Champsocephalus gunnari and Notothenia rossii. The close accord between the model projections and empirical genetic structure demonstrated that passive dispersal during the planktonic early life stages is the dominant influence on patterns and extent of genetic structuring in both species. The shorter planktonic phase of C. gunnari restricts direct transport of larvae between distant populations, leading to stronger regional differentiation. By contrast, geographic distance did not affect differentiation in N. rossii, whose longer larval period promotes long-distance dispersal. Interannual variability in oceanographic flows strongly influenced the projected genetic structure, suggesting that shifts in circulation patterns due to climate change are likely to impact future genetic connectivity and opportunities for local adaptation, resilience and recovery from perturbations. Further development of realistic climate models is required to fully assess such potential impacts.Understanding the key drivers of population connectivity in the marine environment is essential for the effective management of natural resources. Although several different approaches to evaluating connectivity have been used, they are rarely integrated quantitatively. Here, we use a ‘seascape genetics’ approach, by combining oceanographic modelling and microsatellite analyses, to understand the dominant influences on the population genetic structure of two Antarctic fishes with contrasting life histories,  Marine organisms with planktonic early life stages have traditionally been assumed to have high dispersal and weak population genetic structure. However, there is growing recognition of the role of oceanographic features in structuring populations, with frequent uncoupling of dispersal potential, gene flow and genetic differentiation . Theoretically, therefore, it might be expected that N. rossii, with a planktonic duration approximately twice that of C. gunnari, experiences a higher degree of dispersal and greater genetic connectivity.C. gunnari and N. rossii is generally poor and often contradictory. It is thought that C. gunnari forms separate populations on each of the island shelves in the Scotia Sea  were provided by the National Oceanography Centre, Southampton, for the period 1996–2001. This period includes years with relative extremes in atmospheric forcing over the Southern Ocean, associated with extreme phases of large-scale coupled modes of interannual climate variability  from a given source population (columns). To account for egg and larval mortality, the connectivity matrices were multiplied by fixed daily mortality rates of 0.036 and 0.048 d−1 for eggs (ze) and larvae (zl), respectively, integrated over the planktonic periods. There are no data on egg and larval mortality rates for C. gunnari or N. rossii; these mortality rates were inferred from the temperature dependencies described by Pepin (T) of 1°C, representative of annual mean near-surface sea temperatures across the Scotia Sea . The sensitivity of the genetic projection modelling to the imposed mortality rate is considered in Appendix Over a 4-week period centred on the end of the planktonic phase, the position of each model larva was compared with the population boxes associated with each known spawning population Fig.. If a laby Pepin , specifiM is applied to a state matrix Qt containing information on the frequency of alleles of each type in each population at time t, to yield the expected state of the population at time t + 1. This method allows for a sedentary adult phase, with offspring transported according to connectivity matrix M and subsequently merged back into the adult population. Thus, the projection equation isB a diagonal matrix of birth rates for each population, and the top bar indicating row normalization. Using the assumption that individuals are uniquely labelled according to their population of origin by initially fixing populations for different alleles, Q0 = K, with K a diagonal matrix of carrying capacity values for each population. The connectivity matrix M was randomly chosen at each time step from the 5 year-specific matrices. Data on population sizes of the fish species at each spawning site are sparse; thus, the carrying capacities of each site were assumed to be the same. Mean birth rates were derived from empirical fecundity data  or South Shetland Islands (N. rossii)  was used to extract the DNA before PCR amplification of microsatellite loci. Eleven microsatellite loci were amplified for C. gunnari and 9 for N. rossii (detailed in Appendix Muscle tissue or fin-clip samples were collected from adult sii) Fig.. AdditioNa), observed (HO) and expected heterozygosity (HS), locus-specific inbreeding coefficients (FIS), fixation indexes (GST), global and pairwise FST, were also calculated in GenAlex 6.5. To avoid type II errors, the sequential goodness-of-fit method   were used to construct synthetic samples. The final allele frequencies of each iteration were resampled to construct one locus for 100 individuals per location per species, creating a synthetic data set of 100 individuals per location/species with 100 loci. The variance among these 100 synthetic loci is thus due to variability in the frequency of certain migration events in each projection, which result in the presence or absence of certain alleles in each population at the end of the simulation. This variance should be similar to that caused by migration and recombination in real genetic markers. The structure of the synthetic data was also visualized through DAPC.Quantitative comparisons of empirical and projected genetic structure  relative to C. gunnari. Dispersal was highly asymmetric for both C. gunnari and N. rossii for the five study years, with higher values below the diagonal of the connectivity matrix  and r = −0.46 (P = 0.04) for C. gunnari and N. rossii, respectively. Connectivity was lower than expected from geographic distance for N. rossii between proximate sites along the southern Scotia arc, and in particular between the Antarctic Peninsula and South Orkney Islands. Champsocephalus gunnari connectivity declined sharply to very low levels for most sites separated by distances of >∼500 km, but there was higher connectivity than expected from geographic distance between South Orkney Islands and South Georgia, and Elephant Island and South Georgia, compared to site pairs of similar separation.Although predicted connectivity of both species showed a broad decline with increasing distance between sites, both oceanography and life history modified this relationship. C. gunnari and N. rossii, respectively. This difference between species corresponds to theoretical expectations of faster approach to equilibrium in populations connected by higher migration rates  and heterozygosity excess in the other . Locus Nro66 was thus removed from all further analysis. No locus pairs were in significant linkage disequilibrium once corrected for multiple testing.For C. gunnari (P < 0.001), where 12 of 15 sample pairwise comparis-ons were significant after multiple testing correction. The emerging pattern indicated low and nonsignificant values for comparisons within the Antarctic Peninsula, and within South Georgia/Shag Rocks. All comparisons between Antarctic Peninsula, South Orkneys and South Georgia/Shag Rocks were significant (up to P < 0.001). Conversely, no overall significant population genetic structure among samples was found for N. rossii (P = 0.096). The small size of the South Orkneys sample limited the power of tests for genetic differentiation, and the sample was thus excluded from these tests. The remaining comparisons, including Antarctic Peninsula versus South Georgia, were nonsignificant.Significant population genetic structure among samples was found for C. gunnari collected in the Antarctic Peninsula  from those collected in South Orkneys and those from Shag Rocks/South Georgia for both the empirical and synthetic data sets , but suggested an emergence of genetic differentiation between South Orkneys and the other samples  and r = 0.98 (P = 9 × 10−4) for C. gunnari and N. rossii, respectively  and predicted r = 0.89, P = 0.0005). However, neither the projected nor the observed N. rossii . Correlating the residuals from the projected and observed isolation-by-distance relationships allowed consideration of oceanography while allowing for geographic distance. For both species, these correlations were significant, with correlation coefficients of r = 0.93 (P = 9 × 10−5) and r = 0.98 (P = 8 × 10−4) for C. gunnari and N. rossii, respectively. Thus, while both distance between source and destination sites and oceanography significantly affected regional genetic structure in C. gunnari, the effect of spatial variability in oceanographic flows was the dominant influence on genetic structure in N. rossii.For both species, the projected regional genetic structure was in strong agreement with observed structure, with correlation coefficients of By combining empirical genetic data and numerical oceanographic, transport and genetic projection models, we have identified important drivers of population genetic structure in two contrasting Antarctic marine fish species. The numerical models projected the observed population genetic structure of both species with a high degree of accuracy, suggesting that parameters used in the model  were a reasonable approximation of reality. Specifically, the difference between the two species in genetic structure and strength in correlations between genetic structure and physical connectivity indicates an important role of early life history on genetic structure, as previously suggested  which may inflate levels of genetic differentiation. Nevertheless, the sample was retained because the pattern corresponded to the modelled differentiation of the population at South Orkney Islands. Such differentiation is potentially due to the dominant oceanographic flows in the region, which circumvent the South Orkney Islands and directly connect the Antarctic Peninsula with South Georgia. The relative isolation of South Orkney Islands is corroborated by drifter data, which reveal a northward flow of Weddell Sea water across the South Scotia Ridge between Elephant Island and the South Orkney Islands, acting as a dynamic barrier to exchange between populations at the Antarctic Peninsula and South Orkney Islands  and in the long term . Independent, locally adapted populations may react asynchronously to environmental perturbation (Hilborn et al. The projections of genetic structure were highly sensitive to the underlying oceanographic flows, which has potentially important implications for genetic connectivity in a changing world. There is currently significant uncertainty concerning the response of circulation patterns in the Scotia Sea, and in the Southern Ocean more generally, to climatic changes. Warming of the Southern Ocean on decadal timescales has been demonstrated clearly (Boning et al. Champsocephalus gunnari, with a planktonic phase that is approximately only half the duration of N. rossii, exhibited higher levels of genetic structuring, with model results suggesting both isolation-by-distance and stepping-stone models of gene flow, dependent upon the geographic separation of populations and directionality of dominant oceanographic flows. The extent of interannual variability in oceanographic–population genetic structure projections across 5 years endorses the need to incorporate, where possible, a temporal series of samples for comparison, on both localized and regional scales. Population connectivity effected by planktonic dispersal is evidently a key determinant of population recruitment and persistence of Antarctic fishes that inhabit highly seasonal and fragmented environments. Such findings emphasize the vulnerability of such populations to factors likely to disrupt population connectivity, including the impact of elevated temperatures on egg and larval development and planktonic duration, habitat change and over-exploitation. There remains an urgent need to better define the physiological ecology of larvae across changing environments, together with directed functional genomic analyses of the patterns and scale of local adaptation.Here, we have shown that key biological characteristics of the early life-history stages of two Antarctic fishes, when integrated into individual-based models of connectivity and genetic projection models, can yield informative and accurate projections of population genetic differentiation.


