multivariate outliers, which was checked with the R package “mvoutlier” ISF  but significantly lower private allelic richness , and lower unbiased He  and in pair-wise comparisons  and between population pairs . Canoas is in the South of Brazil, while Niteroi is less than 10 km from Rio de Janeiro, where the house sparrows were initially released ; again, Niteroi and Canoas had the highest estD value. Furthermore, when STR was used to infer genetic differentiation between house sparrow populations from Brazil, we found that global STR values were higher than zero  and pairwise STR values were similar to STF and estD . The results from the STF, estD and STR analysis suggest slight genetic population differentiation in Brazil.For Brazil, genetic differentiation was also very low both globally .Pillai trace  = 2.246, df = 55, 245 F = 3.633, p<0.001; Male: Pillai trace  = 1.897, df = 55, 370, F = 4.113, p<0.001). Comparisons of STP with STF show that similar traits in both males and females were usually shaped by selection  and not genetic drift, because lower 95% C.I. for STP were higher than the upper 95% C.I. for STF  was generally higher than expected from neutral genetic markers for similar traits in both males and females. However, our results must be interpreted with caution because of the small number of loci and populations used.The genetic variation in house sparrows from Brazil was only marginally lower compared to populations from the native range in Europe. We found no evidence for recent population bottlenecks or for the occurrence of sequential founder events during the range expansion process. We also found an absence of genetic structuring  among Brazilian populations, implying that expansion occurred with low influence of genetic drift and possibly high population growth. Moreover, we found that populations from Brazil differed morphologically from each other and that phenotypic divergence (ISF) were not different from native European populations, and when six loci were used, we found higher Ar for introduced Brazilian house sparrow populations. Therefore, house sparrow populations in Brazil do not appear to have lost much genetic variation. In support, when genetic diversity estimators from this study were compared with the literature, we found that estimators were not substantially different from what is found in populations from the native range , and unbiased expected heterozygosity (UHe) were lower in introduced Brazilian than native European house sparrow populations. When only three loci were considered, both allelic richness (Ar) and Par were lower for introduced Brazilian populations. These results are consistent with founder effects observed with other bird introductions ve range . AdditioHouse sparrow studies from other introduced ranges have shown mixed results regarding the amount of genetic diversity lost when compared with the native range. For example, introduced populations in Australia and New Zealand exhibit a reduction in the number of alleles, but only the New Zealand populations had lower heterozygosity We found low levels of genetic differentiation among house sparrow populations in Brazil. It seems that out of the six sampled populations only two, Canoas and Niteroi, are genetically different from all other populations. Canoas, which is in the south of Brazil , may be STF variation was not high, suggesting that the influence of genetic drift was low Although Niteroi is very close to Rio de Janeiro, the cities are separated by Guanabara Bay, a large body of water that the house sparrows would need to cross, or alternatively take a longer inland route between the two cities. Perhaps the population of Niteroi had a higher influence of genetic drift when compared to other populations, which could be the case if colonization of Niteroi occurred before house sparrows could reach it via an inland route. Thus, Niteroi may not provide a good representation of the initial founding population of Rio de Janeiro. We also found no isolation by distance for populations, and A possible explanation for the low influence of genetic drift in house sparrow introduced ranges could be that there has not been enough time for genetic drift to take place because most introductions occurred around 1850 STP- STF analysis indicates that divergence of most of the morphological traits was due to selection and not genetic drift, with the exception of wing length and most beak measurements.Morphological divergence was found among house sparrow populations in Brazil and House sparrow populations from other introduced ranges, such as North America STP instead of the more accurate STQ. Our STP estimates therefore cannot rule out environmental or parental  effects on morphological traits. Nonetheless, without genetic differences between the introduced