Triatoma dimidiata is one of the most significant vectors of Chagas disease in Central America and Colombia, and, as in most species, its pattern of genetic variation within and among populations is strongly affected by its phylogeographic history. A putative origin from Central America has been proposed for Colombian populations, and high genetic differentiation among three biographically different population groups has recently been evidenced. Analyses based on putatively neutral markers provide data from which past events, such as population expansions and colonization, can be inferred. We analyzed the genealogies of the nicotinamide adenine dinucleotide dehydrogenase 4 (ND4) and the cytochrome oxidase subunit 1-mitochondrial genes, as well as partial nuclear ITS-2 DNA sequences obtained across most of the eco-geographical range in Colombia, to assess the population structure and demographic factors that may explain the geographical distribution of T. dimidiata in this country.The population structure results support a significant association between genetic divergence and the eco-geographical location of population groups, suggesting that clear signals of demographic expansion can explain the geographical distribution of haplotypes of population groups. Additionally, empirical date estimation of the event suggests that the population's expansion can be placed after the emergence of the Panama Isthmus, and that it was possibly followed by a population fragmentation process, perhaps resulting from local adaptation accomplished by orographic factors such as geographical isolation.T. dimidiata populations are generally in accordance with population expansions that may have been accomplished by two important biotic and orographic events such as the Great American Interchange and the uplift of the eastern range of the Andes mountains in central Colombia.Inferences about the historical population processes in Colombian  Triatoma dimidiata is one of the most important vectors in America, owing to its wide genetic and epidemiological heterogeneity. Colombian T. dimidiata populations occupy eclectic sylvatic ecotopes, but have also been found in dwellings infected with Trypanosoma cruzi, and therefore it is considered  the most important vector in several departments. The current study explores the population structure history of Colombian populations by means of a molecular coalescence approach. The results indicate that the historical population processes in T. dimidiata in Colombia are in accordance with population expansions that may have been accomplished by two important biotic and orographic events such as the Great American Interchange and the uplift of the eastern range of the Andes Mountains in central Colombia. The genetic history as well as the heterogeneity of the populations could be reflected in different responses of the populations to vector control interventions; thus, a local level of entomological vigilance should be implemented to evaluate the intervention results in each region.The Chagas disease vector  A population's demographic history as well as phylogeographic inferences are usually accessed by studying the reconstructed genealogical histories of individual genes (gene trees) sampled from different populations Triatoma dimidiata is considered the major vector of Chagas disease in several Central American countries as well as in various regions of Ecuador and Colombia T. dimidiata occupies a great diversity of habitats, including sylvatic habitats such as palm trees and hollow trees in northern regions, or rock piles, as well as intradomiciliary synanthropic habitats mostly in the country's central Andean departments T. dimidiata shows a strong and significant genetic structure related to its original eco-geographical regions in Colombia T. dimidiata in Colombia was assessed using DNA sequence analysis of the nicotinamide adenine dinucleotide dehydrogenase 4 (ND4) mitochondrial gene, which interestingly suggested a high genetic interpopulation differentiation within Colombia n = 40), representing only a minimal area of the species distribution, a more exhaustive genetic analysis of several communities of Colombian T. dimidiata was performed by using a microsatellite as well as cytochrome c oxidase subunit 1 (CO1) gene T. dimidiata shows more epidemiological relevance and apparently high flow between synanthropic and sylvatic habitats; (ii) the Caribbean Plains (CP) population group, the most widely distributed group from the Caribbean coast to the lowlands of the Central Andean Cordillera, occupying mainly sylvatic habitats; and (iii) the Sierra Nevada de Santa Marta (SNSM) mountain population group, located in the northwestern zone of Colombia, occupying exclusively sylvatic habitats such as palm trees, although a few individuals have also been found sporadically visiting indigenous dwellings and have also been implicated in human Trypanosoma cruzi infections T. dimidiata populations are considered a differentiated form derived from Central American conspecific populations  T. dimidiata in Colombia as well as their position in the phylogeographic picture proposed for the species so far.In a phylogeographic context, according to the evidence addressed by molecular analyses of ITS-2 Analysis of the population genetics in the context of the geographic structure suggests demographic processes that occurred in the past. Thus, while the pattern of variation in mtDNA haplotypes allows one to identify geographical distribution differentiation among groups of haplotypes in several populations, it also supports inferences on demographic events that occurred in the past, such as geographical range expansion and population size, according to coalescence theory. In this way, the change in population size through genealogy is reflected by a haplotype network with a star shape T. dimidiata in Colombia by analyzing ND4 gene nucleotide sequences obtained from 228 specimens in 22 localities; subsequently, several historical demographic tests were conducted using ND4 combined with previously reported CO1 nucleotide sequences In this study, we broaden the knowledge of the spatial structure of the three population groups of Knowledge of population dynamics issues such as geographical dispersion and individual migration between extradomiciliary and domiciliary ecotopes is essential for predicting the success of vector control and surveillance strategies against Chagas disease. In this sense, study of the population structure and demographic history in the most relevant vectors is required for the design of more effective intervention strategies.A total of 228 sequences for the ND4 (624-bp) gene as well as 42 partial sequences (252 bp) for ITS-2 were obtained . Individ5′-TCAACATGAGCCCTTGGAAG-3′) and ND4-R (5′-TAATTCGTTGTCATGGTAATG-3′) primers 2, pH 8.3), 250 µM dNTP, 0.016 µM of each primer, 5 mM MgCl2, and 2 U of Taq DNA polymerase (Promega®). The fragments were amplified with the following thermal cycling conditions: 95°C for 5 min; 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 60 s; 72°C for 10 min.Genomic DNA was obtained from four legs of each insect or from the thorax muscle when necessary . DNA extraction was performed according to a previously reported mosquito DNA-extraction protocol T. dimidiata specimens were obtained when universal primers 5.8S and 28T T. dimidiata-specific primers TdITSF (5′-TGGAAATTTTCTGTTGTCCACA-3′) and TdITS-2R (5′-CTTGCTTTATACAACAAGAAGTA-3′) 2, pH 8.3), 250 µM dNTP, 0.025 µM of each primer, 3 mM MgCl2, and 2 U of Taq DNA polymerase (Promega®). After an initial denaturation of 95°C for 5 min, PCR reactions comprised 35 cycles at 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, followed by a final extension of 72°C for 7 min Because no reproducible and unspecific amplifications in Colombian Forward and reverse sequences from specimens were used to generate a consensus sequence with a previous pairwise alignment using the CLUSTALW algorithm p<0.001) with a permutation test of 1,000 replicates. A median joining (MJ) haplotype network was used to examine inter-haplotype relationships among the 155 haplotypes of the 228 ND4 sequences as well as for 17 partial ITS-2 haplotypes using default parameters in Network 4.6.0.0 software (http://www.fluxus-engineering.com).In the complete data set for ND4 and ITS-2, we evaluated the nucleotide diversity (π), number of haplotypes (h), and haplotype diversity (Hd) using DnaSP v.5.10 T. dimidiata populations suggested in Colombia plus or minus one. The maximized Fct was selected according to the highest significant (p<0.001) value.Spatial analysis of molecular variance (SAMOVA) was performed to estimate the structure among population groups according to pairwise geographical distances between geographical locations by Fct statistical calculations using SAMOVA v.1.0 p<0.001) was assessed by a permutation test of 1,000 replicates.An interpolation-based graphical method was employed to generate a three-dimensional genetic landscape shape (GLS) within the Alleles in Space (AIS) program T. dimidiata was investigated by comparison of mismatch distributions of pairwise nucleotide differences under an expected constant and fluctuating population size in 10,000 generations of coalescent simulations using DNaSP v. 5 n = 86; sequence size, 1,016 bp), and for partial ITS-2 sequences assuming free recombination. Parameters for a sudden demographic expansion were estimated using the sum of squares deviation (SSD) s2The demographic history of the Colombian T. dimidiata using ND4 haplotypes, and combined ND4 and CO1 genes, and then tested for additional haplotype sequences of Central American and Mexican conspecifics for the ND4 (n = 39) gene and ITS-2  fluctuation over time, a Bayesian skyline plot (BSP) analysis  n = 66; . Trees wn = 83) and ND4 (n = 194) haplotypes including Central American and Mexican conspecifics was used to perform a Bayesian inference (BI) Markov Chain Monte Carlo (BMCMC) approach as implemented in the BEAST v.1.6.1 package 6 generations, with a sampling frequency of 1,000. Two independent trees were combined using LogCombiner v.1.6.1, and convergence of parameters in the Bayesian analyses was assessed with Tracer v.1.5, after discarding a 10% burn-in. Finally, a majority rule consensus tree (>0.75 posterior probability node support) was calculated from all trees sampled using TreeAnnotator v.1.6.1 in the BEAST v.1.6.1 package The complete data set of ITS-2 (http://tree.bio.ed.ac.uk). The overall topological match score and a well-supported node match score between IB and ML topologies for both ND4 and ITS-2 markers were calculated using Compare2Trees software Additionally, a maximum likelihood (ML) tree was estimated using the GTRCAT approximation of substitution model, and the best knowledge likelihood tree (BKLT) was selected via bootstrapping  in RAxML-VI-HPC v.2.2.3 All nucleotide sequences are available with GenBank accession codes for ND4: KC489309–KC489463 