Phoca hispida saimensis), which is endemic to the large but highly fragmented Lake Saimaa in southeastern Finland.Small, genetically uniform populations may face an elevated risk of extinction due to reduced environmental adaptability and individual fitness. Fragmentation can intensify these genetic adversities and, therefore, dispersal and gene flow among subpopulations within an isolated population is often essential for maintaining its viability. Using microsatellite and mtDNA data, we examined genetic diversity, spatial differentiation, interregional gene flow, and effective population sizes in the critically endangered Saimaa ringed seal (HE = 0.36) ranks among the lowest thus far recorded within the order Pinnipedia, with signs of ongoing loss of individual heterozygosity, reflecting very low effective subpopulation sizes. Bayesian assignment analyses of the microsatellite data revealed clear genetic differentiation among the main breeding areas, but interregional structuring was substantially weaker in biparentally inherited microsatellites (FST = 0.107) than in maternally inherited mtDNA (FST = 0.444), indicating a sevenfold difference in the gene flow mediated by males versus females.Microsatellite diversity within the subspecies (Genetic structuring in the population appears to arise from the joint effects of multiple factors, including small effective subpopulation sizes, a fragmented lacustrine habitat, and behavioural dispersal limitation. The fine-scale differentiation found in the landlocked Saimaa ringed seal is especially surprising when contrasted with marine ringed seals, which often exhibit near-panmixia among subpopulations separated by hundreds or even thousands of kilometres. Our results demonstrate that population structures of endangered animals cannot be predicted based on data on even closely related species or subspecies. Especially in the absence of incoming migration and gene flow, fragmentation of an already small population may increase its extinction risk by exacerbating demographic and genetic stochasticity in the even smaller subpopulations. Gene flfidelity,5, sociafidelity,7, or infidelity.Studying spatial connectivity and levels of gene flow in rare, elusive, and endangered mammals is difficult using traditional approaches like mark–recapture or telemetry methods. To subsPhoca hispida saimensis) provides an excellent model system for studying how small population size and spatial subdivision influence key genetic and demographic parameters and processes. This endemic ringed seal subspecies derives from marine seals that became trapped in Lake Saimaa in southern Finland after the last glacial period, i.e., nearly 10,000 years ago[th century[c. 300 seals[The landlocked Saimaa ringed seal [Hydrurga leptonyx)[Phoca vitulina)[2, and 0.1 mM of each dNTP (Finnzymes). The thermal cycler profile consisted of initial denaturation at 94°C for 3 min, followed by 30 – 35 cycles of denaturation at 94°C for 30 s, annealing at 50 – 58°C for 30s, and extension at 72°C for 30s, with a final extension step at 72°C for 5 min  following the manufacturer’s instructions. Microsatellite loci were amplified using primers originally designed for grey seal ( grypus)-26, leopeptonyx), and haritulina),29 , Haukivesi area (Nms = 99 and Nmt = 116), Pihlajavesi area (Nms = 43 and Nmt = 61), and Southern Saimaa (Nms = 15 and Nmt = 19).The genotyped individuals originate from different parts of Lake Saimaa and represent a time span of three decades (1980 – 2008). In order to analyse the spatial genetic structure of the population, the specimens were initially divided into four regional samples following the topography of the lake , 1990s (Nms = 48 and Nmt = 54), and 2000s (Nms = 65 and Nmt = 82). A decade was considered an appropriate time span to detect temporal changes, since the estimated generation time of the ringed seal is c. 11 years .Nms = 14), the 1980s (Nms = 54), the 1990s (Nms = 47), and the 2000s (Nms = 54)  (see for detaA), observed (HO) and expected (HE) heterozygosities, and Wright’s inbreeding coefficients (FIS) for each spatial and temporal sample. Departures from Hardy–Weinberg equilibrium and the presence of linkage disequilibrium between pairs of loci were tested with Arlequin and GENEPOP v. 4.1.3[AR) were obtained using the rarefaction method implemented in HP-Rare[AR was estimated separately for each spatial and temporal sample, based on the smallest sample size within each division scheme. Individual observed heterozygosities (HO) were calculated using IR macroN4 ([http://www.zoo.cam.ac.uk/directory/william-amos), and the relationship between heterozygosity and year of birth was tested by performing a linear regression analysis in SPSS Statistics v. 19 (IBM).We used Arlequin v. 3.5.1.2 to estimv. 4.1.3. Estimat HP-Rare. AR was acroN4 , we also ran a hierarchical AMOVA with spatial samples subdivided into temporal subsamples, and vice versa. Significance levels were estimated on the basis of 10,000 permutations. Because FST is influenced by the level of heterozygosity (HS) in the studied subpopulations[Dest[G"ST[Dest also for the mtDNA sequence data using a script[Genetic differentiation among the spatial and temporal samples was investigated using analysis of molecular variance (AMOVA),41 and bulations-44, we uulations,46 to esons[Dest and G"STDest[G"ST), we esta script employina script and apea script packagesa script; statista script. The staa script.K) from 1 to 8, and ran 20 replicate analyses per each K to ensure convergence. Subsequently, we determined the most likely number of clusters by estimating the logarithm of the data probability given each number of K (logP(X|K);[K values estimated using the ad hoc approach of Evanno et al.[Genetic structuring of the population was further studied using Bayesian genotype-assignment approaches. Analyses in Structure v. 2.3.4,55 were gP(X|K);), and ono et al. implemeno et al..K) from 2 to 8, with ten replicates for each K. Plots of the deviance information criterion (DIC) against K were used to identify the most likely number of clusters, which was then used in 100 replicate runs with the admixture model, using the same number of iterations as above. Finally, we averaged results from the ten runs having the highest likelihoods using CLUMPP v. 1.1.2[Because collection locations were known for all but one of the sampled 172 seals, we used TESS v. 2.3.1,59 for av. 1.1.2, and visv. 1.1.2. The iniN = 171) and mtDNA (N = 214) datasets using the kinship coefficient of Loiselle et al.[Nij, a kinship analogue based on genetic distances among haplotypes[et al.[b), were obtained by 10,000 permutations, and standard errors were estimated by jackknifing over loci.The presence of an isolation-by-distance (IBD) pattern among seals within the lake was tested by using SPAGeDi v. 1.3 to contre et al., which ie et al.. For mtDplotypes. Between6 burn-in iterations followed by 9 × 106 iterations with a sampling frequency of 2,000, with Δ parameters of allele frequency, migration rate, and inbreeding coefficient set to 0.15, 0.17, and 0.17, respectively, to achieve the recommended acceptance rates of between 40% and 60%. The analysis was repeated three times with different initial seed numbers to ensure convergence.Migration rates among Lake Saimaa regions were estimated using the Bayesian multilocus genotyping method implemented in BayesAss v. 1.3. We perfet al.[et al.[In order to infer whether dispersal within the lake is sex-biased, we estimated the relative amount of male and female gene flow following the approach of González-Suárez et al., which tet al.). This iet al. prevente[et al., and theHg1.4, Hg3.6, Hg6.1, Hg8.9, Hgdii, and Pvc78) with low frequencies (r < 0.1) in all cases, most likely due to population substructure (see below). Scoring errors suggested for the loci Hg3.6 and Hg8.9 were ruled out by independent manual rechecking of the data by two researchers. In Arlequin, consistent and significant linkage disequilibrium in all temporal samples after a sequential Bonferroni correction was found between loci Hg4.2 and Pvc78, whereas Bonferroni-corrected GENEPOP results indicated no disequilibrium. We consider the latter result more reliable, because the permutational likelihood-ratio test implemented by Arlequin assumes that all loci are in Hardy–Weinberg equilibrium[Genotypes for all 17 microsatellite loci were obtained for 168 individuals, while four individuals lacked data for one locus each .The average number of alleles per locus was 3.5, but the range was broad, so that 16 loci had 2 to 4 alleles, while 16 were found in locus HO = 3.762–0.002*birth year; r2 = 0.030, P = 0.025; Figure FIS estimates increased over the study period from effectively zero for seals born before 1980 to a significantly positive FIS = 0.100 (P = 0.001) in the 2000s (Table No obvious differences in genetic diversity were found among regions Table . The datNe = 14.7 (95% CI 9.9 – 20.7), and similarly low numbers were obtained for different decades, ranging from Ne = 10.7 (6.1 – 17.4) in the 1990s to Ne = 32.7 (18.6 – 66.1) in the 2000s. Estimates based on the temporal approach were somewhat higher, but with broad confidence intervals: 1980s – 1990s Ne = 69 (33 – inf.); 1990s – 2000s Ne = 53 (31 – 171); and Ne = 113 (51 – inf.) over two generations (1980s – 2000s). Effective population sizes within the four main regions ranged from 4.7 to 45.9 , and the result was confirmed by pairwise FST estimates among regions (Table P < 0.001), and all pairwise comparisons were statistically significant (P < 0.01 in each case). Microsatellite differentiation was moderate in comparison to estimates based on mitochondrial control-region sequences, which were consistently higher for overall differentiation  as well as for pairwise FST values , and this was true also for all pairwise comparisons . Hierarchical AMOVA of the two regions with the largest sample sizes (= Haukivesi and Pihlajavesi) likewise showed that, regardless of the order of the hierarchy, most of the differentiation could be attributed to variation among regions and individuals, while the effect of decades was weak and statistically non-significant (results not shown). In contrast, mtDNA-based analyses showed highly significant differentiation among decades  was very weak (ssion in).K = 4 . NotablK = 4  and mtDNA . Pairwise FST estimates showed that Kolovesi is highly differentiated from the rest of the lake . Furthermore, the significantly positive FIS = 0.074, P = 0.0002 of the undivided Haukivesi area disappeared when Main Haukivesi and Kolovesi were analyzed separately   = 0.099) among the five Lake Saimaa regions was only slightly lower than overall microsatellite differentiation (FST = 0.107), and the same was true for nearly all regional pairs  in comparison to marine ringed seal populations, in which microsatellite-based estimates of expected heterozygosities range from HE = 0.80 to HE = 0.89[cf. Mediterranean monk seal (Monachus monachus), HE = 0.40[Monachus schauinslandi), HE = 0.49[Phoca largha), HE = 0.51[Mirounga angustirostris), HE = 0.40[HE = 0.50 ± 0.03;[th-century anthropogenic bottleneck[The Saimaa ringed seal has very low genetic diversity , and he ± 0.03;, see als ± 0.03;). The re ± 0.03;,80, whic ± 0.03;-74 are i ± 0.03;,27,29. Cttleneck,17, but e.g., Finnish wolves (Canis lupus), Ne = 37.8 – 43.0[Oreamnos americanus), Ne = 23.2 – 65.9[Lynx pardinus), Ne = 8.5 – 23.1[Nes of several hundred, estimated by Palo et al.