populations, it seems more plausible that phenotypic plasticity is driving morphological differentiation in some of the traits. However, if selection is indeed responsible for phenotypic divergence, one can expect it to occur in a predictable manner, such as local adaptation to the abiotic environment 2c/h when compared to other traits such as plumage coloration. Therefore, phenotypic differentiation of these traits may not have a very strong adaptive basis for house sparrow populations in Brazil. A similar result was also found for native house sparrow populations from Finland, where only body mass across populations seemed to be adaptive, while other traits  seemed to be shaped by genetic drift Although divergent selection (favouring of different phenotypes in different populations) may be driving morphological divergence of some of the traits, it is difficult to discern whether this pattern is a response to selection (microevolution) or simply a plastic response to the environment. In this study it is more challenging to evaluate this because we used STP−STF analysis. Also, because two of the microsatellites had a high number of alleles, and therefore high-expected heterozygosity that can generate low levels of STF, it is possible that type-I errors of rejecting the null hypothesis STP = STF may have occurred However, we must interpret our results cautiously because of the small number of populations used in our STF was used. However, not all the traits presented STP>STF and traits related to body size  were less robust in the sensitive analysis then plumage traits. Using the STP−STF approach as an initial step allows us to infer that house sparrows should quickly respond to new selective factors they are exposed to in new areas, especially to factors affecting plumage coloration. In addition, future experimental studies should be able to determine if the morphological divergence observed in Brazil is due to microevolution (changes in genotype frequency) or plastic phenotypic responses to environmental conditions.Introduced house sparrow populations from Brazil lost some genetic variation relative to sparrows from the native range in Europe. However, it seems that the expansion process occurred in association with high population growth and possibly gene flow, thus enabling populations from Brazil to retain substantial genetic diversity with little genetic differentiation. However, our results need to be interpreted cautiously because of the low number of markers and populations used. We found significant morphological variation among populations and, overall, morphological divergence was higher than neutral genetic divergence suggesting the action of selection overriding the effect of genetic drift for many of the traits when Figure S1Comparison of phenotypic differentiation (STP – solid line) with the upper 95% confidence interval (C.I.) for neutral genetic differentiation , while the ratio 2c/h was varied from zero to 2. The dashed black lines the 95% C.I. for the STP calculations, while the dashed red line represent the null assumption that c = 2h. Results are for male traits that had critical 2c/h  higher than 0.6.(EPS)Click here for additional data file.Figure S2Comparison of phenotypic differentiation (STP – solid line) with the upper 95% confidence interval (C.I.) for neutral genetic differentiation , while the ratio 2c/h was varied from zero to 2. The dashed black lines represents the 95% C.I. for the STP calculations, while the dashed red line represent the null assumption that c = 2h. Results are for female traits that had critical 2c/h  higher than 0.5.(EPS)Click here for additional data file.Information S1PCR Cycling conditions.(DOCX)Click here for additional data file.


Population stratification is the main source of spurious results and poor reproducibility in genetic association findings. Population heterogeneity can be controlled for by grouping individuals in ethnic clusters; however, in admixed populations, there is evidence that such proxies do not provide efficient stratification control. The aim of this study was to evaluate the relation of self-reported with genetic ancestry and the statistical risk of grouping an admixed sample based on self-reported ancestry.A questionnaire that included an item on self-reported ancestry was completed by 189 female volunteers from an admixed Brazilian population. Individual genetic ancestry was then determined by genotyping ancestry informative markers.Self-reported ancestry was classified as white, intermediate, and black. The mean difference among self-reported groups was significant for European and African, but not Amerindian, genetic ancestry. Pairwise fixation index analysis revealed a significant difference among groups. However, the increase in the chance of type 1 error was estimated to