and ITS-2: KC489292–KC489308.p<0.001 and Hst = 0.0166; p<0.001). Moreover, the ITS-2 marker indicated low variability, mostly in the SNSM region  using ND4 indicates high genetic differentiation among eco-geographical groups. Pairwise Fst was 0.592 (p<0.05) between CP and SNSM, 0.588 (p<0.05) between IAV and CP, and 0.512 (p<0.05) between IAV and SNSM.The ND4 gene analysis at both haplotype and nucleotide diversity levels showed a statistically significant differentiation index among the three eco-geographical groups , which are likely to be rare or recent in a population, were widely distributed in the haplotype network according to the three eco-geographical groups suggested , whereasT. dimidiata indicated a significant maximized Fct at k = 3 , supporting the eco-geographical structure previously evidenced. Collection sites from the Boyacá, Santander, and Huila departments comprised the first group (congruent with the IAV region), sites from Bolívar, Antioquia, Norte de Santander, Córdoba, and Cesar formed the second group (congruent with the CP region), and Magdalena and La Guajira sites formed the third (the SNSM region). Genetic landscape shape interpolation analysis showed that the spatial distribution of haplotype diversity across Colombia was not uniform, as indicated by the presence of peaks and valleys , suggesting that nonsignificant clustering of the haplotypes (based on pairwise genetic distance) within each of the 10 spatial classes tested could be inferred , indicating no isolation-by-distance model fit that could explain the geographical structure of T. dimidiata in Colombia  around the central geographical distances of the classes, and showed a significant autocorrelation when spatial classes ranking between ∼525 m and ∼625 m . This reColombia .ND4: Rag = 0.003, p>0.05; SSD = 0.003, p>0.05; ND4+CO1: Rag = 0.001, p>0.05; SSD = 0.003, p>0.05; and ITS-2: Rag = 0.019, p>0.05; SSD = 0.002, p>0.05). Thus, the possibility that the expansion model fits cannot be rejected. In addition, significant values for Fs and R2 in the neutrality tests used for detecting population expansion were found in the three sequence data sets, indicating clear signals of population growth in T. dimidiata in Colombia  of ND4, and combined ND4 and CO1 genes, indicated that  present . Moreove present , and the present .T. dimidiata genetic groups The overall topological match score between IB and ML approaches was moderate for both ND4 (65%) and ITS-2 (49.6%), but high match node scores (>75%) were obtained between them for the well-supported clades observed using both markers . This reThe topologies built using the partial ITS-2 showed three main clades , congrueMoreover, in the ND4 phylogeny four well-supported monophyletic clades showing a high match node score (>95%) were found . The fouT. dimidiata population history. The geographical structure patterns obtained using mtDNA sequences were congruent with previous results T. dimidiata expanding into Colombia, but this structure was not related to the isolation-by-distance model explaining genetic differences in the three main population groups. Instead, a strong signal arguing for a sudden change in population size and range expansion undergone in the recent past after the emergence of the Isthmus of Panama , thus suggesting a possibly Mesoamerican origin of Colombian populations. However, the phylogenetic result obtained with the ND4 gene  and nucleotide (Kst) diversity levels indicates that the differential distribution of genetic variability is placed among the IAV, CP, and SNSM regions. Additionally, the results of spatial genetic structure analyses  make at least two main conclusions possible: (i) there is a heterogenic geographical distribution of genetic diversity and (ii) none of the correlations among genetic divergence and geographical distance explains the geographical group structure of a population.T. dimidiata could be one of many probable causes. An additional observation can be made about this distribution. While T. dimidiata populations in SNSM and IAV occupy mostly diverse mountain and premountain ecosystems located around 1,000 masl, CP is for the most part composed of extended lowlands with dry and warm zones at lower altitudes, given that the departments of the CP region extend from the Colombia–Panama border to the foothills of the west mountain range of the northern Andes.Spatial interpolation of genetic diversity in Colombia shows a bimodal curve-shaped distribution with SNSM and IAV having higher diversities than the CP region, indicating that several microevolutionary processes have been involved in the genetic diversity divergence among population regions, where possible disruptive segregation of ancestral populations of T. dimidiata cannot be understood separately from its history. Unlike the previous hypothesis on the possible isolation of population groups due to geographical distance explaining the genetic differences identified T. dimidiata diversity.The current eco-geographical structure of Colombian T. dimidiata.With the emergence of the Isthmus of Panama, an important paleozoogeographical event occurred as a product of the Great American Interchange between North America via Central America to South America and vice versa for fauna during the Piacenzian Click here for additional data file.Table S2T. dimidiata used in this study. Map numbers and geographic origin are detailed in ITS-2 Haplotype distribution of Colombian (DOCX)Click here for additional data file.Table S3T. dimidiata used in this study. Map numbers and geographic origin are detailed in ND4 haplotype distribution of (DOCX)Click here for additional data file.


Phytophthora plurivora, which is not subject to regulations, is a plant pathogen frequently found on a broad range of host species, both in natural and artificial environments. It is supposed to be native to Europe while resident populations are also present in the US. We characterized a hierarchical sample of isolates from Europe and the US and conducted coalescent-, migration, and population genetic analysis of sequence and microsatellite data, to determine the pathways of spread and the demographic history of this pathogen. We found P. plurivora populations to be moderately diverse but not geographically structured. High levels of gene flow were observed within Europe and unidirectional from Europe to the US. Coalescent analyses revealed a signal of a recent expansion of the global P. plurivora population. Our study shows that P. plurivora has most likely been spread around the world by nursery trade of diseased plant material. In particular, P. plurivora was introduced into the US from Europe. International trade has allowed the pathogen to colonize new environments and/or hosts, resulting in population growth.Human activity has been shown to considerably affect the spread of dangerous pests and pathogens worldwide. Therefore, strict regulations of international trade exist for particularly harmful pathogenic organisms.  Global trade has dramatically increased the chances of pathogens to be spread artificially around the world, on or with traded goods Batrachochytrium dendrobatidis, where global trading of African clawed frogs (Xenopus laevis) for laboratory purposes resulted in dispersal of the pathogen Magnaporthe oryzae, Ascomycota) which cause severe losses of harvests of wheat and barley Plasmopara viticola) and hop (Pseudoperonospora humili) Recent invasive diseases with dramatic consequences have gained a lot of attention, e.g. the worldwide amphibian dieback caused by the chytrid fungus Armillaria mellea that was introduced to South Africa by Dutch settlers more than 300 years ago, but remained undetected until recently Nonetheless, there are also invasive pathogens with less dramatic consequences that may be difficult to detect. This applies, for example, to invasive pathogens that infect native host species, which are present at low frequencies in specific ecosystems and/or only play a marginal ecological role. Furthermore, pathogens that have established in a non-native ecosystem a long time ago and have, in the meanwhile, become part of this ecosystem are not necessarily considered invasive anymore. Historic records may be helpful, as it is the case for the root fungus Cryphonectria parasitica) in North America and Europe Fraxinus sp.) dieback caused by the ascomycete fungus Hymenoscyphus pseudoalbidus in Europe Phytophthora species , e.g. Sudden Oak Death Phytophthora species Phytophthora species in forest ecosystems is probably P. ramorumPhytophthora species are still poorly understood.Several invasive diseases are well known and are being increasingly reported in forest ecosystems worldwide Phytophthora plurivora, one of the four species of the former P. citricola species complex, is a widespread pathogen in different environments in Europe Fagus sylvatica) and oak species  in the Eastern and North Central United States P. plurivora has only been sporadically found in plantations and nurseries, e.g. in Australia and South Africa. Due to its regular recovery from natural ecosystems in Europe and its rare recovery in other continents, P. plurivora is generally assumed to be native to Europe. However, to this date scientific evidence is missing to confirm this hypothesis.Phytophthora plurivora is considered to be a homothallic species P. plurivoraPhytophthoraP. ramorum, P. infestans, and P. cinnamomi). Therefore, little detailed information is available about the consequences of a homothallic mating system on the expected genetic diversity of Phytophthora populations.With the advent of molecular tools it is now possible to assess whether a pathogen is invasive and determine the most probable center of origin P. plurivora population and to use a coalescent approach to determine the demographic history of this species as well as the degree of gene flow among geographic populations. Specifically, we addressed the following questions and related hypotheses: (i) do microsatellite and sequence data support a homothallic mating system in P. plurivora? We hypothesized that in that case no traces of recombination should be present in the sequenced genes and that microsatellite data should reveal a high degree of inbreeding; (ii) what are the main directions of gene flow within the global P. plurivora population? We hypothesized that the US populations originated from Europe; (iii) is the European P. plurivora population geographically structured? Because of the frequent association of P. plurivora with nursery trade, we hypothesized that no significant geographic structure is present in Europe; and (iv) what kind of demographic trend  is detectable in the global P. plurivora population? Given the detection of P. plurivora in different environments and geographic ranges, we would exclude a population reduction as a demographic trend and hypothesize that the population is either stable or expanding. To answer these questions, we genotyped and sequenced