[et al.[Ne after exclusion of Kolovesi individuals (Table Ne estimates yield a ratio of Ne/Nc = 0.17 – 0.36. The lower end of this range is consistent with the average ratio observed in wild populations (0.16), and the higher end would comply with the higher-than-average Ne/Nc ratios observed in small threatened populations[Unfortunately, it seems that the answer to this question is "No": Estimates of total and regional effective population sizes were very low, ranging roughly between 5 and 113 Table . Compara8 – 43.0, Western2 – 65.9, and Ibe5 – 23.1. It shoulo et al. and Valtl.[et al., are farl.[et al.), as is ulations.Nes apparently are not enough for safeguarding the existing diversity: our detailed analysis based on the birth years of the sampled individuals detected slowly declining individual heterozygosity in microsatellite loci during the past few decades  is remarkable considering the short distances among the main basins , and geographic differentiation is weak also in grey seals, in which Graves et al.[FST values only between Baltic and North Sea breeding colonies (FST = 0.068 – 0.097), while differentiation within the Baltic was clearly lower (FST ≤ 0.023). In general, interpopulation genetic differentiation in marine seals is often negligible – or at least lower than that observed within Lake Saimaa – even across thousands of kilometres[FST on overall heterozygosity[et al.[FST = 0.005) and Martinez-Bakker et al.[FST = 0.0086) into G"ST values, based on the reported FST s, mean values of HS, and numbers of sampled populations. The converted estimates  resulting from these studies, both of which include populations on different continents, are still below the overall differentiation among regions within Lake Saimaa (G"ST = 0.121 – 0.196).Our results demonstrate that ringed seal subpopulations inhabiting the main regions of Lake Saimaa are differentiated with respect to both autosomal and mitochondrial variation . Howeve et al. showed tes et al. found Salometres-74,90-92zygosity,44 meanszygosity and Meir[et al. , in which remaining populations are restricted to mountaintops separated by unsuitable lowlands[cf.[Nd) well below the census size[As can be expected based on the high spatial differentiation, interregional migration estimates Table  were vere.g.,,11). Acrlowlands. Althouglowlands, serves nds[cf.), but ousus size.FST and Dest) and a smoother isolation-by-distance pattern than did mtDNA [Microsatellites exhibited lower levels of interregional differentiation .cf.[cf.[cf.[The critically endangered ringed seal subspecies endemic to Lake Saimaa is genetically impoverished and fragmented into several partially isolated subpopulations. Microsatellite diversity within the subspecies is the lowest recorded so far within the order Pinnipedia and, worryingly, our analyses reveal an ongoing downward trajectory in both microsatellite (this study) and mtDNA variation. Althougcf.,99). Thecf.[cf.-102). Hecf.[cf.,103,104)The level of genetic differentiation among animal populations is a function of multiple factors, including time, effective population sizes, degree of geographic separation, and species-specific dispersal ability and propensity,106. Henhttp://datadryad.org),http://dx.doi.org/10.5061/dryad.5j754. The mtDNA sequence dataset is available from Genbank (http://www.ncbi.nlm.nih.gov/genbank), accession numbers: JX109584–JX109833.The microsatellite dataset supporting the results of this article is available in the Dryad repository  from Structure runs. (B) Structure results post-processed with the Evanno approach; ΔK values for each K. (C) Deviance information criterion (DIC) scores computed by TESS plotted against K.Population structure analysis of individual Saimaa ringed seals using Structure and TESS. (A) Mean log-likelihood for each number of Click here for file


Callithrix marmosets give us a unique glimpse of genetic hybridization effects under distinct natural and human-induced contexts. Here, we use a 44 autosomal microsatellite marker panel to examine genome-wide admixture levels and introgression at a natural C. jacchus and C. penicillata species border along the São Francisco River in NE Brazil and in an area of Rio de Janeiro state where humans introduced these species exotically. Additionally, we describe for the first time autosomal genetic diversity in wild C. penicillata and expand previous C. jacchus genetic data. We characterize admixture within the natural zone as bimodal where hybrid ancestry is biased toward one parental species or the other. We also show evidence that São Francisco River islands are gateways for bidirectional gene flow across the species border. In the anthropogenic zone, marmosets essentially form a hybrid swarm with intermediate levels of admixture, likely from the absence of strong physical barriers to interspecific breeding. Our data show that while hybridization can occur naturally, the presence of physical, even if leaky, barriers to hybridization is important for maintaining species genetic integrity. Thus, we suggest further study of hybridization under different contexts to set well informed conservation guidelines for hybrid populations that often fit somewhere between “natural” and “man-made.”Animal hybridization is well documented, but evolutionary outcomes and conservation priorities often differ for natural and anthropogenic hybrids. Among primates, an order with many endangered species, the two contexts can be hard to disentangle from one another, which carries important conservation implications.  While animal hybridization is historically regarded as an “evolutionary dead-end” , contempet al. [et al. [Allendorf et al.  perhaps  [et al.  highlighet al. [The challenges described by Allendorf et al.  certainlet al. , 12). Hoet al. , 13. Whiet al. . Thus, iCallithrix) provides a unique opportunity to study interspecific breeding within separate natural and anthropogenic contexts. Callithrix contains six species, C. jacchus, C. penicillata, C. geoffroyi, C. kuhlii, C. flaviceps, and C. aurita, with the last three species being threatened or near threatened (www.iucnredlist.org). This is a young genus, aged at about 2.5 million years, with C. penicillata and C. jacchus diverging as sister species less than 1 million years ago [Callithrix [Callithrix jacchus and C. penicillata have also been introduced together in areas outside of their respective distributions, and into the native ranges of other Callithrix species. Marmoset hybridization occurs at many of these natural and anthropogenic contact points , 22CalliC. penicillata x C. jacchus hybrid zone in NE and SE Brazil, respectively, using the mitochondrial DNA control region (mtDNA CR) [C. penicillata x C. jacchus natural and anthropogenic hybrid zones using a large genome-wide panel of autosomal microsatellites. These autosomal markers complemented the mtDNA data [C. jacchus x C. penicillata hybrid zones? (2) Do levels of genetic admixture and introgression differ between natural and anthropogenic marmoset hybrid zones? (3) What are the evolutionary and conservation implications of natural and anthropogenic marmoset hybridization?We have previously characterized genetic diversity and introgression at a putative natural and anthropogenic tDNA CR) . Here, wDNA data , which wC. penicillata populations and expanded previous C. jacchus genetic data. We characterized individual admixture within the natural hybrid zone as biased toward one parental species or the other, likely the result of a large river system flowing through this zone and limiting interspecific gene flow. Interestingly, fluvial islands within this river system may serve as gateways for bidirectional gene flow across the species border. On the other hand, marmosets essentially formed a hybrid swarm in the anthropogenic zone and showed intermediate levels of admixture. The anthropogenic hybrid zone admixture and swarm patterns have probably resulted from the absence of a strong physical barrier to limit interbreeding between introduced C. jacchus and C. penicillata.Our large microsatellite dataset gave first time estimates of autosomal genetic diversity for any wild C. jacchus and 44 C. penicillata, in both wild and captive (outbred) populations of each species. We also sampled 89 animals within two putative C. jacchus x C. penicillata hybrid zones. Phenotypic differences between the parental species and their hybrids are described in Malukiewicz et al. [C. jacchus samples were collected in 2005 and graciously donated by Dr. Maria Adélia Borstelmann de Oliveira  and Juazeiro, Bahia (BA) make up a putative natural hybrid zone referred to as the “PJ natural zone” and a detailed view of capture locations within this zone is shown in s et al. .C. jacchus and C. penicillata present in the area since at least the mid-1980s [The municipalities of Silva Jardim and Rio Bonito make up an anthropogenic hybrid zone in Rio de Janeiro state, abbreviated here as the “RJ anthropogenic zone.” Capture locations within the RJ anthropogenic zone are detailed in id-1980s . The RJ in utero exchange of stem cells by twins [et al. [C. kuhlii, with epithelial tissue having some of the lowest levels (12% chimeric) and blood-derived tissue having some of the highest levels (50% chimeric). We carried out a preliminary microsatellite genotyping analysis in C. jacchus using blood and epithelial tissues to determine whether the latter would be suitable for microsatellite genotyping of marmosets sampled during our study.Marmoset chimerism  occurs as a result of frequent twinning in these primates and by twins . As a reby twins . Ross et [et al.  showed tFor this preliminary analysis, the tissue was donated by the New England Primate Research Center and DNA was obtained via a standard phenol-chloroform extraction protocol. We genotyped 10 unrelated individuals at two microsatellite loci, caja1 and caja5 . The twoC. jacchus and 41 C. penicillata. These 44 loci were amplified in 15 multiplex reactions  and expected heterozygosity (HE) were determined with GENODIVE [Locus allele frequencies for the two parental species and within hybrid zones were calculated with GENEPOP 4.2 . The “exparisons . FSTAT 2parisons  was usedsons [IS  for eachGENODIVE .E across a large range of r. Thus, we did not make corrections for any of the within-population analyses discussed above.Possible presence of null alleles within the dataset was examined by MICROCHECKER , which aST values that are corrected and unbiased for null alleles [ST value is based on Weir [ST statistics, based on the stepwise mutation model for microsatellites, and 10,000 permutations.We next examined population differentiation between the two study species as well as subpopulations found on the respective northern and southern sides of each hybrid zone. FREENA can calculate F alleles , and we  on Weir  and inclC. jacchus and C. penicillata admixture, STRUCTURE 2.3.4 [et al. [et al. [a priori assumption that the number of clusters (K) is two, i.e. that there are two ancestral populations contributing to the gene pool in either hybrid zone. Based upon the simulation studies of Vaha and Primmer [q <0.90 for that individual .We applied two complementary Bayesian clustering approaches to determine levels of RE 2.3.4  and BAPSRE 2.3.4 –47, with [et al. , who fou [et al. , we made Primmer , we consCallithrix penicillata individuals were indicated by