be 14%.Self-reporting of ancestry was not an appropriate methodology to cluster groups in a Brazilian population, due to high variance at the individual level. Ancestry informative markers are more useful for quantitative measurement of biological ancestry. Although the biogeography of some groups is culturally and genetically fixed, other groups have experienced substantial recent admixture with ancestors from widely divergent regions. That is the case in the Brazilian population, which is genetically characterized by differing degrees of admixture of 3 parental populations .3–8 Self-reported ancestry has been described as a method that is highly correlated with genetic population structure in well defined, stratified ethnic groups, such as Europeans, Africans, and Asians.7–9 However, in cases of admixed populations, both self-reported ancestry and anthropometric traits used as proxies, such as skin pigmentation, are believed to be unreliable methods of determining ancestry,3–5,8 which suggests that molecular markers based on genetic clustering should be used to reduce the potential inaccuracies of population stratification.3,6,10The debate on how genetic studies should be controlled for population stratification has encompassed several methodologies, including self-reported ethnicity and genetic ancestry markers.11–15 However, the recent use of molecular markers to determine genetic ancestry has revealed wide genetic heterogeneity in admixed Brazilians.5,16–22 One problem in performing association studies of admixed populations that are assessed solely by self-reported ancestry as a proxy of ethnic group is the possibility of spurious association with false-positive or false-negative results.5–7,23–26 Thus, the aim of this study was to evaluate the relation of self-reported with genetic ancestry and the statistical risk of grouping an admixed sample by using self-reported ancestry.Many association studies have classified ethnic groups by means of subjective assessment by the interviewer, evaluation of anthropometric traits, genealogical examination, and self-reported ancestry.27 All sampled individuals signed an informed consent form, and the research protocol was approved by the University Ethical Committee.Samples were obtained from 189 postmenopausal women  who had volunteered as part of a healthcare program developed by the Universidade Católica de Brasília, located in the Center-West Region of Brazil . The volunteers answered a lifestyle questionnaire that included a multiple-choice question on self-reported ancestry, based on the method used by the Brazilian Institute of Geography and Statistics (IBGE) national census survey.28–30  and shrimp alkaline phosphatase (SAP) enzymes to eliminate nonincorporated dNTPs and primers. Finally, the minisequencing reaction was performed using the SNaPshot Multiplex minisequencing kit reaction mix , and the products were analyzed on the ABI 3130 XL Genetic Analyzer (Applied Biosystems) in an ABI 3700 POP-6 polymer. Genotypes were called using GeneScan Analysis Software, version 3.7 (Applied Biosystems) and Genotyper version 3.7 (Applied Biosystems). The detailed optimized multiplex single-base extension protocol, with reactant concentration and PCR thermocycling conditions, has been reported elsewhere.21,31–33For assessment of individual genetic ancestry, we selected 13 Ancestry Informative Markers (AIMs) that have differential allele frequencies among European, African, and Amerindian parental populationsST) analysis, which was performed using GenAlex software.34 The FST measures population differentiation based on the heterozygosity of genetic polymorphism data is calculated using the formula, FST = (HT − HS) × HT−1, where HT is the expected heterozygosity in the total population and HS is the observed heterozygosity in a subpopulation.35 The fixation index can range from 0.0 (no differentiation) to 1.0 (complete differentiation) and theoretically varies from little (0.0 to 0.05) to moderate (0.05 to 0.15), great (0.15 to 0.25), or very great genetic differentiation (>0.25).Allelic frequencies were obtained by direct counting, along with pairwise population Fixation index . Briefly, the log likelihood function is maximized for the admixture parameter of up to 3 parental populations using a priori known allele frequencies and estimates the individual ancestry probability from a predetermined number of analyzed genotypes of an admixed individual. The MLE approach was implemented in the software program IAE3CI; the detailed statistics have been described elsewhere.P value of 0.05 or lower was considered statistically significant. Statistical analysis was performed using SPSS software version 13 .Basic descriptive statistics and 1-way analysis of variance (ANOVA) with the post-hoc Games-Howell test to adjust for