P. plurivora isolates derived from Europe and the US.The main objective of this study was to quantify the genetic diversity of the global Phytophthora plurivora isolates from 16 countries  were analyzed. The Swiss isolates originated mostly from our culture collection, whereas all other isolates were kindly provided by colleagues. As P. plurivora is not a quarantine organism subject to phytosanitary regulations by the Swiss plant protection ordinance , a sampling or import permit is not required.In the present study, a total of 359 ITS) region and/or the nuclear beta tubulin (btub) gene following the procedure described in Blair et al. P. citricola s.s., CBS 18125 – P. pini, CBS 124087 – P. plurivora and CBS 124094 – P. multivora) obtained from the CBS-KNAW fungal biodiversity center’s culture collection (http://www.cbs.knaw.nl/databases/) to confirm species identity.Prior to our analyses, the species identity of all isolates was confirmed by sequencing the ribosomal internal transcribed spacer  following the published protocol The 359 isolates were genotyped at 11 ST, rBarD, and allelic richness, see below), the 221 isolates were combined into 12 geographic populations in order to increase the sample sizes of each respective population, i.e. US-East Coast (EC), US-West Coast (WC), United Kingdom (UK), Finland (FIN), France (F), Belgium and the Netherlands (BNL), Germany (D), Eastern Europe , Poland (PL), and Hungary (H)), Alps (with samples from Switzerland (CH) and Austria (A)), Balkans  and Slovenia (SLO)), Italy (I), and Turkey  and expected (HE) heterozygosities were calculated using the software GENETIX A but is independent from the number of loci considered, was calculated using the R package POPPR ST; 1000 randomizations) and pairwise FST-values Population history. The hypothesis of a population expansion was tested using the Microsoft Excel macro KGTESTS In addition to testing for expansion, populations were also tested for evidence of a bottleneck using the software BOTTLENECK v. 1.2.02 Gene flow. The software BAYESASS v. 3.0 Genotype clustering. Population assignment tests were carried out using STRUCTURE v. 2.3.3 ITS and btub, for a subset of 37 isolates, selected to represent all clusters detected by the STRUCTURE analysis, five other genes were sequenced. These genes were the four nuclear genes enolase, heat shock protein 90 (HSP90), TigA gene fusion protein (TigA), the tryptophan biosynthesis gene 1 (trp1), and the mitochondrial gene cytochrome c oxidase subunit I (cox I). PCR products were amplified using the specific primer sets Fm84 and Fm83 cox I, Enl_for and Enl_rev enolase, HSP90_F1, HSP90_F3, HSP90_R1, and HSP90_R2 HSP90, Tig_for, Tig_F2, Tig_rev, and G3PDH_rev TigA, and Trp1F1, Trp1R1, Trp1F2, and Trp1R2 trp1. PCR reactions were conducted following the respective reference. For detailed methodology see In addition to Phytophthora Database (http://www.phytophthoradb.org). For details see Table B of File S1.DNA sequences have been submitted to NCBI Genbank (accession numbers KF443812-KF444041) as well as to http://code.google.com/p/jmodeltest2/) and DT-MODSEL http://mesquiteproject.org).JMODEL TEST , and the following substitution models were chosen: TN93 for btub, HSP90 and enolase, HKY for ITS, trp1 and TigA. A UPGMA starting tree was chosen as a tree prior and priors were linked for all trees. Furthermore, the option “coalescent: constant size” and a chain length of 250,000,000 (25% burn in) were selected. The prior for the clock rate was set to lognormal.The software BEAST v. 1.7.2 P. plurivora population, a total of 109 alleles were detected across the 11 microsatellite loci. All loci were polymorphic with two (locus PpMs11) to 30 (locus PpMs01) observed alleles. Allelic richness (Ar) and private alleles (Pa) were highest for populations US West Coast , France , and Eastern Europe . The lowest Ar and Pa values were observed in the US East Coast population  values ranged between 0.03 (Turkey) and 0.18 (France), with a mean value of 0.08 across all 12 populations. The index of association rBarD was significantly different than zero in six geographic populations , which indicates a significant deviation from random mating. In the other six populations, based on the index of association statistics the hypothesis of random mating could not be rejected.In the entire ST-statistic analysis revealed an overall population differentiation of FST = 0.102. FST-values between pairs of populations varied between 0.01 (BNL and UK) to 0.38 (BNL and TR), but none of the FST values were significant .FSignificant contemporary gene flow from Belgium and the Netherlands (BNL) to both the US East (EC) and West Coast (WC) was detected for the microsatellite data using BAYESASS. Furthermore, contemporary gene flow could be detected from BNL to the UK and to France (F). Additionally, significant migration from Germany to the Alps (Switzerland), to Eastern Europe (Czech Republic), to the Balkans (Slovenia) and to Turkey could be detected .TigA (7 haplotypes), followed by the nuclear gene regions btub and HSP90 (6 haplotypes), and by the mitochondrial gene cox I . The least variation was observed for the nuclear gene regions enolase (2 haplotypes) and trp1 (3 haplotypes). In all gene regions, a dominant haplotype was detected that was accompanied by a few, less common haplotypes. In all genes, no clustering of haplotypes according to their geographic origin was observed . On the other hand, for btub and trp1 US isolates are only characterized by the most common, central haplotype , but evidence for recombination was also detected in these two loci  increased more or less constantly until K = 10, without reaching a clear plateau . Therefoisolates . The firisolates  mainly cThe UPGMA Phenogram based on the Nei’s unbiased genetic distance P. plurivora population by conducting within-locus k tests. A significant number of negative k values were observed for 10 of the 11 loci  for the pooled European samples and for all 20 US samples pooled . The inter locus g statistic was higher than 1 in both populations , which is indicative of no population expansion.BOTTLENECK analysis using the Wilcoxon test revealed no significant bottleneck signals either in the US (p = 0.38) or in the European (p = 0.91) population for the microsatellite data. The mode shift test showed a normal L-shaped distribution for the European population and a shifted mode, which is indicative of a bottleneck, for the US population. A signature of recent population expansion was detected in the European as well as in the US Conducting Bayesian phylogenetic multi-gene analyses  mating system P. plurivora low levels of recombination should be present in the sequenced genes and that microsatellite data should reveal a high degree of inbreeding. Our results provide evidence of low levels of recombination given that five out of seven genes show no recombination (exception: TigA with two recombination events and HSP90 with one event) and a significant excess of homozygosity at all microsatellite loci. These results are further supported by our rejection of the null hypothesis of no linkage among loci in most populations as measured by the index of association.Like most P. alni subsp. uniformisP. sojaePhytophthora species that have been investigated so far using population genetic tools. However, P. alni subsp. uniformis and P. sojae present important ecological differences compared to P. plurivora. The first is most likely exotic to Europe and, thus, the low genetic diversity and the homozygosity excess observed at some loci may be a consequence of the fixation or loss of specific alleles after its introduction into the new continent. The latter is a pathogen of soybean in agricultural fields whose origin remains uncertain P. plurivora is expected to be native to Europe and the significant deficit of heterozygosity detected at all loci could support this hypothesis. In fact, if we consider that in self-fertile, diploid species heterozygosity should decrease by 50% in each generation Phytophthora species.High degrees of homozygosity were previously observed in Phytophthora exists. The presence of two genes (TigA and HSP90) showing clear traces of recombination events might suggest that in P. plurivora outcrossing, although rare, is or was possible. The potential to outcross was previously described for the homothallic species Pythium ultimum (oomycete) and for P. sojaeDifferent modes of homothallism are described in literature, including two nuclei in one spore, two either linked or unlinked mating types in one nucleus, two mating types in one nucleus that undergo mating type switching, or only one mating type present at all Phytophthora species are plant parasites and, thus, for survival rely on a host. For host species, vegetative reproduction and self fertilization is considered disadvantageous as clonal hosts, lacking genetic variability created through recombination, face an evolutionary disadvantage. On the contrary, outcrossing hosts may escape the pressure imposed by coevolving parasites through sexual recombination  we did not detect any sign of recent bottlenecks. Conducting two specific tests for microsatellite data using the software BOTTLENECK, however, we obtained contradictory results; on one hand, the Wilcoxon test indicated no visible traces of bottleneck, whereas on the other hand, the mode shift test suggested a significant bottleneck. The structure of the data set and the assumptions for the two tests may explain this particular situation. A bottleneck might not be detected if sample sizes are small, the individuals used are not entirely representing the populations, or if the population subject to a bottleneck is not completely isolated. Additionally, one assumption for these tests is that loci are under Hardy Weinberg equilibrium, which was not the case in the present study. Hence, larger samples sizes, especially for the US populations, would be needed to increase the chance of detecting bottleneck effects with microsatellite markers. In order to draw definite conclusions about the centre of origin of P. plurivora, further samples from other continents should be analysed, as the present study only includes samples from Europe and North America. Furthermore, the possiblity of an initial introduction to Europe from elsewhere and then from Europe to the US should also be considered.Microsatellite data also indirectly support a European origin of P. plurivora has been introduced into the US from Europe, most probably from Belgium and the Netherlands. No significant gene flow was detected between the two US populations, which suggests that two independent introductions from Europe most likely occurred. In mainland Europe, Germany seems to have been the most important P. plurivora source for the Alps, the Balkans, and Eastern Europe. Populations in Finland and Italy show low levels of gene flow with other European P. plurivora populations, but this might be associated with