Popflag = 1, C. jacchus individuals were identified by Popflag = 2, and all individuals sampled from hybrid zones were identified by Popflag = 0. The PFROMPOPFLAGONLY option was turned on for allele frequency estimation only based on the parental species. Admixture levels of hybrids were estimated for hybrid zone individuals under the admixture ancestry model, which assumes that some fraction of an individual’s genome comes from the two parental clusters. This model was used with the default setting of an inferred alpha initially set to 1.0. The default correlated allele frequency model was used for all runs, and each run consisted of 80,000 burn-in steps followed by 8,000,000 MCMC iterations. The ten runs were checked for consistency in summary statistics and convergence of parameter values. Average q-values for hybrid samples across the independent runs were determined with the full search algorithm of CLUMPP 1.1.2 [We set up 10 independent runs in STRUCTURE under the USEPOPINFO model to allow for use of pre-defined parental species groups to aid classification of hybrid samples following conditions per run with MIGRPRIOR = 0.05 . PP 1.1.2 . DISTRUCPP 1.1.2  was usedPP 1.1.2  found thC. jacchus and C. penicillata clusters of individuals from outside of marmoset hybrid zones. Genotypes of pure individuals were used to define allele frequencies. Each BAPS run was executed with the following conditions: minimum population size was set to the default size of 5, there were 300 iterations per run, 200 reference individuals, and the number of iterations for admixture estimations of reference individuals was set at 10. Five independent BAPS runs were carried out and consistency of ancestry estimation for hybrid zone samples was verified between run replicates. Null allele corrections were not applied to BAPS analyses, as simulations studies by Chapuis et al. [Second, we used BAPS by conducting an admixture analysis based on pre-defined s et al.  found thq. We simulated 10 STRUCTURE datasets and 5 BAPS datasets composed of all reference samples, 10 F1 hybrids, 10 F2 hybrids, 10 C. jacchus x F1 backcross hybrids, and 10 C. penicillata x F1 backcross hybrids. All simulated hybrid classes were created with HYBRIDLAB 1.0 [Because the assumed priors and efficiency, or proportion of correctly identified hybrids out of a total number of actual hybrids in a sample , of the DLAB 1.0  and analDLAB 1.0 .We also employed one non-Bayesian method to study the partition amongst hybrid and pure groups by using GENODIVE to carry out a principal component analysis (PCA). The PCA used a covariance matrix based on individual multilocus genotypes as presented in C. penicillata loci and 5 out of 44 C. jacchus loci were in LD. MICROCHECKER indicated the possible presence of null alleles for almost all loci out of HWE in C. penicillata, but within-species pooling of samples from isolated captive and geographical subpopulations may have also caused loci to be out of HWE . Null allele presence indicated by MICROCHECKER and Walhund effect for C. jacchus loci may also explain some of the significant heterozygote deficiencies. In the hybrid zones, 12 out of 44 loci in the PJ natural zone and 6 out of 44 loci in the RJ anthropogenic zone were in LD. All loci out of HWE had positive FIS values indicating heterozygote deficiencies in pure and hybrid populations. MICROCHECKER flagged most hybrid zone loci not in HWE for the likely presence of null alleles. For the PJ natural zone, pooling of samples from two parapatric and genetically differentiated forms separated by a putative dispersal barrier (the São Francisco River) may explain part of the observed significant heterozygote deficiency. In the RJ anthropogenic zone, pooling of several subpopulations separated by highway BR-101, also a putative dispersal barrier, may explain observed Hardy-Weinberg disequilibrium.Deviations from HWE varied among loci within the parental species and the hybrid zones  Tables. genome.ucsc.edu), and those located on the same chromosome are expected to be far enough apart to minimize the chance of physical linkage within the C. jacchus and C. penicillata genetic backgrounds. Nonetheless, significant LD was found in some pairwise locus comparisons out of a total of 946 comparisons within each group as follows: (1) pairs of markers where each locus was found on a different chromosome totaled 29 in C. penicillata, 8 in C. jacchus, 34 in the PJ natural zone, and 14 in the RJ anthropogenic zone, (2) and marker pairs with both loci located on the same chromosome totaled 1 in C. penicillata, 3 in C. jacchus, 3 in the PJ natural zone, and 11 in the RJ anthropogenic zone. Demographic factors such as population structure may help explain inflated levels of LD, particularly in the case of C. jacchus and C. penicillata where we pooled samples from different captive and geographic origins. For the RJ anthropogenic zone, the high number of physically linked loci in disequilibrium may indicate that they continue to segregate together within their ancestral genetic background if the founder populations originally possessed limited allelic diversity at these loci.The majority of markers used in this study map to different chromosomes of the common marmoset published genome  and allelic richness  at these microsatellite loci than C. jacchus . HE levels were broadly similar between the two species, but for many C. penicillata loci HO levels were lower than expected, while HE and HO were similar for C. jacchus. Measures of genetic diversity in the PJ natural zone were similar to values seen in the parental species. The number of observed alleles and allelic richness within the RJ anthropogenic zone was the lowest out of the four groups, but HE end HO were comparable to the other three groups. Loci with lower HO than HE are loci with a significant heterozygote deficit and those that may contain null alleles. Thus allele drop-out may have caused deflation of HO relative to HE at such loci, especially in C. penicillata.An averaged summary of various genetic diversity measures for pure and hybrid groups is shown in C. jacchus and C. penicillata were found. Callithrix penicillata had on average 4.84 (or 44.6%) private alleles per locus and C. jacchus had 2.65 (or 31.6%) private alleles per locus. Most private alleles in C. penicillata were found together in along a continuous range of sizes. C. jacchus private alleles were found as intermittent singletons among non-private alleles within a continuous range of shared allele sizes. The remaining alleles overlapped in the two species but allele frequencies differed interspecifically. Alleles present in the hybrid zones were a mosaic of those alleles found in the parental population, but the PJ natural zone contained a much larger representative sample of parental alleles than did the RJ anthropogenic zone. The PJ natural zone had an average of 1.52 (or 18.3%) private alleles per locus, mostly as singletons and the RJ anthropogenic zone had an average of 1.36 (or 20.1%) private alleles per locus, also mostly singletons.Allele frequencies uncorrected and corrected for null allele presence across the 44 microsatellite loci are shown in ST values corrected and uncorrected for null allele presence were similar for each pairwise comparison . AMOVA analysis revealed that a significant portion  of genetic variation occurred at the species level (32% RST-based). The northern and southern sides of the respective hybrid zones also showed significant pairwise levels of genetic variation  at a similar level observed for parental populations. AMOVA indicated that 26% of variation in the PJ natural zone was found between northern and southern subpopulations . In the RJ anthropogenic zone, 14% of genetic variation was found between populations separated by highway BR-101 .Fmparison . CallithC. jacchus and C. penicillata. On the other hand, high genotypic similarity between C. jacchus and PJ natural hybrid zone marmosets was observed along the first component of the bivariate plot. The RJ anthropogenic hybrid zone population was very similar to C. penicillata, especially along the second component.In the PCA , the firC. penicillata ancestry with q = 1.0 indicating full ancestry and q = 0.0 indicating no C. penicillata ancestry. All runs conducted in STRUCTURE were concordant for C. jacchus and C. penicillata admixture levels in hybrid individuals, and results averaged across 10 runs by CLUMPP are shown in q-values for the parental species reference samples, so those individuals were not included in the plot shown in q-values of hybrid individuals are shown in q<0.90, indicating a strong possibility that most of these marmosets were C. penicillata x C. jacchus hybrids. The average admixture coefficient for the entire PJ natural zone was q = 0.36, ranging from 0.14 to 0.79. Marmosets showed on average q = 0.75 on the south side of the river within the PJ natural zone, and an average of q = 0.26 on the north side of the river. Thus, this indicated that the former possess mostly ancestry from C. penicillata and the latter possess ancestry mostly from C. jacchus. However, these data also strongly suggested bi-directional gene flow and introgression of C. jacchus and C. penicillata across the PJ natural zone. Ten PJ natural zone individuals had 90% q-value confidence intervals that overlapped with the q-values indicative of non-admixed individuals. More specifically, the q-value confidence intervals of 7 marmosets sampled north of the river dropped into range of full C. jacchus ancestry and 3 marmosets sampled south of the river dropped within range of full C. penicillata ancestry. The average admixture for marmosets in the RJ anthropogenic zone was q = 0.69, with a range of 0.53 to 0.86. Average q-values on the north side of this zone were 0.75 and on the south side average values were 0.60, thus overall, ancestry on both sides of the RJ anthropogenic zone tended more towards C. penicillata than C. jacchus. Nine RJ anthropogenic zone marmosets, all from the northern side, showed 90% confidence interval ranges that fell into the range of q-values expected for pure C. penicillata.For all cluster analyses, admixture coefficients are relative to C. jacchus. The average BAPS coefficient in the PJ natural zone was q = 0.20, ranging from 0 to 0.68, and the average RJ anthropogenic zone BAPS coefficient was q = 0.53, with a range from 0.31 to 0.77. As with STRUCTURE estimates for the north PJ side, ancestry tended towards C. jacchus (average q = 0.09) and on the south side ancestry was biased towards C. penicillata (average q = 0.63). BAPS q-values, in contrast to STRUCTURE, indicated pure C. jacchus ancestry for 18 individuals on the north PJ natural zone side. Overall, BAPS admixture analysis again suggested bidirectional gene flow and introgression between C. jacchus and C. penicillata across the PJ natural zone. The average north-side RJ anthropogenic zone BAPS q-value was 0.61, while the south-side average was 0.41, a trend concordant with that of STRUCTURE q-values. No marmosets within the RJ hybrid zone were of pure C. jacchus or C. penicillata ancestry according to BAPS q-values.Admixture coefficients calculated by BAPS were concordant between replicate runs, but BAPS coefficient values for all hybrid zone individuals  were lowC. jacchus and C. penicillata multilocus genotypes, plus 10 each of F1 hybrids, F2, hybrids, and backcross hybrids