unequal variances were used to determine the relation between genetic ancestry distribution and self-reported ancestry groups. A A total of 192 participants completed the study, but only 3 were of self-reported Amerindian ancestry. Due to the lack of statistical power, these 3 women were not considered in the analysis, and 189 participants remained for study, as previously described. The questionnaire responses indicated that the sampled population had similar prevalences (41.8%) for 2 groups, white and intermediate . Blacks represented 16.4% of the sample. No individual reported Asian descent in the present research.ST analysis revealed little difference between the white and intermediate groups (FST = 0.022), a moderate difference between the intermediate and black groups (FST = 0.138), and great differentiation between the black and white groups (FST = 0.225); all P-values were significant.Allelic frequency for the 13 genotyped SNPs is described in Table The genetic ancestry of each self-reported ancestry category and the total sample was estimated Table . The ranP = 0.05).One-way ANOVA for comparison of means in conjunction with the Games-Howell post-hoc test revealed significant mean differences among self-reported groups for European and African ancestries, but not for Amerindian ancestry Table . Althoug27 The inferred ancestry estimated here is comparable to those of other published studies of the Center-West Brazilian population,17,21 with only slight differences in European and African ancestry proportions, which were probably due to sampling issues.The Federal District is a modern urban center and the capitol of Brazil. It has a population of migrants from several regions of Brazil. The 2007 National Household Sample Survey reported a distribution of self-reported ancestry in the Federal District that was very similar to that of the present study, differing only in the prevalence of blacks.38 However, when the population ancestry estimates, standard deviations, and variances of the present study were compared with those of a different sample of Center-West Brazil that was assessed using a set of 28 ancestry markers,21 the values did not statistically differ between samples (P = 0.49), especially with regard to individuals of Amerindian ancestry .21In this study, the statistical power of the 13 AIM panel might have been insufficient to accurately assess ancestry in an admixed population.Allelic frequency and F-statistic estimates significantly differed among groups. It is noteworthy that the differences in allelic frequencies between the corresponding ancestry-related populations  were remarkably divergent. This was the case for CYP3A4 in EUR-white (δ = 0.473) and for rs1871534 in AFR-black (δ = 0.710), which highlights the admixture among these groups. The proportions of genetic ancestries in the intermediate group were similar to those of the total sample and differed only in variance. Therefore, the range amplitude and variance of ancestry at an individual level were too large for self-reported ancestry to be considered a suitable proxy for homogenic clustering, although the differences in their means were statistically significant.5 and in an urban population sample from Rio de Janeiro (49% non-African ancestry).39 The intermediate group described here had estimates closer to those of the white group, as was the case for the urban population of Rio de Janeiro.39 Alternatively, in a study of a sample from a rural community,5 the intermediate group was closer to blacks, revealing an important issue, namely, that groups with equal self-reported proportions can have different genetic ancestry profiles, especially if the samples are from communities with different levels of urbanization. Those features were also demonstrated in other Brazilian population samples that assessed ancestry by using maternal (mtDNA) and paternal (Y-chromosome) molecular markers.20,22,40In addition, we observed an overlap in the range of genetic ancestry values among groups, which suggests that individuals with the same proportions of admixture could include themselves in any ethnic category. The confidence interval revealed that the risk of this occurring simply by chance was 14%, considering the European and African ancestry estimates. It is worth mentioning that the group self-reported as black had a proportion of non-African ancestry exceeding 53%. In previous studies of the Brazilian population, African ancestry did not exceed non-African ancestry, but a large proportion was observed in a sample of the rural community of the Southeastern Brazilian state of Minas Gerais (48% non-African ancestry)41 The results of that study can be compared with the categories in the present study. An overlapping range in ancestry estimates was also reported, in which the distribution of