the small samples size of these specific populations. Due to significant differences in sample sizes between countries, we cannot exclude a bias in the migration analysis towards countries with large sample sizes. Therefore, to definitely confirm our hypotheses about gene flow among and from European countries to North America, a larger number of samples from both forests and nurseries from each country should be analysed. Our findings concur with previous studies that found that nursery trade is an important source for the spread of plant diseases. According to Liebhold et al. P. ramorum has been dispersed over long distances by means of nursery trade and shipments of infected host plants across the US and the US-Canadian border, as well as from Europe to the US. The Netherlands, Belgium and Germany are important producers and exporters of ornamentals worldwide P. plurivoraP. plurivora spread within Europe and from Europe to the US through infected plants coming from these three countries may therefore be well founded. Several US isolates considered in this study originated from natural environments  and residential areas, indicating that P. plurivora has already become established in the wild, both on the East and West Coast of the US. In a study on the fungal wheat pathogen Mycosphaerella graminicolaP. plurivora has spread mainly through nursery trade, we could hypothesize that the gene flow detected occurred during the past 50 to 100 years. This time frame coincides with global commercial trade expansion and when nursery trade between European countries as well as between Europe and the US has flourished.Based on our analyses, P. plurivora population in Europe. The European isolates analyzed belonged to four divergent microsatellite clusters. Only three and two of them were present on the West and East Coast of the US, respectively. The lineage completely missing in the US was associated with P. plurivora isolates obtained from German and Swiss nurseries, as well as with a few isolates originating from Turkish and Slovenian forests. Noteworthy, all isolates recovered from alder (Alnus sp.) stands belonged to the same lineage and were detected both in Europe and the US West Coast. By performing inoculation test, it would be possible to verify whether P. plurivora genotypes from this specific lineage are better adapted to alder species than genotypes from the other lineages.According to the high levels of gene flow observed, no clear pattern of geographic structure could be detected within the P. plurivora. Moreover, they show that this species was most likely introduced from Europe (Belgium and the Netherlands) to both the East and West coasts of the US. In mainland Europe, P. plurivora may have primarily been spread from German nurseries. The importance of human-mediated international trade for the dispersal of this pathogen is also reflected by the lack of geographic structuring in the P. plurivora population sampled in this study. None of the four genetic clusters detected among the analyzed samples corresponded to a specific geographic region. A recent population expansion was detected, which might be due to the human mediated establishment of P. plurivora in new environments or on new host plants. While strict regulations exist for quarantine organisms in international plant trade , indigenous pathogens and pests are usually not subject to such policies. Our analyses, however, indicate that the international plant trade is a major pathway of spread also for non-invasive plant pathogens. Therefore, we would like to emphasize that the unintentional spread of dangerous but non-regulated organisms with diseased plant material should not be underestimated. Similarly to quarantine organisms, non-regulated pathogens may negatively affect local ecosystems and long-term consequences for biodiversity still remain unknown.In conclusion, our genetic analyses support a homothallic mating system in the plant pathogen File S1Includes supplementary tables A, B, C and D. Table A: Origin and supporting information on the Phytophthora plurivora isolates used in the STRUCTURE analysis. Table B: Origin, gene bank accession numbers and supporting information on the Phytophthora plurivora isolates used in the coalescent analyses. Table C: FST values between all population pairs of European and US Phytophthora plurivora isolates. Table D: Supplementary information on the Phytophthora plurivora isolates used in the Haplotype Network analysis.(DOCX)Click here for additional data file.Figure S1Gene genealogy for Phytophthora plurivora. Coalescent-based gene genealogy for the three genes (a) ITS (37 sequences), (b) cox I (33 sequences) and (c) btub (32 sequences) generated using GENETREE bub (c) all haplotypes diverged at the same time. For (c) the oldest haplotype was selected by GENETREE based on likelihood scores. Note: At position 824 of the cox I region there was a incompatible site, leading to 5 haplotypes above, while for the analyses in (TIF)Click here for additional data file.Figure S2Additional information on STRUCTURE analysis. Membership to STRUCTURE Phytophthora plurivora isolates sampled from 16 study populations. (a) Population assignment barplots, (b) Change (mean over 10 runs ± standard deviation) of L(K) (i.e. posterior probability of the model according to the number of genetic clusters tested); (c) change in Δ K (i.e. ad hoc quantity related to the order rate of change of L(K) with respect to the number of genetic clusters tested). The chosen number of clusters was K = 4 (red frame).(EPS)Click here for additional data file.


Oreochromis niloticus) populations, across their natural distribution in Africa. While its large biogeographic distribution can cause genetic differentiation at the paleo-biogeographic scales, its restricted dispersal capacity might induce a strong genetic structure at micro-geographic scales.Reconstructing the evolutionary history of a species is challenging. It often depends not only on the past biogeographic and climatic events but also the contemporary and ecological factors, such as current connectivity and habitat heterogeneity. In fact, these factors might interact with each other and shape the current species distribution. However, to what extent the current population genetic structure reflects the past and the contemporary factors is largely unknown. Here we investigated spatio-temporal genetic structures of Nile tilapia (RST = 0.38 - 0.69). This result suggests the predominant effect of paleo-geographic events at macro-geographic scale. In addition, intermediate divergences were found between rivers and lakes within the regions, presumably reflecting relatively recent interruptions of gene flow between hydrographic basins (ST R= 0.24 - 0.32). The lowest differentiations were observed among connected populations within a basin (ST R= 0.015 in the Volta basin). Comparison of temporal sample series revealed subtle changes in the gene pools in a few generations (F = 0 - 0.053). The estimated effective population sizes were 23 - 143 and the estimated migration rate was moderate (m ~ 0.094 - 0.097) in the Volta populations.Using nine microsatellite loci and 350 samples from ten natural populations, we found the highest genetic differentiation among the three ichthyofaunal provinces and regions  (O. niloticus in Africa. The effects of paleo-geographic and climatic events were predominant at macro-geographic scale, and the significant effect of geographic connectivity was detected at micro-geographic scale. The estimated effective population size, the moderate level of dispersal and the rapid temporal change in genetic composition might reflect a potential effect of life history strategy on population dynamics. This hypothesis deserves further investigation. The dynamic pattern revealed at micro-geographic and temporal scales appears important from a genetic resource management as well as from a biodiversity conservation point of view.This study revealed clear hierarchical patterns of the population genetic structuring of  Further periods ,7-9. Spee.g. [e.g. [Cichlid fishes are well known examples of complex population genetic structure among African ichthyofauna. Lake-wide studies have revealed the impact of paleo-historical lake level fluctuations on genetic diversity (e.g. ). Small g. [e.g. ). To datg. [e.g. .Oreochromis niloticus , is an interesting model-species to study the interactions between intrinsic and extrinsic factors on the structure of the natural populations from a local and temporal to a broader biogeographic scale. This economically important fish has one of the largest natural distributions among African fresh-water fishes, covering the entire Nilo-Sudanian province , the Ethiopian Rift Valley province, the Kivu province, north Tanganyika province (Ruzizi) and the Northern part of the East African Rift Valley. This species shows an exceptional capacity of adaptation, which allowed its colonisation of a wide range of habitats from small forest rivers to large drainage and lakes, as well as alkaline pools with hot springs [The Nile tilapia,  springs ,14. The  springs  largely O. aureus into O. niloticus populations restricted to West Africa, whereas the 2 species are also sympatric in the Nile River. This introgression has certainly happened during the drastic water level fluctuations of the Pleistocene [i.e. Oreochromis spp.) within the natural distribution of O. niloticus has been reported so far [Due to its great interest for aquaculture and fisheries, the Nile tilapia and a few other tilapia species have been introduced outside their natural distributions . Introduistocene . But neid so far .i.e. maternal mouth-brooding and guarding) [As most of the cichlid fish, the Nile tilapia exhibits interesting and complex life history traits, and especially well-developed social behaviour ,14,17-19uarding) . These lsensu lato), whereas the Sudano-Sahelian region experienced dramatic climatic fluctuations with alternating humid and dry phases [From the paleo-geographic point of view, Africa has experienced severe hydrogeographic modifications since the Pleistocene. The East African Rift valley has been subject to many tectonic disruptions of the water basins with inversion of the course of some rivers in the Nile basin  to understand the current population genetic structure of O. niloticus in its natural habitat range in Africa and (2) to evaluate to what extend the population genetic structure was shaped by the paleo-geographic events and the current geographic connectivity at the different spatio-temporal scales. We also discuss potential roles of life history of the species in the species-range population genetic differentiation.In this study, we investigated the spatio-temporal genetic structure of ten natural populations of O. niloticus were sampled from ten geographic sites across Africa to maximise the representation of the biodiversity of the species, naturally spread over the vast Nilo-Sudanian and the