for each species. q-value threshold. Further, 90% confidence intervals for C. jacchus and C. penicillata backcross hybrids stayed within our q = 0.90 threshold for hybrid classification. Thus our dataset had 100% power to detect F1, F2, and backcross hybrids of both species using the STRUCTURE clustering algorithm. q-values and q-value ranges for simulated BAPS runs averaged across 5 repetitions per simulated dataset. BAPS showed a 100% efficiency of correct hybrid assignment for F1, F2 and C. jacchus backcross hybrids. BAPS calculated full C. penicillata ancestry for 1 out of 10 C. penicillata backcross hybrids in 2 out of 5 datasets, giving it an average of 96% efficiency of correct assignment for this hybrid class. STRUCTURE q-values tended to be higher than BAPS q-values. However, our reference data set of pure individuals allowed both programs to estimate, on average, appropriate admixture values expected for F1, F2, and backcross hybrids. STRUCTURE and BAPS estimated admixture levels within the PJ and RJ hybrid zones were proportionally similar to simulated early and later generation hybrids.Simulation files contained the full set of pure C. penicillata and C. jacchus interbreeding at a natural and anthropogenic hybrid zone using a large panel of 44 autosomal microsatellite loci and reference samples from both parental species. While the reference dataset did not contain any fixed species-specific alleles at any locus between the two study species, there were allele frequency differences between species and each species possessed private alleles. Given the very recent divergence date estimated by Perelman et al. [C. penicillata and C. jacchus, diagnostic loci between the species may emerge with time. Our microsatellite panel combined markers from several previous studies of wild and captive C. jacchus , 30C. pez et al. . NonetheC. jacchus to the north of the São Francisco River and C. penicillata to the south [C. jacchus, and subpopulations to the south exchange genes mostly with parental C. penicillata populations. Thus, this pattern suggested an important evolutionary role for the São Francisco River as a barrier to gene flow between C. jacchus and C. penicillata.PJ and RJ hybrid zone genetic diversity indices based on autosomal markers were broadly similar to those found in the two parental species. Nonetheless, cluster analyses as well as PCA allowed for a deeper insight into admixture within the two hybrid zones. The general observed geographical distribution of admixture patterns within the PJ natural zone matched the geographical boundary of he south . Observeet al. [C. jacchus and C. penicillata; albeit our autosomal data showed two-way genetic introgression and the mtDNA data showed one-way introgression across the PJ natural hybrid zone , and there was a dominant C. jacchus ancestry component in these individuals. Grouping patterns within the PCA corroborated our PJ natural zone cluster data. Since our genetic sampling within the PJ natural zone focused primarily on the C. jacchus north side of the São Francisco River, we most likely overrepresented the C. jacchus ancestry component within the PJ natural zone as a result of an unintended sampling bias. Most likely, an extended sampling along the south bank of the São Francisco River would support the observed pattern of majority C. penicillata autosomal ancestry for marmosets found on that side of the PJ natural zone.Cluster admixture coefficients showed that individuals sampled on the southern side the PJ natural zone had mostly C. jacchus and C. penicillata admixture, with a bias towards C. penicillata ancestry. The RJ anthropogenic zone contained, on average, a somewhat lower number of alleles and allelic richness than that observed in either parental population, possibly a consequence of the lack of direct gene flow between this zone and parental populations. PCA and cluster analysis indicated that the ancestry of marmosets found within the RJ anthropogenic zone was closer to C. penicillata than C. jacchus. Surprisingly, heterozygosity levels within the RJ anthropogenic zone were comparable to those of parental populations, despite the isolated location of this zone in relation to the natural distribution of C. jacchus and C. penicillata. Malukiewicz et al. [C. penicillata ancestry than that of C. jacchus within the RJ artificial hybrid zone  were found found in the RJ artificial hybrid zone [It is sometimes assumed that populations of introduced animals are depauperate of genetic variation due to the effect of drift on small founder populations, but multiple introductions can boost genetic diversity in introduced populations , 56. Givrid zone  and the rid zone . On the C. jacchus or C. penicillata, whereas the highly admixed subpopulations of the RJ anthropogenic zone possess a more unimodal genotype distribution. Compared to the RJ anthropogenic zone, we also observed much higher levels of Hardy-Weinberg disequilibrium and LD within the PJ natural zone. The conditions observed within the PJ natural zone are similar to what Arias et al. [Heliconius erato venus and Heliconius erato chestertonii butterfly hybrid zone, which the authors presented as strong evidence of incipient speciation in these taxa. Reproductive isolation between various Callithrix species is not complete [Genetic signatures of hybridization can vary for each hybrid zone as evolutionary and ecological dynamics can also differ between any set of interbreeding taxa. Bimodal hybrid zones at geographical contact points between different populations contain individuals that are genotypically similar to one or the other parental taxa with few intermediates, usually requiring evidence of strong LD and indicating strong assortative mating and pre-zygotic reproduction barriers . Intermes et al.  observedcomplete , but theCallithrix given the historical geographic range separation of marmoset species described in Rylands et al. [Callithrix. Thus, natural geographical barriers probably serve as important buffers against levels of hybridization that would erode the genetic integrity of separate marmoset lineages. Overall, we observed a lack of such gene flow barriers within forest fragments of the RJ anthropogenic zone, where we essentially observed complete collapse of genetic integrity within C. penicillata and C. jacchus. We note that the northern/southern autosomal population structure marmoset of the RJ anthropogenic hybrid zone suggests that BR-101 is a likely phyiscal barrier to gene flow across the zone. The physical isolation of marmoset populations among different forest fragments (as discussed above) also creates gene flow barriers within the RJ anthropogenic hybrid zone. However, when we look at the high level of admixture within individual sample locations on either side of the RJ hybrid zone . These areas include ranges of the three previously mentioned threatened Callithrix species. Callithrix jacchus and C. penicillata may pose an ecological as well as genetic threat to other marmoset species, since they are more morphologically specialized than other marmosets to exploit disturbed habitats [Leontopithecus rosalia) [et al. [Other areas where habitats , which chabitats . In the rosalia) . These l [et al.  indicateet al. [Our data showed that hybridization is likely a natural part of the evolution of marmosets, yet it can also threaten the genetic integrity of marmoset species under human-induced conditions. As recommended by Allendorf et al. , the disS1 Data(TXT)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


Malus sylvestris (L.) Mill). However, the conservation status of this species remains unclear in Europe, other than in Belgium and the Czech Republic, where it has been declared an endangered species. The population density of M. sylvestris is higher in the forests of the upper Rhine Valley (France) than in most European forests, with an unbalanced age-structure, an overrepresentation of adults and a tendency to clump. We characterize here the ecology, age-structure and genetic diversity of wild apple populations in the Rhine Valley. We use these data to highlight links to the history of this species and to propose guidelines for future conservation strategies. In total, 255 individual wild apple trees from six forest stands (five floodplain forests and one forest growing in drier conditions) were analysed in the field, collected and genotyped on the basis of data for 15 microsatellite markers. Genetic analyses showed no escaped cultivars and few hybrids with the cultivated apple. Excluding the hybrids, the genetically “pure” populations displayed high levels of genetic diversity and a weak population structure. Age-structure and ecology studies of wild apple populations identified four categories that were not randomly distributed across the forests, reflecting the history of the Rhine forest over the last century. The Rhine wild apple populations, with their ecological strategies, high genetic diversity, and weak traces of crop-to-wild gene flow associated with the history of these floodplain forests, constitute candidate populations for inclusion in future conservation programmes for European wild apple.The increasing fragmentation of forest habitats and the omnipresence of cultivars potentially threaten the genetic integrity of the European wild apple ( Malus sylvestris (L.) Mill.), a small fruit tree of the Rosaceae family, is a light-demanding, pioneer species of European forests. It is found in a broad range of latitudes between 370 and 640 N, soil environmental conditions and forest habitats, from deciduous  to mixed (deciduous and conifer) The wild apple (Fagus sylvatica L.), providing other flood-tolerant, light-demanding hardwood species, such as oak (Quercus robur L.), ash (Fraxinus excelsior L.), white poplar  and elm (Ulmus minor Mill.) with an opportunity to spread Unlike most upland forests, floodplain hardwood forests along large rivers provide favourable conditions for the establishment and reproduction of the European wild apple. Floods strongly limit the expansion of the most competitive European trees, such as beech  in the landscape have made crop-to-wild introgression more likely M. sylvestris across Europe. Genetic analyses across the geographic distribution range of this species have revealed the presence of three main populations, displaying high levels of genetic diversity in Europe: one covering a large area of Western Europe and the other two covering limited areas in Eastern Europe, one in the Balkans and the other in the Carpathian Mountains i.e., Belgium and the Czech Republic), which have classified M. sylvestris as an “endangered species”, leading to the dedication of greater resources to in situ and ex situ conservation programmes Interest in wild apple has recently grown, because population sizes for this tree have fallen considerably, particularly over the last two decades. The increasing fragmentation of forest habitats and the omnipresence of the cultivated apple  Floodplain forests, which are dominated by light-demanding canopy trees, are candidate areas for the targets of European wild apple conservation programs. In the Upper Rhine Valley (France), the European wild apple grows preferentially in these floodplain forests, along the Rhine and its tributaries Malus sylvestris in the forests of the Upper Rhine Valley, France. These forests are potential candidate areas for inclusion in