African ancestry overlapped across 12% for all 3 color categories (range of ancestry estimates: 0.27–0.35). In the present sample, this range was considerably higher (0.19–0.48), which encompassed 48% of the sample. For European ancestry, the overlap accounted for 63% of the sample in a range between 0.41–0.78, while for Amerindian ancestry, 95% of individuals were in the overlapping range (0.0–0.42) for all 3 categories.A related study of Puerto Ricans who self-classified ancestry/color groups found statistically significant differences in genetic ancestry among 3 groups .4 in which the family history would be assumed to be more reliable. In the same way, interviewers might misclassify an individual for whom they do not know the ancestral family history. Indeed, different interviewers have classified the same individual into different groups.23,42 These examples illustrate how self-identified ethnicity might not be sufficiently accurate for use in biomedical research, as it is primarily a sociocultural construct.42 Considerable variation exists because ethnicity is essentially constructed under social circumstances that consider many cultural traditions.1,42,43 In admixed populations, individuals might feel that they belong to a certain ethnic group for cultural reasons or beliefs; however, their genealogy might consist of an unknown admixture.1,2 Although our sample comprised only women, we did not evaluate such effects on self-declared ancestry, as it may have more sociological than biological meaning.42,43 From a sociological and anthropological point, a person’s biological ancestry might have no relationship to their self-identification with a cultural group, but such ancestry might be of great importance in clinical research.The reliability of self-classification can be poor, even among a proband and siblings,5,24,44 Several strategies can be effective in controlling heterogeneity equivalence. For example, individual ancestry estimates can be used to match admixed case-control groups.45 They can also be used in cross-sectional studies as covariates to adjust for a population stratification effect.24,31,33 The use of ancestry informative markers to estimate individual ancestry is an effective and reliable solution to correct the effects of heterogeneity.In conclusion, determination of ethnicity based on self-reported ancestry is vulnerable to misclassification and should be avoided in scientific research. Therefore, the concept of ethnic group and self-declared ancestry are not synonymous in biomedical research and must be replaced with scientific measurements that have biological meaning, such as individual ancestry estimated by DNA markers.


Limnoperna fortunei by characterizing the genetic structure in native and introduced ranges in Asia and South America. We surveyed 24 populations: ten in Asia and 14 in South America using the mitochondrial cytochrome c oxidase subunit I (COI) gene, as well as eight polymorphic microsatellite markers. We performed population genetics and phylogenetic analyses to investigate population genetic structure across native and introduced regions. Introduced populations in Asia exhibit higher genetic diversity (HE = 0.667–0.746) than those in South America (HE = 0.519–0.575), suggesting higher introduction effort for the former populations. We observed pronounced geographical structuring in introduced regions, as indicated by both mitochondrial and nuclear markers based on multiple genetic analyses including pairwise ФST, FST, Bayesian clustering method, and three-dimensional factorial correspondence analyses. Pairwise FST values within both Asia  and South America  were lower than those between continents . Fine-scale genetic structuring was also apparent among introduced populations in both Asia and South America, suggesting either multiple introductions of distinct propagules or strong post-introduction selection and demographic stochasticity. Higher genetic diversity in Asia as compared to South America is likely due to more frequent propagule transfers associated with higher shipping activities between source and donor regions within Asia. This study suggests that the intensity of human-mediated introduction vectors influences patterns of genetic diversity in non-indigenous species.We explored possible links between vector activity and genetic diversity in introduced populations of  Blackburn et al. The distribution and genetic structure of introduced populations can exhibit complex patterns Dreissena polymorpha in North America exhibit lower but not severely diminished haplotype diversity relative to putative source populations in the Black Sea Genetic variation in introduced populations also depends on the structure of the source population The nature of transmitting vector can define the extent of propagule pressure