Ethiopian ichthyofaunal provinces [Natural populations of rovinces ,30. In oO. niloticus, representing the Nilo-Sudanian ichthyo-province, including Sudano-Sahelian  and Nilotic regions (Turkana and Nile basins) and the Ethiopian Rift Valley province (Awash basin) [O. niloticus: O. n. niloticus (in West-Africa and Nile) O. n. vulcani (in Lake Turkana), O. n. cancellatus (in the main part of Awash basin) and O. n. filoa (in hot springs of the Awash basin)  ,31. Accoh basin) , this reMicro-geographic and temporal sampling on the Volta basin was conducted at three sampling localities along the hydrographic basin, of which two were sampled successively multiple times between 2001 and 2003  and heterozygosity (Ho = 0.17 - 0.34). All other Nilotic and Sahelo-Sudanian populations presented an overall higher level of genetic diversity , except for Niger revealing an intermediate level of diversity .The populations of the Awash basin showed the lower gene diversity based on both allelic richness . The test remained significant after Bonferroni correction for the two populations from the Volta basin constituted of temporal sub-sampling . Both populations showed significantly positive FIS values, suggesting a deficit of observed heterozygotes . The deficit of heterozygotes could be due to either i) null alleles, ii) a spatial Wahlund effect, or iii) non-random mating. After exclusion of the three loci potentially possessing null alleles (see detail in Methods section), however, four samples (i.e. two in Kpandu and two in Nynuto) still exhibited significant positive FIS values. Thus, null alleles are not the main cause of the disequilibrium. The possibility of the spatial Wahlund effect, namely artificial substructure from the pool of different fishing catches within the temporal samples, was tested using AMOVA. No significant differentiation among seine catches was detected , suggesting the absence of Wahlund effect at this level. To investigate a possibility of non-random mating within population and cohorts, we analysed the level of relatedness among individuals at both i) site and ii) temporal levels, and compared the distribution of the observed pairwise relatedness index (rxy) to simulated distribution expected under panmixia. At the site level, Nyinuto population presented an overall lower level of relatedness  than expected under panmixia, however the population from Kpandu did not differ from the null expectation. Furthermore, the majority of the temporal samples showed significant difference in level of relatedness than expected in a randomly mating population in both populations: two temporal samples showed higher average pairwise relatedness (i.e. KpN1 & NyF3) and one showed a higher variance of relatedness distribution (i.e. KpM2), while one sample showed a lower variance of relatedness distribution  according to the Evanno correction [At macro-geographic level, factorial correspondence analysis (FCA) discriminated three distinct groups of populations along the first two factorial axes  of the test suggested by Hardy et al. [STR, see Methods) is the most pertinent to describe genetic differentiation at macro-geographic scale The highest population differentiations were found between regions (Table P < 0.001). The alternative AMOVA model, with populations clustered according to taxonomy, showed comparatively less fitted variance partitioning, with lower percentage of variance explained by the clustering factor (47.3%). Although this result was highly significant, a larger part of the genetic variance was attributed to the between-populations within group component (9.1%). When samples were clustered by hydrographic basins, 49.8% of the genetic variability was partitioned among groups, and 3.5% was detected among populations within basins , revealing intra-basin heterogeneity.The significance , while Koka appeared more isolated, especially from Metahara . Within the Sudano-Sahelian region, 24.8% of the variance was observed between hydrographical basins (Table RSC = 0.002) indicating certain homogeneity within basins. Within the Volta basin, almost all of the genetic variance (~100%) was found within populations with very little differentiation between sample sites , while 90.6% for the Sudano-Sahelian region (n = 5). Within Ethiopian Rift valley province, the multivariate analysis widely separates the three populations across the first factorial plane  and 7.4% for Nyinuto .The highest genetic differentiation was found between geographic sites in the Volta basin  was estimated based on two focal populations in the Volta basin (Kpandu & Nyinuto). We used three different methods, based either on temporal variations of allele frequency within a population, compared the global gene pool of the basin [Ne) within population [Ne and the first temporal method (NeCLOSED) assume absence of immigration to estimate e N[Neopen) relaxes this assumption and assume connectivity between populations to jointly estimate e Nand immigration rate (m) [e Nranged between 31-143 for Kp and 23-34 for Ny. While different methods provided different confidence intervals , real e Nwould likely be in or around the above ranges (see discussion in [m) were similar for both populations .The effective population size   Table 6Ne was esssion in ). Estimai.e. 20-5 My BP) to the Pleistocene , numerous paleo-geographic (e.g. causing disruption of river drainages) and paleo-climatic events (e.g. humid and dry phase cycles), by changing the connectivity between the different hydrographical systems, have deeply affected African ichthyofauna [i.e. the Awash system), (2) the Nilotic region  and (3) the Sudano-Sahelian region (i.e. West-African basins) , and further remaining significant molecular variance among populations within basins (3.5%). This pattern is consistent with those observed in the few other fish species widespread across the Nilo-Sudanian area and studied by genetic markers, Tilapia zillii [Oreochromis aureus [Clarias gariepinus [From the Miocene  - compahyofauna . The prea zillii , Oreochrs aureus  and Clarriepinus . In partriepinus ,14, presriepinus ,24,40.O. n. cancellatus in the river and cold-water lakes (e.g. Lakes Hora and Koka), and O. n. filoa endemic to the hot spring areas (e.g. Lake Metahara). The lower genetic distance between Hora and Metahara (RST = 0.07) compared to Koka (RST = 0.43 and 0.47 respectively) does not reflect their taxonomic status (i.e. paraphyly of O. n. cancellatus). In addition, the description of O. n. filoa is mostly based on the reduced number of some meristic characters , which might be subject to developmental plasticity [O. n. filoa and O. n. cancellatus are two ecotypes, rather than distinct subspecies. However, further specific experiments, especially according to environment-induced developmental plasticity and mate choice preference, should be conducted to confirm this hypothesis.The Ethiopian populations exhibited the lowest levels of polymorphism within each population and the largest genetic divergence from the other ones Tables . The isoasticity . These rasticity ,43 supposensu lato, disconnection occurred between the Nile river and the currently endorheic Lake Turkana basin about 8,000 years ago [O. n. niloticus and O. n. vulcani respectively [RST = 0.26 - Figure In the Nilotic system ears ago ,44. Indiectively . HoweverO. aureus to O. niloticus involving the whole West African area, but not reported in any Nilotic population [The Sudano-Sahelian basins were isolated from the Nile River by multiple paleo-geographic events between 7,500 and 6,000 years ago . Althougpulation .The Chad-Chari system appears to have played a key role between the Nilotic and Sudanian biogeographic entities, providing an unidirectional connectivity for the ichthyofauna from the Nile to the West African drainages, via the Niger, during the periods of growth of the mega-paleo-Lake Chad ,24,40,45As expected, no signature of anthropogenic impact on population differentiation was identified in this study. Paleo-geographic and climatic events appear to be important factors for the current pattern of the genetic structure of Nile tilapia populations within its natural distribution across Africa.= 0.02 ± 0.02) and Nyinuto (= 0.03 ± 0.03) was lower than between sample sites , from the upstream Kou River, to the central Lake Volta (Kpandu) and downstream Volta River (Nyinuto). This suggests their recent differentiation and/or non-interrupted gene flow. The genetic differentiation within temporal series in Kpandu (e Nin Kpandu than in Nyinuto (Table m), obtained jointly with Neopen [e Nestimates together with relatively small confidence intervals indicates a good coherence and reliability of our estimations [e.g. variation of water level and salinity - E. Bezault pers. obs.), and/or more isolated, than a central basin population. Despite the potential limit associated to our sampling strategy, our e Nestimates consistently showed relatively small effective population size of the natural Nile tilapia populations relative to expected census population size  and a non-negligible dispersal rate.Our estimates of the effective population size from three different approaches consistently showed larger to Table . The esth Neopen , provideimations ,46. The FIS observed in both populations and the significant relatedness detected in some temporal samples . A shoaling behaviour involving relatives could for instance explain such a pattern. This behaviour has been observed in other populations of O. niloticus [Sarotherodon melanotheron [i.e. potential shoal) within temporal samples. A more direct effect of a non-panmictic reproductive system should be considered. Polygynandric mating system, generally exhibited, by maternal mouthbrooding cichlids, as O. niloticus, is generally associated with sexual selection and pronounced between-individual variations in reproduction success [The hypothesis of the effect of specific social and reproductive behaviour of the Nile tilapia emerges from the positive iloticus ,48 as wenotheron ,47. Thesnotheron . Furthernotheron . In the  success ,20. Such success . The rapPseudocrenilabrus multicolour victoriae, presenting relatively large distribution area with geographic structure being the main driver of population structure and gene-flow [i.e. 2 years), which has been associated with drastic fluctuation of hydrographic regime [P. m. victoriae in this study compared to effective population size estimated here for O. niloticus. Unfortunately no census population size estimate for any of these species is available to effectively compare the eN/N ratio, however, in the case of P. m. victoriae it cannot be ruled out that the larger effective population size observed is influenced by flooding regime.Furthermore the non-negligible dispersal rate detected in the present study opens up new research perspectives on cichlids. While sophisticated reproduction strategy and extended parental care are expected to increase territoriality and consequently