future wild apple conservation programmes. With this in mind, our main objectives were: (1) to evaluate the chances of the current populations surviving and (2) to determine the genetic characteristics of the wild populations of the Rhine Valley, by checking for the presence of hybrids or feral individuals and evaluating the genetic diversity of the wild apple. On the basis of the results obtained, we aim to reconstruct the history of this species over the last century and to provide information useful for future conservation actions in this region.We investigated the ecology, age structure and genetic diversity of The study site is located in the Upper Rhine Rift Valley, in a rift, on the French side of the river . Trees wi.e., syntaxa) called “alliances”. Similar alliances may be grouped in “orders” and orders in vegetation “classes” Quercus petraea (Matt.) Liebl; Q. pubescens Willd) and hornbeam (Carpinus betulus L.). The understorey is relatively light and rich in Rosaceae species .The mean altitude of the Hardt forest terrace is 230 m. The climate is more continental than at the sites further north, with a mean annual rainfall of only 600 mm and 1800 hours of sunshine per year th century, straightening and embankment works modified the hydrological and sedimentary processes considerably, with a general deepening of groundwater levels. The main consequence of these changes was an increase in the surface area covered by mature hardwoods , which had previously been restricted to high terraces close to the river. The second phase occurred in the middle of the 20th century (1940–1960), with the canalisation of the river. This totally eliminated flooding, except on small islands. This elimination of flooding and stabilisation of ground water fluctuations resulted in the hardwood canopy becoming more uniform, with a large increase in the density of sapling carpets, which were previously selected by temporal anoxia during floods. Early successional forests, dominated by the Salicaceae , are now restricted to a few old channels The mean altitude of the Holocene floodplain is 148 m. The climate is temperate and oceanic, with a mean annual temperature of 11°C and a mean annual rainfall of 730 mm. The number of hours of sunshine varies between 1600 and 1700 per year th century. A period of intensive management  then followed from the 1970s to the 1980s and 1990s, particularly in certain forests, such as that at the Daubensand site. Management practices have since shifted towards other forestry practices, and this has resulted in a forest of heterogeneous age, with small clear-cuts, natural regeneration and woody vines. Natural reserves have been created in the Rhine forests, mostly bordered by dikes, in which the complex architecture of hardwoods remains visible. Wild boar and roe deer populations are abundant in the Rhine Valley, facilitating the active dispersal of wild apple fruits from late August to late September. We chose to study these protected areas here.The forests of the Rhine Valley were coppiced until the mid 20On both sides of the river, over a distance of 180 km, 45530 ha of the Upper Rhine Valley have been classified as a Ramsar site. The Rhine forests (both the floodplain and the Hardt forest) have also been integrated into the Natura 2000 conservation network in France.There were many orchards in the Rhine Valley before the 1970s, covering at least one fifth of the total area of cultivated land. Cultivated apple trees were also planted with other fruit trees, as landmarks, and along forest edges. They were traditionally used for fruit consumption and distilling apple brandy, as well as for decoration. These different uses account for the large number of cultivars grown. Since the 1970s, some of these orchards have been replaced by maize crops.This species has a long juvenile period, lasting five to six years. The flowers are hermaphrodite and pollinated predominantly by bees and flies (Syrphidae) In the Rhine forests of the Holocene plain and the Hardt, this species reproduces every year. Apple production is nevertheless highly variable, depending on the meteorological events of the previous spring and summer temperatures Pseudomonas sp.) or fungi and, ultimately, mistletoe . These individuals are therefore considered to be in bad health. Wild apples are often colonized by large woody vines , which climb up to the crown. These vines need good light conditions to ascend the trunks in understoreys. Their presence, together with that of large apple trees, thus provides an indirect evaluation of the suitability of the local light microclimate, mostly in gaps or ecotones. When ivy, which is more shade-tolerant than other climbers, is present alone, the under storey is often illuminated from the side Adult trees present various sizes and morphological forms. They may have upright trunks  and someDendrochronological studies in the Erstein natural reserve   have shoWe have obtained permission for collecting wild apple leaves from the Conservatoire des Sites Alsaciens, which is the authority responsible of the natural reserves of Erstein and Rhinau, manager of the Erstein natural reserve and the Rhinau natural reserves.The other locations did not require specific permission because forests are not protected but we have collected the leaves with the help and consent of local foresters from the Office National des Forêts, manager of these forests. These field studies did not involve endangered or protected species.N = 80), Marckolsheim (N = 47), Rhinau (N = 3), Daubensand (N = 3), Erstein (N = 80) and Illkirch (N = 42) forests. The small number of individuals from the Rhinau and Daubensand forests reflects the small number of wild apple trees present at these sites. This scarcity of wild apple is due to highly hydromorphic conditions at Rhinau and local intensive forest management in Daubensand. The geographical coordinates (latitude and longitude) of each site and the DBH of each individual were recorded.We collected data for 255 trees growing at six different sites in the forests of the Rhine Valley: the Hardt , according to the manufacturer’s instructions. Microsatellites were amplified by multiplex PCR, as described by Cornille et al. Malus domestica to the European wild apple has been demonstrated M. domestica cultivars previously identified as displaying no introgression from the European wild apple  M. sylvestris dataset (N = 255). We retained only individuals assigned with a membership coefficient of more than 0.7 to the M. sylvestris genepool for further genetic analyses (N = 246).Gene flow from the cultivated apple EH) and observed (OH)), Weir & Cockerham F-statistics, deviation from Hardy-Weinberg equilibrium and genotypic linkage disequilibrium. Allelic richness and private allele frequencies were calculated with ADZE software, for a sample size of 4 (2×2 chromosomes) After removing the individuals identified as potential hybrids, we used GENEPOP 4.0 K distinct clusters. The underlying algorithms attempt to minimise deviations from Hardy-Weinberg and linkage disequilibria within each cluster. We used an admixture model with priors. Six independent analyses were carried out for each number of clusters K (1≤K≤6), with 100000 MCMC iterations after a burn-in of 100000 steps.We used the individual-based Bayesian clustering methods implemented in STRUCTURE 2.3.3 N = 175 trees) and the Würmian terrace (N = 80) was assessed with Student’s t-tests.The significance of differences in DBH between the floodplain forests . We then performed a hierarchical clustering analysis (HCA). The squared Euclidean distance dissimilarity measure and the Ward linkage method were applied to determine profiles. We then checked for uniform variance and normality of the distribution and carried out a series of one-way ANOVAs to investigate the differences between categories for the variables studied. The significance level was set at P<0.05. We identified four categories, which we used in subsequent analyses.Ecological data were collected at a finer scale, in the natural reserve of Erstein (180 ha). Almost all the individuals present in this forest  were studied. We investigated possible associations between size, health status, morphological forms and the presence of woody vines, by collecting the following data: DBH, health status (a tree was considered as (i) in good health if upright and devoid of fungi, mistletoe, or/and dead axes; (2) in bad health when leant and reiterated axes. These trees were attacked by fungi or mistletoe and often harbour holes and dead axes), morphological plasticity, presence of Spatial correlation was assessed on the basis of geographical data, using the four categories identified, in a Mantel test. This test assessed the correlation between a similarity matrix containing the four categories of wild apples and the converted geographical distance matrix (Si = 1-Di).i.e., individuals assigned with a membership coefficient of more than 0.3 to the M. domestica genepool) among the entire sample of trees from the population of the Rhine Valley. The nine hybrid individuals were located in the Erstein (1), Hardt (7) and Illkirch (1) forests. These hybrids were not included in subsequent genetic analyses.STRUCTURE analysis detected nine hybrids , indicating an age of almost or more than 100 years. With the limited number of loci studied, it was difficult to determine in which generation introgression had occurred, but our findings clearly indicate that the hybrids were fertile and that their presence could therefore lead to the presence of wild introgressed individuals in forests. In the Hardt, all the hybrids were juveniles aged between two and five years. Five of these hybrids were located along paths running within the forest, whereas two others were located within the interior of the forest.P<0.001). ISF was low, suggesting random mating within each population and a weak population structure . Heterozygosity was higher in the southern populations (Hardt), decreasing towards the north . Frequentructure .priors, the individuals of the floodplain populations  were evenly distributed between the different clusters  were geographically dispersed, and saplings (from 50 cm to 1 m high) were found only in the Hardt forest. The tallest trees  and the trees with the greatest DBH  were found in the natural reserve of Erstein (vs Würmian terrace) and found a significant difference in DBH between these two groups, with higher values obtained for trees from the floodplain forests (mean ± standard deviation: 23.5±8.3) than for those from the Hardt forest (18.5±6.8) (Student’s t  =  P<0.05).We measured 255 individuals at the six populations: about 35% of the trees had a DBH between 10 to 20 cm, and an additional 35% had a DBH between 20 and 30 cm. Larger trees (DBH of 44 to 62 cm) were much rarer . Juveniles  differed from Category 1 for one factor: the absence  of the most light-demanding woody vine, Clematis vitalba. Category 3 (38%) contained stressed, parasitized trees, with multiple reiterations. Trees of all diameters (and ages) were observed. Ascending vines were rare. Category 4 (14%) was small, and contained healthy trees with upright trunks but without vines.Cluster analysis for the Erstein forest revealed the presence of four distinct ecological categories of wild apples . Categorr statistic (−0.0779) and the corresponding two-tailed P values (0.001) obtained for the 10000 permutations