and the genetic composition of introduced populations received from the source region Limnoperna fortunei, a freshwater mytilid native to mainland China, Korea, Cambodia, Indonesia, Laos, and Vietnam Limnoperna thereafter expanded its distribution very rapidly into Uruguay, Paraguay, Brazil and Bolivia, traveling an average of 240 km per year In this study, we explore genetic consequences of global spread of the golden mussel L. fortunei via discharged ballast water L. fortunei was introduced to Japan via aquaculture as a ‘fellow traveler’ with stocked Asian clams Corbicula fluminea imported from China L. fortunei could be introduced in Asia via at least two possible vectors , whereas only a single vector  appears possible for invasions in South America. Consequently the likelihood of admixed introduced populations should, be lower in South America than in Asia. This pattern serves as the basis of our first hypothesis: introduced populations of L. fortunei in South America will be genetically impoverished relative to introduced ones in Asia. Introduced populations in Asia (Japan and Taiwan) are separated by geographical barriers (marine water) whereas introduced South American populations have spread upstream from the putative initial invasion site in the Río de la Plata estuary along the Paraná, Uruguay, and Paraguay rivers. This pattern serves as the basis of our second hypothesis: gene flow is more limited among introduced Asian populations than among South American ones, and, as a result, genetic differentiation is more pronounced among introduced Asian populations. To test these hypotheses we used the cytochrome c oxidase subunit I (COI) gene and microsatellite markers to study genetic structure of this species in both continents.Japan, Taiwan, Korea, and parts of China are considered at high risk for biological invasion on the basis of ship traffic volume, while the opposite is true for South America No specific permits were required for the described field studies in Asia and South America. The species collected is an invasive pest in South America, Japan, and Taiwan and is not protected throughout this range. Sampling points did not include protected or private lands.Limnoperna fortunei was sampled from 24 locations in Asia and South America, distributed across both native and introduced regions .Sequences were aligned using CodonCode Aligner 2.0  and then manually edited. The possibility of doubly uniparental inheritance (DUI) of mitochondrial genome observed in other Mytilidae species was tested and ultimately excluded according to the method described by Gillis et al. L. fortunei from Japan were retrieved from GenBank (Accession nos. AB520611– AB520627) and included in phylogenetic analyses.A parsimony haplotype network with 95% connection probability n), haplotype diversity (h), and nucleotide diversity (π), were estimated using DnaSP 5.0 ST with the Tamura-Nei substitution model implemented in ARLEQUIN version 3.1 a priori expectations according to their geographical origin into two groups: i) all South American populations versus all populations from Asia; and ii) based on their geographic region within Asia  and within South America (samples were divided based on river basins into six regions: I Upper Paraguay (CO), II Paraná River , III Uruguay River (UR), IV Río Tercero (RT), V Sao Gonçalo (SO), and VI Paraná Delta - Río de la Plata .The number of haplotypes (A), allelic frequency (F), allelic richness (Ar), observed heterozygosity (HO), and expected heterozygosity (HE) were measured using FSTAT v.2.9.3 Ar), which is an estimate of allelic diversity adjusted by the lowest sample size, was calculated using Fstat. Genetic differentiation among populations was examined by FST using ARLEQUIN. Similar to ФST, sequential Bonferroni corrections were used to adjust the significance level for multiple comparisons FST and the interpretation of population differences, we also calculated one of the corrected FST –like indices Jost’s D Genetic diversity indices including the number of alleles  was tested from one to 24  and ten independent runs for each K value were set at 106 Markov chain Monte Carlo (MCMC) iterations, with an initial burn-in of 105. We followed the method of Evanno et al. K. We assessed possible hierarchical genetic structure by conducting separate Bayesian analyses on populations from South America (K from 1 to 14), and Asia (K from 1 to 10), plus Taiwan and Japan (K from 1 to 5). A three-dimensional factorial correspondence analysis (3D-FCA) was performed using GENETIX v. 4.05 To further investigate population genetic structure of L. fortunei. Of the 16 Japanese haplotypes retrieved from GenBank, eight were identical to those detected in this study. The haplotype frequency map revealed a high level of geographic structure . All