prevent adult migration, our results suggest that the exceptional adaptive plasticity and riverine affinity of the tilapia could also be seen in its dispersal leading to larger migration rate than more specialised (stenotypic) cichlid species from East African Great Lakes ,20,51. Tene-flow . It has c regime . Larger O. niloticus, the genetic pattern observed from large geographic to temporal scales first reflects the hierarchical impact of paleo-geographic and climatic events as well as the impact of hydrographic connectivity on population genetic structure. Furthermore, the hierarchical pattern of genetic differentiation seems to reflect only bio-geographic events, without revealing any signature of anthropogenic impact. Compared to the most studied, highly radiating cichlid lineages, the morphologically and ecologically generalist species, as especially the tilapiines, [The distribution and pattern of genetic diversity of a species over its natural range is the result of multiple and interlinked factors, which acted through space and time. For the Nile tilapia, apiines, ,19, mighO. niloticus from ten geographic sites across Africa  were visually checked according to specific morphological characters [O. niloticus at the nuclear genome level [We collected 350 samples of ca Table . Our samaracters  and confaracters ,42,43. Saracters ,25,26, wme level .O. n. niloticus, O. n. vulcani, O. n. cancellatus and O. n. filoa) according to their eco-morphological characters [The sampling locations covered six hydrographic basins in Africa, belonging to the Sudano-Sahelian  and Nilotic  regions of the Nilo-Sudanian province and Ethiopian Rift valley (Awash basin) ichthyofaunal province ,30,31 anaracters .i.e. biased sampling among relatives), each temporal sample was composed of individuals taken from multiple seine catches.Analysis of micro-geographic and temporal variation was focussed on the Volta basin, where temporal sample series were collected repeatedly over a period of 18 months in two of the three geographic populations: Kpandu and Nyinuto (Table i.e. Kpandu and Nyinuto). (2) The micro-geographic and temporal analyses were conducted at the scale of the entire Volta basin, including the samples from the 3 sites along the basin (n = 206): one sample from the Kou River (upstream Volta basin), four temporal samples from Kpandu (on the Lake Volta) and three temporal samples from Nyinuto (downstream on the Volta River)  The macro-geographic analyses were conducted including all samples from the ten geographic populations considered  and non-biased expected (Hnb) heterozygosities were calculated for each locus separately and across all loci using GENETIX 4.03 [STAT 2.9.3 [FIS, was estimated across all loci, and statistical significance was assessed with permutation tests (n = 1000) using GENETIX. Corrections for multiple tests were performed using the sequential Bonferroni procedure [The total number of alleles (A), observed  sampling issues (e.g. Wahlund effect) or (2) impact of specific life history traits including a low dispersal or any social behaviour leading to a particular mating system. However, technical problems, such as null alleles, can also affect FIS. Thus, we first ran MICRO-CHECKER[FIS (data not shown).Heterozygote deficits, as indicated by O-CHECKER to estimRLEQUIN 2.0 [FIS values by calculating the degree of relatedness within samples using the pairwise relatedness index rxy of Queller & Goodnight [DENTIX[xy (Mrxy) in a sample was compared to that expected for a panmictic population. The observed variance of rxy (Vrxy) was also compared with the expectation from a panmictic population to test for the presence of multiple family groups within samples (which would increase the variance of rxy) [et al. [i.e. Kp or Ny) [We used analysis of molecular variance, AMOVA , implemeQUIN 2.0  to test oodnight  implement [DENTIX. The obs of rxy) ,65. For p or Ny) .a priori. Successive correspondence analyses were performed to group populations from the largest to the finest geographic scales investigated.Factorial correspondence analyses were performed using R  and the STRUCTURE v.2.3.1[i.e. respectively 11 for macro-geographic and 9 for micro-geographic and temporal analysis). The optimal number of clusters was assessed based on correction proposed by Evanno et al. [Similarly, genetic population clustering was assessed using E v.2.3.1,33 and co et al. .F-statistics [μ >m), this parameter generally is used to compare populations at small geographic scale. In contrast, the analogous R-statistics [et al. [F-statistics and R-statistics in the most objective way. We applied this test implemented in SPAGeDi [R-statistics or F-statistics) were computed using ARLEQUIN. The averaged genetic differentiation calculated over pairwise value between or within group of populations were noted Genetic parameters, providing a measure of the genetic differentiation existing between two populations, can be calculated using different estimators (for a discussion see ). The F-atistics , based oatistics  based on [et al.  to choos SPAGeDi  for bothFurther, AMOVA  was alsoeN) and migration rate (m) based on the spatio-temporal samples from the Volta basin. In order to reduce the sampling biases due to the small sample size, we excluded a temporal sample with the smallest sample size  from the estimation, and then retained only larger samples (n ≥ 28). We used three different approaches to estimate e N(see discussion in [eN-CLOSED) assumes no immigration [eN-open), relaxes this assumption and allows the joint estimation of e Nand m [Neopen of a given focal population, we pooled the samples from the other populations from the basin  to mimic the allele frequencies in the source population of all potential immigrants. The third approach (LD-eN), is based on the linkage disequilibrium method [EESTIMATOR[e Nwas calculated for each temporal sample and then averaged over them. A point estimate and the 95% confidence intervals were obtained for each population by each method.We estimated effective population size . ssion in , but theigration , assumptNe and m . For theNe and m . For them method  and implESTIMATOR. In thisEB, PB, XR, HA and JFB designed the research within the framework of the PhD project of EB; EB, YF, XR and AT collected the samples and data; EB and PB analysed the data; EB, PB, XR and HA led the writing. All authors read and approved the final manuscript.We would like to thank Eddie Abban and the team of the Water Research Institute (Ghana), Abebe Getahun (University of Addis Ababa), Pascal Bonnet & Géraud Laval (CIRAD/ILRI), the Office des Parcs Nationaux du Sénégal, Gabriele Hörstgen-Schwark (University of Göttingen), and Brendan McAndrew (University of Stirling) for their cooperation in obtaining samples. We also thank Stéphane Mauger, Gaëlle Chupot, René Guyomard (INRA), and Claire Billot (CIRAD) for their laboratory assistance; Agathe Bezault for the illustrations; Wendy Brand-Williams (INRA) for English improvement, and especially, Nicolas Poulet (ONEMA) for his help in statistical analyses and discussions about demography, Bernard Chevassus (INRA) and Mark Jobling (University of Leicester) for stimulating discussions, and Dylan Fraser , Irene Keller (EAWAG), Marta Barluenga , the editor Michael Hansen and the anonymous reviewers for their constructive comments. This study was supported by grants from the CIRAD, INRA & MENRT (France) to EB.Genotyping protocol. Detailed microsatellite genotyping protocol.Click here for fileGenetic diversity information per loci and population. Tables of genetic diversity estimators calculated per loci and population.Click here for fileAdditional Structure Analyses. Genetic Structure analysis at a) macro-geographic and b) micro-geographic and temporal scales, including plots of evolution of log-likelihood and individual cluster assignment for the different number of clusters K.Click here for file


Microcosmus squamiger (Ascidiacea) using microsatellite loci, mitochondrial DNA sequence data and 11 worldwide populations. Discriminant analysis of principal components, clustering methods and approximate Bayesian computation (ABC) methods showed that the most likely source of the introduced populations was a single admixture event that involved populations from two genetically differentiated ancestral regions - the western and eastern coasts of Australia. The ABC analyses revealed that colonisation of the introduced range of M. squamiger consisted of a series of non-independent introductions along the coastlines of Africa, North America and Europe. Furthermore, we inferred that the sequence of colonisation across continents was in line with historical taxonomic records - first the Mediterranean Sea and South Africa from an unsampled ancestral population, followed by sequential introductions in California and, more recently, the NE Atlantic Ocean. We revealed the most likely invasion history for world populations of M. squamiger, which is broadly characterized by the presence of multiple ancestral sources and non-independent introductions within the introduced range. The results presented here illustrate the complexity of marine invasion routes and identify a cause-effect relationship between human-mediated transport and the success of widespread marine non-indigenous species, which benefit from stepping-stone invasions and admixture processes involving different sources for the spread and expansion of their range.In recent years, new analytical tools have allowed researchers to extract historical information contained in molecular data, which has fundamentally transformed our understanding of processes ruling biological invasions. However, the use of these new analytical tools has been largely restricted to studies of terrestrial organisms despite the growing recognition that the sea contains ecosystems that are amongst the most heavily affected by biological invasions, and that marine invasion histories are often remarkably complex. Here, we studied the routes of invasion and colonisation histories of an invasive marine invertebrate  Selective forces and demographic processes have shaped community composition and biogeographic patterns of the world's ecosystems over millions of years of evolution Genetic studies have been recognized as crucial to uncover the pathways of the introduction of non-indigenous species (NIS), colonisation histories, and the origin of these introductions Most research studies which have successfully implemented ABC methods have focussed on terrestrial ecosystems, analysing species in their native environment The majority of the species responsible for marine invasions are from lower trophic levels Microcosmus squamiger , to infer the colonisation histories and routes of invasion of its world populations. We obtained a dataset comprising microsatellite and mitochondrial DNA (mtDNA) data from populations located within the vast distributional range of M. squamiger, and analysed them using a variety of