indicated that the four categories, taken together, were not randomly distributed in the forest. We tested the four categories separately against geographical distance: Category 4 was randomly distributed, whereas Categories 1, 2 and 3 were geographically constrained.The Mantel The current ecological clustering pattern at the wild apple sites of the Erstein forest can be interpreted in terms of specific local history and environmental factors:Clematis vitalba in particular. These Clematis vitalba vines have now reached heights of 40 m and widths of more than several tens of meters, with trunks of more than 15 cm in diameter. Hedera helix was also favoured by the light conditions and by the cracked bark of the largest wild apple tree trunks. The extensive network of climbers growing in the canopy has prevented canopy closure for decades, limiting the overtopping of wild apples.C1. This category established in large gaps. Before river management, such gaps were numerous because floods accelerate tree fall. After river management, the massive death of canopy elms following the spread of Dutch elm disease in the 1970s and 1980s also created large gaps. These large gaps were highly suitable for the establishment of both wild apples and light-demanding woody vines, such as Clematis vitalba suggests poorer light conditions, due to smaller gaps , partial canopy closure or the presence of edges deep in the forest (between channels and the forest and between paths and the forest). However, there is nevertheless sufficient light for wild apple trees to grow straight up and for ivy to establish itself in the crown. This situation was found to be more frequent in the forest along the ancient river channels of the Rhine, in the eastern part of the reserve. In managed forests, human practices have essentially reproduced the heterogeneity of the canopy topography , and respect for native trees has also preserved many wild apple sites.C2. The absence of Hedera helix may or may not be present. Such situations were found in the best preserved part of the forest, rich in large, old canopy trees.C3. The wild apple trees of this category are in poor health because of poor local light conditions  and other unknown reasons . C4. This category results more from the age structure of wild apple populations in this region rather than a given ecological situation. All the individuals of this category are younger than those in the previous categories. They may have colonized sites with partially closed canopies or small temporary gaps or clear-cuts. These growth conditions allow the vertical growth of the trunk and are found at random sites in many parts of the forest. The absence of ivy is not an indication of low light conditions, but of the smoothness of the bark, making it difficult for the adventitious roots to establish a hold.These ecological categories provide new insight into the factors driving the population dynamics of the European wild apple in the Rhine Valley:Wild apple ecology. Beyond the local presence of specific communities of species associated with the different categories found , the differences in size between the floodplain and the Hardt populations may provide insight into the types of environment most suitable for wild apple in terms of water and nutrient resources. The moist and nutrient-rich soils of the floodplains seem to be highly suitable for rapid plant growth, contrasting markedly with the drier Hardt environment. Nevertheless, further ecophysiological investigations are required to determine the ecological preferences of wild apples.Local site-specific  historical events. There were probably a large number of suitable sites for wild apple 150 years ago because 1) regular flooding severely limited woody plant regeneration Successive floods in past decades may explain the unbalanced age structure of the populations. Following canalisation of the river, competition between seedlings increased, due to both the elimination of flooding and the spread of Dutch elm disease, which killed many elms, thereby creating large gaps in the canopy. In the meantime, forestry practices shifted from small to large clear-cuttings and plantations. These changes had deleterious effects on wild apple populations, decreasing sapling establishment and adult growth, and increasing stress in adults due to overtopping, except in large gaps created by the death of elms or extensive clear-cutting. The ecological conditions of the Erstein forest have thus resulted in a rarity of seedlings and saplings. A few 1 m- to 3 m-tall saplings were found in the driest parts of the forest, along paths. A few seedlings were also found in the dense carpets of saplings of other species, but they were heavily browsed and rapidly disappeared Local high-disturbance events that temporarily open the canopy, such as high winds, fire and human practices, have also helped to maintain the light-demanding wild apple in these forests. More open, anthropogenic landscapes provide this species with suitable sites for survival.The wild apple has thus developed a particular colonising strategy in deciduous forests, due to its low growth capacity and poor shade tolerance (resulting in scarcity within the forest and a tendency to grow in clumps), leading to an unbalanced age structure with many very old adults. This strategy accounts for the broad distribution of this species in Europe and the large differences in population density as a function of the light regime of the understorey and natural and anthropogenic disturbance or stress regimes.The high heterozygosities of the Rhine and Hardt forests are consistent with the results of previous studies wild apple populations in Belgium STRUCTURE analysis revealed an absence of genetic structure across the Rhine forest. These results are consistent with the obligatory cross-pollination system of apples, preventing local endogamy, and the strict endozoochory by mammals over large territories. Large-scale dispersal increases opportunities for finding suitable forests in which individuals can reproduce at higher rates and sapling mortality is lower. The weak population structure revealed that, despite the high level of clumping observed in all forests, individuals from the same clump were not more closely related genetically than individuals from different clumps..The ecological data indicated that the Hardt populations had a very different history from the floodplain populations. Indeed, before the dikes were built, hardwoods were also regularly destroyed by major floods  i.e., 1 or 2 weeks) between cultivated and wild apples th century, may account for the old age of the few hybrids we detected. It is also possible that the domesticated apple cannot tolerate the ecological constraints of the Rhine forest, limiting its presence in this area. Cultivars originated from Malus sieversii (Ledeb.) M. Roem from the Tian Shan Mountains Malus sieversii is a very demanding species, and the main component of fruit-tree forests of these regions. These forests, characterized by an open canopy, are found on the moist slopes and alluvial plains of the mountain rivers, at altitudes of between 800 and 1500 m above sea level, in humus-rich soils Several factors may account for the rarity of introgressed wild apples in the forests of the Rhine Valley: the sample size used, which was restricted to a narrow geographical area, and the only partial overlap in flowering time  have made a major contribution to these difficulties. We propose here a possible reconstruction of the principal events leading to the current situation as concerns wild apple in the Upper Rhine floodplain forests:Historically, and even during prehistory, before human management of the river, the wild apples lived in hardwood forests towards the outer edges of the floodplain. These populations were regularly destroyed by major floods, but subsequently colonized the floodplain from source sites, such as the Hardt forest in particular, through dispersal by large mammals.Once the dikes had been built, flood strength decreased, favouring the expansion of hardwoods. This period was ideal for the wild apple, particularly in the zones between two dikes, in which the hardwoods were still flooded, as it limited the penetration of shade-tolerant species and seedling survival, and forest management was dominated by a combination of coppicing and high trees. Most of the current population of wild apples probably dates from this period, with establishment and reproduction occurring from 1850 to 1960.After canalisation, the opportunities for establishment decreased, and many saplings and adult trees died. However, the light regime in the understoreys remained adequate for the maintenance of an adult population in gaps, forest ecotones, clear-cuts created by intensive forestry and overtopping situations in closed forests.Since the establishment of regulations protecting the ecosystem of the Rhine Valley in the 1990s, the forests have been managed less intensively, and wild apples are not cut anymore, with their survival even favoured locally by the cutting of overtopping trees. However, competition remains fierce in the sapling carpet, due to the absence of flooding. The age structure of the population thus remains unbalanced and many of the apples in the most productive parts of the plain may soon become senescent.However, hybrids and cultivars are clearly disadvantaged in these forests, and so the genome of the wild apple is thus well preserved.In the Hardt forest, ecological conditions for wild apples have changed little, other than an increase in fragmentation of the area due to paths, roads, meadows and cultivated fields, facilitating the penetration of hybrids. Hybrids may thus be more numerous in this forest than in the floodplain, and this higher frequency of hybrids may also reflect lower levels of interspecific competition than in the humid conditions of the floodplain.i.e., at least 100 ha) and preferably circular in shape, according to our observations in the Rhine floodplain The most effective way to preserve this species at the European scale would be to recognise this species as threatened and to protect all forest types naturally rich in wild apples against fragmentation. An essential condition is that these forests must be wide enough . The nine dendrochronological sequences shown in this diagram illustrate the growth characteristics of wild apples in the floodplain forest of Erstein. The horizontal line, which represents a growth ring with a width of 1 mm, provides a benchmark for assessing periods of growth and no growth. The circle drawn to the left of each sequence indicates the estimated pith location, as coring to the pith could not be done because of the hardness of the wood. The first five to ten years of difficult growth are generally followed by 70 years of vigorous growth and then a final 40 years or so of slower growth, as evidenced by very thin rings (example of KUHAU-029). The great variations in growth for each of the analysed wild apples made it impossible to establish correlations among their dendrochronological sequences. These sequences were thus positioned and drawn with their terminus in 2006. Unpublished report from Patrick Gassmann (2007), Laboratoire de dendrochronologie, Office du Patrimoine et de l’Archéologie, Laténium, Espace Paul Vouga 7, CH-2068 Hauterive, Switzerland.(TIF)Click here for additional data file.