haplotypes were recovered after we used only female mussels (F-type haplotypes), suggesting that doubly uniparental inheritance (DUI) is not characteristic of tructure . Native tructure , Table 1tructure . Twenty-tructure .The Bayesian phylogenetic tree showed a shallow structure lacking apparent phylogeographic structure . We obsen = 9) in population CH3 sampled from mainland China, while only two haplotypes were recovered from population TW2 collected from Taiwan. Mean haplotype diversity (h) and nucleotide diversity (π) in Chinese populations were 0.611 and 0.007, respectively, higher than those observed in invaded areas. Comparable values for South America were 0.595 and 0.004, while those in introduced populations in Asia were 0.439 and 0.004, respectively. Populations from Taiwan (TW1 and TW2) exhibited the lowest haplotype and nucleotide diversity  ranged from 5 to 9.5, while mean expected heterozygosity (HE) and observed heterozygosity (HO) ranged from 0.518 to 0.858, and from 0.128 to 0.528, respectively (HE) and mean allelic richness (Ar) were higher in Asia as compared to South America . Many loci (150 of 192) deviated from Hardy-Weinberg equilibrium (HWE) and all exhibited heterozygosity deficiency  and South America (0.046) were lower than those between the continents (0.257). Almost all FST values were significant in Asia and between Asia and South America while most of the FST values were non-significant in South America. In Asia, pairwise FST values were lower inside each geographic region  as compared to the overall value (0.067) for the continent . We observed relatively high genetic differentiation between neighbouring populations in South America, for example, FST = 0.072 between EC and UR (separated by ∼ 50 km), while some geographically distant populations exhibited relatively low FST values, for example, 0.004 between CO and QU (separated by ∼ 2000 km). Similar to FST values, Jost’s D values were lower among populations in South America (D = 0.00–0.074) than in Asia (D = 0.024–0.404), and between Asia and South America (D = 0.351–0.726)  . GeneticP<0.001; .K = 2) were supported when Asia (K = 2). Two populations from Taiwan , and one Japanese population (JP1) were grouped together, while the other two Japanese populations (JP2 and JP3) were clustered into another group  and nuclear markers (microsatellites) to contrast the geographical distribution of genetic diversity of L. fortunei has been introduced more than once and/or in large inocula (i.e. high propagule pressure). This conclusion is consistent with Tominaga et al.’s (2009) L. fortunei was introduced to Japan via aquaculture as a ‘fellow traveler’ with stocked Asian clams, C. fluminea, imported from China L. fortuneiL. fortunei to neighboring countries Expected heterozygosity (0.667–0.746) for introduced populations in Japan and Taiwan was higher than that in introduced South American ones (0.519–0.575). Both groups exhibited lower expected heterozygosity as compared to native populations in mainland China and Korea (0.701–0.858). The high number of haplotypes at the COI locus recovered from Japan, coupled with relatively high heterozygosity, suggests that n = 3), but rather high level of allelic richness (Ar = 7.3) and heterozygosity (HE = 0.7052) at microsatellite loci (Ne) relative to nuclear DNA owing to its maternal inheritance and haploid nature. Therefore, the mitochondrial genome is expected to be more sensitive to bottleneck events than the nuclear genome Ciona intestinalisL. fortunei introduction from Japan through aquaculture appears to be low. L. fortunei has not yet been reported in USA or Canada. While aquaculture remains a possible vector for introduction to Taiwan, it is more likely active in Japan. The low haplotype diversity observed in Taiwan could be also result from inhospitable environmental conditions in primary introduction areas. It is also possible that the putative source population carries similar level of genetic diversity. Korea was also represented by a low number of haplotypes, and high nuclear allelic diversity, relative to native populations in mainland China (We observed a low number of mtDNA haplotypes in Taiwan (ite loci . MtDNA hnd China . HoweverD. rostriformis bugensis) and zebra mussels (D. polymorpha) L. fortunei can be transported both up- and downstream through recreational boating, natural inland currents, and seasonal flooding, temporal and/or spatial Wahlund effect could account for the heterozygosity deficit, although inbreeding and selection cannot be completely dismissed.Heterozygosity deficit was found in 77.6% of all analyzed microsatellite loci, including native populations in mainland China and Korea . This paL. fortunei in each geographic region by a saltwater dispersal barrier, with inter-region gene flow limited to