analytical tools, including the ABC methods. We specifically compared different scenarios to understand: (1) whether the introductions were independent or not, (2) the origin of the colonisers, and (3) the sequence of the different introduction events worldwide.Here, we studied a sessile marine invertebrate, Microcosmus squamiger is native to Australia M. squamiger outside its native range might in fact have been from along the southern African coast. Millar Microcosmus exasperatus in surveys conducted between 1950 and 1956 along this coastline. However, considering that: 1) Both M. squamiger and M. exasperatus are widespread species that can easily be confused M. exasperatus has never again been reported along the coast of South Africa M. squamiger , 9.5 - 8.3 µL H2O, 1 U Taq polymerase (Promega) and 1 µL DNA. An initial denaturation at 94°C for 5 min was followed by 30 cycles consisting of a denaturation step at 94°C for 1 min, an annealing step at 53 to 57°C . We amplified by PCR six polymorphic loci  that had been isolated for this species IS) and estimated its significance (10000 permutations).We used the GenAlex programme v. 6.1 D estimator D has been advocated to overcome some of the shortcomings of conventional statistics such as FST or GST, as it performs better when the goal is to estimate genetic differentiation ST estimates for comparison, while a randomization test was used to test the existence, or lack thereof, of significant genetic differentiation for each population pair across all loci. These analyses were done using the Arlequin v. 3.5 programme P-values using the sequential Bonferroni method We assessed pairwise population differentiation using the microsatellite dataset and the In order to have a visual assessment of between-population differentiation, we performed a discriminant analysis of principal components (DAPC) adegenet package for R We performed DAPC using the The programme STRUCTURE v. 2.3 was used to detect the number (K) of genetically homogeneous populations in our microsatellite dataset M. squamiger, we designed a series of sets of evolutionary scenarios and analysed them with ABC methods using the DIYABC v. 1.0.4.41 programme In order to obtain relevant and detailed information of the routes of invasions and colonisation histories of M. squamiger has two overlapping generations per year based on what is know about its life cycle M. squamiger from surviving in this area. At the beginning of the Holocene, the global climate became warmer M. squamiger to thrive in this area. Consequently, we considered that the populations included in this study could have originated ca. 10000 years BP  to estimate the relative posterior probability  of each scenario with a logistic regression For all sets of scenarios we used 106 diploid individuals for N, Nau and Nu; Prior set 3) stepwise mutation model for microsatellite loci; and Prior set 4) no insertion-deletion mutation rate in microsatellites flanking regions.We assessed the sensitivity of different priors that included different effective population sizes and mutation models for the combined dataset of microsatellites and mtDNA. For this, we performed simulations using the first set of scenarios, which focussed on whether or not the colonisations within the introduced range were independent. Different prior sets were used to test the robustness of demographic estimates as follows: Prior set 1) standard priors as described in IS values were significant in all cases except for Bunbury  and three sites in Spain  . The mean allelic richness was significantly higher in native than in introduced populations . However, when we analysed the numbers of mtDNA COI haplotypes t-test, t = −1.62, P = 0.16). Both native and introduced populations showed similar microsatellite heterozygosity  and mtDNA haplotype diversity . When we compared the observed and expected gene diversity of the microsatellite alleles found in each population, the programme BOTTLENECK M. squamiger microsatellite dataset.A total of 268 individuals were genotyped using six microsatellite loci. The number of alleles per locus ranged from 4 at the MS7 locus to 18 for the MS10 locus . No linkrcelona) . The sigrcelona) . The poprcelona) . On averD in pairwise comparisons was smaller among introduced  than among native (0.227±0.058) populations, and the same was found for the FST estimates . After correcting for multiple tests, 38 out of 55 D values indicated significant genetic differentiation  as implemented in the DIYABC programme (data not shown). The results from the first set of scenarios convincingly showed that the most likely scenario was the one that included non-independent colonisations across ed range , Fig. 4Aed range , Fig. 4Bed range . To resoRegarding the third set of scenarios (i.e. sequence of worldwide introductions), no scenario could be conclusively selected because the confidence intervals of the scenarios with the highest posterior probabilities overlapped with the intervals from other scenarios . HoweverThe demographic parameters obtained for the most supported scenario (i.e. the scenario with the highest posterior probability) of each set of scenarios are shown in M. squamiger showed that the introduced populations have elevated levels of genetic diversity, similar to those found in native populations. The role of genetic diversity during the colonisation process has been at the heart of recent debates in invasion biology research M. squamiger indicated that the spread of introduced populations has not been limited by reductions of genetic diversity both in terms of nuclear (this study) and mtDNA The results of the present study are consistent with a scenario of high population connectivity among introduced populations and absence of genetic bottleneck. The analysis of microsatellite data of D or FST values, as well as with STRUCTURE and DAPC. Moreover, this genetic barrier was also found using FST statistics on mtDNA data from the same populations M. squamiger are needed to ensure a fine-scale analysis of this pattern.Strong genetic differentiation was detected among Australian populations using microsatellite loci, with a major genetic break between the population found on the eastern coast and the two western populations. This genetic differentiation was observed by pairwise comparisons, either using D and as FST) than those found when comparing native populations. Only 43% (D) and 29% (FST) of the pairwise comparisons among introduced populations showed significant differentiation in terms of allelic frequencies. A higher genetic similarity among introduced populations than among native ones was also shown by the clustering of these populations in the DAPC and STRUCTURE. This similarity among introduced populations was also reflected by the presence of some loci of low frequency alleles that were only shared among introduced populations  in inter-basin  comparisons were low , but much higher than those in intra-basin comparisons (0.006±0.005), which indicates that the Strait of Gibraltar (and associated intense shipping) plays a relevant role in structuring these populations.We found a strong link between the populations of the NE Atlantic Ocean and the Mediterranean Sea, while California and South Africa appeared as genetically separated regions. The close genetic relationship between the NE Atlantic Ocean populations and those from the Mediterranean Sea could be a matter of geographical proximity that enhanced connectivity both through commercial and recreational shipping In the present study we revealed important characteristics of the introduction process of a widespread marine species. Firstly, we obtained consistent evidence of the non-independent nature of the world colonisations. Subsequently, we found that an admixture event between populations from the east and west Australian coasts was most likely responsible for shaping the genetic composition of the introduced populations of the species. In addition, this admixture event resulted on an unsampled population that served as a stepping-stone between native and introduced populations. Finally, we found that the sequence for the introduced range followed the historical taxonomic records, with the first colonisations occurring in the Mediterranean Sea and South Africa, followed by California and thereafter in the NE Atlantic.We showed that the combined use of multilocus data obtained from samples of both native and introduced populations with new analytical methods allows for discrimination among complex evolutionary scenarios, and is a powerful approach to understand invasion histories of widespread marine organisms. The translocation of coastal marine species from one distant region to another is increasing Figure S1Set of scenarios used to infer the colonisation histories of Microcosmus squamiger using approximate Bayesian computation analyses: A) Independent vs non-independent colonisations, B) Origin of colonising populations, C) Sequence of worldwide introductions. The Y-axis indicates the prior time of events  as in (DOC)Click here for additional data file.Figure S2Plot of IncK as described in Evanno et al.  as a function of the number of clusters (K) across the 20 runs: (A) for the whole dataset, (B) for the native populations and (C) for the introduced populations.(DOC)Click here for additional data file.Table S1Prior distribution parameters describing the set of scenarios investigated for Microcosmus squamiger. The time priors were constrained (t1<t2<t3<t4<t5<t6) and comprised of split or admixed events. Nau: effective population size of the ancestral populations, Nu: effective population size of the unsampled bridgehead population, N: effective population size of each cluster of introduced populations, SNI: single nucleotide indel. All populations had the same prior regarding their effective population size.(DOC)Click here for additional data file.Table S2List of allele frequencies found in each population of Microcosmus squamiger. Population abbreviations as in (DOC)Click here for additional data file.Table S3IS) and its significance (significant results in bold) for each locus in each population of Inbreeding coefficient  per locus are indicated.(DOC)Click here for additional data file.Table S4Probability of Wilcoxon signed rank test to detect bottlenecks for the introduced populations of Microcosmus squamiger. Mutation model abbreviations are as follows: IAM - Infinite alleles model and TPM - Two-phase model.(DOC)Click here for additional data file.Table S5Demographic parameters obtained for the most supported scenario in each set of scenarios using microsatellite markers (see ). Demographic parameter abbreviations as in (DOC)Click here for additional data file.Table S6Robustness of prior choice in discriminating among introduction scenarios  and inferences of demographical parameters. All values correspond to the highest probability scenario in each prior set (i.e. Scenario 3).(DOC)Click here for additional data file.