Megastigmus schimitscheki (Hymenoptera: Torymidae) in France. The explanatory power was tested by: (1) survival analysis of empirical data on M. schimitscheki presence, absence and year of arrival at 52 stands of the wasp's obligate hosts, Cedrus (true cedar trees); and (2) Approximate Bayesian analysis of M. schimitscheki genetic data using a coalescence model. The Bayesian demographic modeling and traditional population genetic analysis suggested that initial invasion across the range was the result of long-distance dispersal from the longest established sites. The survival analyses of the windborne expansion patterns derived from a particle dispersal model indicated that there was an informative correlation between the M. schimitscheki presence/absence data from the annual surveys and the scenarios based on regional-scale wind data. These three very different analyses produced highly congruent results supporting our proposal that wind is the most probable vector for passive long-distance dispersal of this invasive seed wasp. This result confirms that long-distance dispersal from introduction areas is a likely driver of secondary expansion of alien invasive species. Based on our results, management programs for this and other windborne invasive species may consider (1) focusing effort at the longest established sites and (2) monitoring outlying populations remains critically important due to their influence on rates of spread. We also suggest that there is a distinct need for new analysis methods that have the capacity to combine empirical spatiotemporal field data, genetic data, and environmental data to investigate dispersal and invasion.Understanding how invasive species establish and spread is vital for developing effective management strategies for invaded areas and identifying new areas where the risk of invasion is highest. We investigated the explanatory power of dispersal histories reconstructed based on local-scale wind data and a regional-scale wind-dispersed particle trajectory model for the invasive seed chalcid wasp  Alien invasive species are a major threat to species survival and can significantly alter entire ecosystems  in France and C. brevifolia and C. libani in its native range of Asia Minor. Introduced populations of the wasp can have a direct impact on the reproductive success of their obligatory host by reducing viable seed production by up to 87% , a group which presents a major ecological and economic threat in Europe Roques . MegastiC. atlantica stands have been conducted since 1978, and in 1994, it revealed the first specimens of M. schimitscheki. The wasp was introduced to France only once, between 1990 and 1993, from Cyprus to the region of Mont Ventoux through clandestine C. brevifolia seed trade for ornamental purposes. French populations were founded from an extremely restricted number of individuals that realized a host shift from C. brevifolia to C. atlantica  analysis in the ABC-based program DIYABC . The wasps lay their eggs inside cedar ovules by inserting their ovipositor through young cone scales, and the larval instars develop by consuming the seed  stand, three mature cones were randomly collected at 2 m above the ground on 10 randomly selected cedar trees before seasonal cone disarticulation to estimate seed infestation rate by Megastigmus spp. In the laboratory, cones were disarticulated and seeds colonized by either of the two Megastigmus species were identified by Faxitron numerical X-ray radiography , grouped according to year and site of collection, and stored under natural conditions. Adults emerging in the spring were determined to species, sexed and stored in 100% ethanol for genetic analysis , 1 μL H2O, 5 μL QIAGEN Multiplex PCR Master mix 2× , and 1 μL of 2 μM primer mix. PCRs were conducted as two multiplex reactions using genus-specific microsatellite primers , allelic richness (AR), and inbreeding coefficient (FIS). The significance of FIS-values was assessed at the 5% confidence level using strict Bonferroni corrections. Allelic richness was estimated using rarefaction with a sample size of nine individuals (minimum sample size per plot). The components of variance in allelic frequencies among study sites and among individuals were estimated using a hierarchical AMOVA in ARLEQUIN v.2 . Accordingly, we calculated both FST and RST in ARLEQUIN as estimators of genetic differentiation between groups and populations. All loci were tested for significant deviation from Hardy–Weinberg equilibrium in ARLEQUIN. Null allele (NA) frequencies were estimated for each locus in each population according to the Expectation Maximization algorithm in FreeNA  and RST / (1 − RST) between all pairs of sampling plots, computing the regression slope of those values against the logarithm of pairwise geographic distance (blogFST or blogRST), and comparing these slopes to their frequency distribution under the null hypothesis of spatial genetic randomness obtained by 5000 permutations of individuals among populations (Hardy and Vekemans RST = FST), we used the test in SpaGeDi  indicated that diurnal temperatures in southern France in May always remain above the thermal threshold of adult flight activity (ca. 16°C) and that the annual number of days with precipitation varied from zero to three. We therefore assume that the influence of these factors is negligible in this study.M. schimitscheki as observed in the annual surveys. The cumulative exposure to “treatment” was the annual arrival of M. schimitscheki individuals at the site, which was modeled as the yearly HYSPLIT particle point count in the grid cell of each study site (see below). There was no equivalent quantitative value that could be derived from the CLIMATIK data so we did not conduct a survival analysis of the local-scale wind data.We used survival analyses to test whether long-distance dispersal from the point of introduction or short-distance “stepping-stone” dispersal better explained observed patterns of colonization. Survival analysis is a statistical method typically used to analyze the probability of death in biological organisms exposed to a treatment ) (see Table S5 for details).The explanatory power of the three scenarios above was tested using time-dependent survival analysis. We tested the effect of yearly particle point count in the grid cell of each study site on the rate at which establishment of migrants occurred at that study site. The only explanatory variables for the survival analysis were based on counts of the regional-scale HYSPLIT particle trajectories. Our data were interval-censored, meaning that we did not observe the exact time at which first colonization occurred but could only observe that it occurred within an interval of years. We used time-dependent covariates  M. schimitscheki colonized through long-distance dispersal, independently of wind patterns; (2) M. schimitscheki colonized through short-distance dispersal, based on a stepping-stone model calibrated by regional wind data; (3) regional-scale and higher altitude wind data explain observed patterns of colonization; (4) local-scale and near ground-level wind data explain observed patterns of colonization. These four demographic scenarios are presented as coalescent “gene genealogies,” similar to phylogenetic trees but representing the process whereby individuals from an established population emigrate and establish a new population, . No single nucleotide in-del mutations were allowed in the simulations, the Generalized Stepwise Model of SSR mutation was assumed, and the mean SSR mutation rate ranged between 1 × 10−4 and 1 × 10−5. According to Chapuis et al. ; the wind data from the nearest CLIMATIK weather station were used for each source site Fig. , and thoM. schimitscheki population size increases with time as 12 years after introduction local abundance of the wasp was found to be tenfold higher in the introduction area than in newly colonized areas  for foundation time of the Mont Ventoux population (t1) in the models. The other dates of divergence were fixed and not estimated. We modeled the Mont Ventoux population as the result of recent admixture between two populations to take into account the observed genetic diversity in the initial population on Mont Ventoux and allow for the possibility that the population was not in Hardy–Weinberg equilibrium. A list of both demographic and time parameters and prior distributions used to model scenarios is summarized in Table S7.Defining the prior distribution of parameters is a key step in ABC analysis. Distributions should include all of the values which are considered possible and prior information can be based on previous knowledge of some parameters ² between all pairs of populations. This allowed us to avoid an overestimation of scenario fit to our data  or allelic richness (A) were similar in the fourteen populations, with overall means of He = 0.486 and A = 3.557 Table . The spaFST / 1 − FST against the logarithm of distance blogFST = 0.028 (95% CI: −0.043 to 0.050) or RST / 1 − RST against the logarithm of distance blogRST = 0.080 (95% CI: −0.072 to 0.092). The confidence intervals are based on a permutation procedure, which randomizes the spatial locations of individuals. The test using randomization of allele sizes was not significant, suggesting that stepwise mutations did not make an important contribution to genetic differentiation , and therefore, RST was not a better measure of differentiation than FST in this study.The variation in pairwise population differentiation with spatial distance revealed no pattern of isolation by distance. The overall increase in differentiation with distance was not significant, as shown by the regression slopes of K ranging between 1 and 10, both with and without sampling plot location to assist clustering and with and without admixture. The K value with the highest posterior probability was consistently K = 1, indicating that STRUCTURE did not detect genetic differentiation between the study sites.The STRUCTURE analysis was run with P-value <0.0001 for all scenarios), meaning that the colonization of a site by a wasp occurred more rapidly when the covariate (here the particle point count in the grid cell of each study site according to each scenario) increased. The scenario involving long-distance dispersal from the introduction site  showed that in most years the greatest proportion of wind travelled toward the southeast. Moreover, over the seventeen year period from 1994 to 2010, the number of days during which wind travelled to the southeast was twice to three times as many as the number of days in which wind travelled in any one of the other directions (Table S9). This result coupled with the observation that the majority of the infested stands are located to the east or southeast of Mont Ventoux  suggest that founder effects or genetic drift may be important, and gene flow is limited. Moreover, the AMOVA analyses showed that the genetic structure is at the population level and no substructure exists at higher levels. Together, the results from the analysis of genetic structure could support range expansion through long-distance dispersal. It should also be noted that populations that are growing in effective population size may show low genetic drift and relatively stable allele frequencies even with genetic isolation, at least in the short term and depending on the genetic profile of the propagule pool  as the main source of all emigrants  may be more meaningful for the construction of dispersal models.M. schimitscheki appears to regularly disperse over distances greater than 10 km. Given that this distance is greater than the known active flight capacity of the genus, this dispersal is likely to be passive. (2) Our analyses of two wind datasets did not produce supported scenarios in the ABC analysis, but given the environmental importance of wind in this region, and the southeastern-tending pattern of M. schimitscheki dispersal, wind is still likely to be the dispersal vector. Developing methods to analyze wind as a dispersal vector remains a rich opportunity for research, for example, Nathan et al. Overall these results suggest that (1) M. schimitscheki population dynamics in relation to within-site biotic factors, such as host plant seed masting cycles and interspecific competition with the conspecific M. pinsapinis, will help refine our ability to focus monitoring and control efforts for this invasive wasp.Both the survival analysis and the DIYABC analysis found that long-distance dispersal based on regional-scale, high-altitude wind activity best explained the annual presence/absence data from the study sites. This result suggests that initial invasion across the range may be the result of long-distance dispersal and supports previous research which found such patterns in closely related wasps (e.g., Jarry et al. M. schimitscheki and other seed and cone predators.The population genetic analyses found little evidence of genetic structure, supporting the single introduction proposed by Auger-Rozenberg et al. . HoweverWe suggest that current field survey methods detect populations only once they are relatively large and already serving as sources of emigrants. Monitoring programs aimed at detecting populations at an early stage of establishment would therefore need to use alternative survey techniques such as a suction trap network. Such a network could also help elucidate the effects of meteorological conditions on insect dispersal (e.g., Sanders et al.