human-mediated translocation of propagules. In addition, distinct sources of introduction can drive genetic differentiation among regions. We observed high genetic differentiation between South American and Asian populations (FST = 0.180 – 0.306). High genetic differentiation has been reported in other freshwater invasive mussels, including D. polymorpha (FST = 0.006 – 0.263) and D. rostriformis bugensis (FST = 0.008 – 0.267) L. fortunei, to upstream areas due to ship-mediated translocation appears responsible for the patchy post-establishment spread of the species in South America L. fortunei populations violated the HWE assumption for STRUCTURE analyses. However, similar results were observed using another method (i.e. 3D-FCA) without this assumption.In Asia, both mtDNA and microsatellite markers exhibited lower genetic differentiation within geographic regions as compared to that among regions. This finding is supported by a higher percentage of variance allocated to among groups as compared to among populations within groups AMOVA; . JapanesL. fortunei. It appears that a recent geographic expansion of L. fortunei throughout its native distribution can explain of its low phylogeographic structure. This finding suggests that even in the native region, populations may still be expanding to areas not previously populated. Vector activity such as ship traffic between local ports in native region (e.g. mainland China and Korea) may have contributed to recent expansion of L. fortunei across its native range The parsimony network analysis and Bayesian phylogenetic reconstruction revealed a close relationship among haplotypes of 4 ships from countries considered native for L. fortunei, whereas Argentina received only 26 ships. Second, Taiwan and especially Japan may benefit from an aquaculture transfers from adjacent Asian countries, notably mainland China. Third, given the comparatively short distance between Asian countries, hull fouling could effect local or regional spread. L. fortunei larger than 20 mm can tolerate anoxia for up to 18 days at 20°C L. fortunei in these regions.Our study suggests that introduced golden mussels carry less allelic diversity in South America than in Asia. A number of factors may contribute to this pattern. First, data from global ship traffic between 2005–2006 L. fortunei, D. polymorpha and D. rostriformis bugensis share similar ecological and biological characteristics including planktonic larvae and a sessile, benthic adult stage. These life history traits suggest similar vectors could effect their human-mediated spread . Also, golden mussel can be transported as a ‘fellow traveler’ in aquaculture D. polymorpha and D. rostriformis bugensis in North America and Eurasia L. fortunei provides a good basis for comparison of the genetic characteristics of the three mussel species (L. fortunei (40 haplotypes) as compared to 11 found for D. polymorphaD. rostriformis bugensisD. polymorpha, the Great Lakes appear to have served as a ‘hub’ for subsequent expansion across North America Although taxonomically distinct,  species . When us species . The higL. fortunei’s introduction in both Asia and South America likely involved multiple introductions and high propagule pressure, resulting in populations with high genetic diversity relative to sampled native populations in Asia. Introduced populations exhibiting lower genetic diversity (South America) likely received lower propagule pressure relative to those with higher diversity (Japan). Our genetic survey shows how human-mediated introduction of NIS can create genetic complexities across introduced locations. Our study evaluates possible links between vector activity and genetic composition of a nuisance NIS at a global scale, and highlights the utility of incorporating population genetics and vector activity data to understand species dispersal patterns Our findings suggest that Figure S1Values of ΔK calculated as in Evanno et al.for detecting the biologically relevant clusters of Limnoperna fortunei collected from all 24 locations (A) and Asia (B).(TIF)Click here for additional data file.Table S1Limnoperna fortunei, sampled from 24 locations across the global range in East Asia and South America. A, number of alleles; Ar, allele richness; HO, observed heterozygosity; HE, expected heterozygosity; PHW, exact P-value for Hardy-Weinberg equilibrium test. The significance after sequential Bonferroni correction was bolded.Genetic diversity at eight microsatellite loci for the golden mussel, (DOC)Click here for additional data file.Table S2Dreissena polymorpha, quagga mussel Dreissena rostriformis bugensis, and golden mussel Limnoperna fortunei.Comparison of microsatellite-based genetic features of the three highly invasive freshwater mussels, zebra mussel (DOC)Click here for additional data file.