Clistocoeloma sinense, designated as an endangered species due to the limited suitable saltmarsh habitat available, have decreased accordingly, being now represented as small remnant populations. Several such populations in Tokyo Bay, have been recognised as representing distributional limits of the species. To clarify the genetic diversity and connectivity among local coastal populations of Japanese Clistocoeloma sinense, including those in Tokyo Bay, mitochondrial DNA analyses were conducted in the hope of providing fundamental information for future conservation studies and an understanding of metapopulation dynamics through larval dispersal among local populations. All of the populations sampled indicated low levels of genetic diversity, which may have resulted from recent population bottlenecks or founder events. However, the results also revealed clear genetic differentiation between two enclosed-water populations in Tokyo Bay and Ise-Mikawa Bay, suggesting the existence of a barrier to larval transport between these two water bodies. Since the maintenance of genetic connectivity is a requirement of local population stability, the preservation of extant habitats and restoration of saltmarshes along the coast of Japan may be the most effective measures for conservation of this endangered species.During recent decades, over 40% of Japanese estuarine tidal flats have been lost due to coastal developments. Local populations of the saltmarsh sesarmid crab  Many estuarine ecosystems worldwide have been severely disrupted by human activities, such as overexploitation, pollution and land reclamation Clistocoeloma sinense Shen, 1933 is distributed throughout estuaries in subtropical and temperate eastern Asia, Japan The saltmarsh sesarmid crab Clistocoeloma sinense along the Japanese coast utilized mitochondrial DNA (mtDNA) analyses in order to clarify the genetic diversity and relationships/connectivity among local populations of the species. Based on the investigation, we discuss a focus on conservation strategies for the geographically isolated Tokyo Bay populations.This investigation of local population genetic structures of the endangered saltmarsh crab Clistocoeloma sinense was conducted by hand-digging on saltmarshes at 8 localities in inner Tokyo Bay and 13 localities on saltmarshes along the western Japanese main Islands from Shizuoka Prefecture to Saga Prefecture, and on Okinawa Island, thereby covering the entire geographical range of Clistocoeloma sinense in Japan. In total, 418 samples of Clistocoeloma sinense were collected from the 8 Tokyo Bay localities and 9 other localities  values based on uncorrected sequence differences FST/1–FST) against geographic distance (minimum coastline distance) between all pairs of sampling localities, using the Isolation by Distance Web Service (Version 3.23) The number of mtDNA COI haplotypes, and the haplotype and nucleotide diversities were calculated for each local population of n = 418, h) and nucleotide (π) diversity values tended to be very low at most localities, with values ranging from 0.0714 to 0.6087 and from 0.000135 to 0.001392, respectively. The Katsuura River population  exhibited the greatest genetic diversity (h = 0.6087 and π = 0.001392), followed by the population from Yoshida River  (h = 0.5421 and π = 0.001146). In contrast, almost all of the Tokyo Bay populations were characterized by low genetic diversity . The parsimony network of the mtDNA COI haplotypes of Clistocoeloma sinense consisted of two major haplotypes  and seven minor haplotypes, separated from the major haplotypes by a single mutational step . Pairwise FST values  and had the following pattern: (i) differentiation between Tokyo Bay populations  and central Honshu populations ; (ii) differentiation between central Honshu populations  and southern Shikoku and Kyushu populations , excluding the Yoshida River population ; and (iii) no differentiation between the Tokyo Bay populations  and southern Shikoku and Kyushu populations , excluding the Yoshida River population .The AMOVA analysis revealed significant overall population differentiation .In the demographic analyses , Tajima’Clistocoeloma sinense in estuarine saltmarshes located from central Honshu to Kyushu showed low levels of genetic diversity, as measured by haplotype and nucleotide diversities, compared with those of other intertidal brachyuran crabs  geographical limits of the species, were characterized by lower genetic diversity than the other local populations. Similar genetic patterns have been previously reported for other marine invertebrate populations representing distributional limits in Japanese waters, viz. the tideland snail FST analysis between the (i) Tokyo Bay populations and central Honshu populations and (ii) central Honshu populations and southern Shikoku and Kyushu populations, but not between the Tokyo Bay populations and southern Shikoku and Kyushu populations. According to the Isolation by Distance analysis .The AMOVA analysis revealed significant overall population differentiation. Differences were apparent by the analysis , howeveron range . To veriClistocoeloma sinense, indicating limited gene flow between distant local populations, despite their planktonic larval development Marine species are generally characterized by metapopulation structures that have a large population size, high dispersal capacity during the pelagic larval stages and extensive distribution Chaenogobius annularisDitrema jordaniDeiratonotus cristatusPeriophthalmus modestusEriocheir japonicaGaetice depressusChiromantes dehaaniIlyoplax pusillaThe genetic differentiation between geographically distant local populations in Tokyo Bay and Mikawa Bay, demonstrated herein, parallels similar examples of genetic differentiation, also shown by mtDNA analysis, in other marine animal species occurring in Tokyo Bay (including Sagami Bay) and along the coast of western Honshu, viz. the intertidal goby Clistocoeloma sinense has only three zoea and one megalopa stage Clistocoeloma sinense may have a relatively short planktonic life, it is likely that they remain near their parental population, with little chance of extensive dispersal. This may well have resulted in the genetic differentiation apparent among the regional populations. In the enclosed water body of Tokyo Bay, however, little genetic differentiation was apparent among the discrete local populations, suggesting that strong gene flow occurred between them via larval dispersal. Short distance larval dispersal in an enclosed water body may be adaptive for the maintaining of small local populations as a regional metapopulation.Each of the aforementioned intertidal brachyuran crabs have four or more zoea and one megalopa stages Clistocoeloma sinense were found along the coast between Tokyo Bay and Mikawa Bay (a distance of ca. 250 km), despite the existence of small saltmarshes habitat in some estuaries on Shizuoka Prefecture Chiromantes dehaani and Eriocheir japonica are, in contrast, distributed along this stretch of coastline, in Sagami Bay or Suruga Bay Clistocoeloma sinense is estuarine habitat specialist, utilizing only the saltmarsh habitat in the highest intertidal zone. The discontinuity of local populations of some other habitat specialists, apparently also a consequence of the steep rocky shore lined Izu Peninsula Chaenogobius annularis, Ditrema jordani, and Deiratonotus cristatus. Thus, suitable habitat continuity along the coast is important to Clistocoeloma sinense for the maintenance of local population networks of marine and brackish water habitat specialists.No local populations of Clistocoeloma sinense is suggestive of recent gene flow between the two populations. Unintentional human mediated introduction of the crab to Tokyo Bay from Kyushu is another possibility, as suggested by the genetic analysis of Japanese populations of the venerid bivalve Phacosoma japonicumPhacosoma japonicum appear to have been accidentally transported to Tokyo Bay from Ariake Bay (Western Kyushu) together with juvenile short neck clams intended for aquaculture on the tidal flats. Unintended introductions of animal species from foreign countries, including Phacosoma japonicum together with short neck clams, have been reported elsewhere, including the tidal flat purse crab Philya pisum and other tidal flat animals Clistocoeloma sinense, however, introduction mediated by short neck clam aquaculture is unlikely as these two species live in quite different habitats; the short neck clam lives in sandy sediment on middle to lower tidal flats whereas the crab lives in muddy sediment under higher salt marsh vegetation. Furthermore, aquaculture of transplanted short neck clam began post 1980s in Tokyo Bay Clistocoeloma sinense have been found in 1952 The similar genetic structures of the widely separated Tokyo Bay and Kyushu populations of Pyromaia tuberculata, Carcisus estuarii, Phithropanogeus harrisii and Acantholoblus pacificus, following their transportation in ballast water and/or ship hull fouling, have been found since 1980 Clistocoeloma sinense. The possibility of introduction of Clistocoeloma sinense in ballast water is also low, since most domestic Japanese ships before the 1950s did not have ballast water tanks. Accordingly, populations of Clistocoeloma sinense may have been established naturally before at least 1950, but reasons for the genetic similarity between the Tokyo Bay and Kyushu populations remain unknown. Furthermore, the low genetic diversity and demographic analysis results for these populations suggest that these largely separated local populations may have been established coincidently by the introduction of larvae from unknown another habitat. The haplotype similarity between the Tokyo Bay and Kyushu populations may have resulted from naturally occurring genetic drift. The present data, however, is insufficient for inferring population connectivity. Because microsatellite DNA analysis is likely to be a useful tool for estimating contemporary gene flow among populations, such should be considered in future studies aiming to elucidate the metapopulation structures of Clistocoeloma sinense.Ballast water and ship hull fouling are thought to be major vectors for the introduction of non-indigenous marine animals Clistocoeloma sinense, such being basic for future conservation and management of local populations of this species in Japanese waters. The Tokyo Bay regional populations of Clistocoeloma sinense may warrant special conservation status, as they are already exhibiting relatively low levels of diversity. By way of comparison, the tidal flat snail Cerithidea cingulata, formerly with a distribution pattern similar to that of Clistocoeloma sinense, occurring in both Tokyo Bay and Mikawa Bay until the 1990s, was not found in Tokyo Bay or along the coastline between the two bays Cerithidea cingulata populations still extant in Mikawa Bay probably cannot disperse to Tokyo Bay due to the lack of suitable interjacent habitat, as in the case of Clistocoeloma sinense.These findings provide the first substantial genetic information on the endangered sesarmid crab Clistocoeloma sinense through larval dispersal. However, the preservation of extant habitats, restoration of saltmarshes and creation of new suitable habitats around the coastline of Tokyo Bay would likely be the most effective measures for conserving this endangered saltmash species.There would appear to be few barriers to the continuation of gene flow between the Tokyo Bay populations of Figure S1Isolation by distance of Clistocoeloma sinense samples. Genetic distances (FST/1– FST) plotted against geographical distances  without data of Tokyo Bay .(TIF)Click here for additional data file.