Syncerus caffer) exhibits extreme morphological variability, which has led to controversies about the validity and taxonomic status of the various recognized subspecies. The present study aims to clarify these by inferring the pan-African spatial distribution of genetic diversity, using a comprehensive set of mitochondrial D-loop sequences from across the entire range of the species. All analyses converged on the existence of two distinct lineages, corresponding to a group encompassing West and Central African populations and a group encompassing East and Southern African populations. The former is currently assigned to two to three subspecies  and the latter to a separate subspecies (S. c. caffer). Forty-two per cent of the total amount of genetic diversity is explained by the between-lineage component, with one to seventeen female migrants per generation inferred as consistent with the isolation-with-migration model. The two lineages diverged between 145 000 to 449 000 years ago, with strong indications for a population expansion in both lineages, as revealed by coalescent-based analyses, summary statistics and a star-like topology of the haplotype network for the S. c. caffer lineage. A Bayesian analysis identified the most probable historical migration routes, with the Cape buffalo undertaking successive colonization events from Eastern toward Southern Africa. Furthermore, our analyses indicate that, in the West-Central African lineage, the forest ecophenotype may be a derived form of the savanna ecophenotype and not vice versa, as has previously been proposed. The African buffalo most likely expanded and diverged in the late to middle Pleistocene from an ancestral population located around the current-day Central African Republic, adapting morphologically to colonize new habitats, hence developing the variety of ecophenotypes observed today.The African buffalo ( Alcelaphus buselaphus) and the common warthog (Phacochoerus africanus) comprise three lineages associated with West-Central, Eastern and Southern Africa Connochaetes taurinus), the topi , the greater kudu (Tragelaphus strepsiceros), and the wild dog (Lycaon pictus) comprise at least two lineages associated with Eastern and Southern Africa Kobus kob), the African lion (Panther leo), the roan antelope (Hippotragus equinus), the waterbuck (Kobus ellipsiprymnus), the savanna elephant (Loxodonta africana) and the bushbuck (Tragelaphus scriptus) Aepyceros melampus) which has a distinct South-West African lineage Hippotragus niger) which has two East African lineages, a South-West lineage and a Southern lineage i.e., climatic oscillations), paced by earth’s orbital variations The phylogeographic pattern of most of the savanna mammals distributed across Africa can be partitioned into two to four main lineages. These lineages are typically associated with a West-Central, Eastern, Southern and/or South-East African distribution. For example, the hartebeest (Syncerus caffer) at a continental scale. The African buffalo is a good model because, together with a few other large taxa such as e.g. the elephant and the giraffe, it is distributed throughout most of sub-Saharan Africa. The species is primarily found in savannas, but also occurs in other habitats, including clearings in the rainforest belt S. c. nanus), which occurs in the West and Central African rainforests, shows adaptation to forest life S. c. caffer) of the South-East African savannas is about twice the size of the forest buffalo, with large downward curved horns and a brownish to black color. The third and fourth subspecies–not recognized by all authorities–are the West African savanna buffalo (S. c. brachyceros) and the Central African savanna buffalo , which are more difficult to distinguish morphologically. They occupy the Sahelian savanna, characterized by a low net above ground primary production compared to the East and Southern African savannas (±15% lower) S. c. caffer and S. c. nanus in captivity, these being the two subspecies with the more divergent morphological characteristics nanus and the caffer phenotypes have been reported, with S. c. caffer having 52 chromosomes and S. c. nanus having 2n = 54 to 56 chromosomes Phylogeographic studies that accurately reflect the pattern of spatial genetic variation require access to large sample collections distributed across the entire range of the target species. Perhaps because of the difficulty in obtaining samples over such a large geographic area, as well as the inaccessibility of remote areas in Africa, it is rare to find studies involving more than 200 samples or which cover the entire distribution of the model species. To improve our understanding of African biogeography, we studied the spatial genetic structure of the African buffalo (S. c. caffer), using various molecular markers such as mitochondrial DNA, autosomal microsatellites, Y-chromosomal microsatellites and the histocompatibility complex (MHC) DRB3 gene S. c. caffer and S. c nanus/S. c. brachyceros. No significant genetic differentiation was found between S. c. nanus and S. c. brachycerosAlthough several genetic studies on African buffalo have already been conducted, these were generally focused at smaller spatial scales, mainly in East and Southern Africa (D-loop (control region) sequences. The control region is the most variable part of the mammalian mtDNA genome In the present study, a detailed genetic analysis of the African buffalo across its geographic range was performed using mitochondrial i.e., nanus ecophenotype , brachyceros ecophenotype , aequinoctialis ecophenotype  and caffer ecophenotype .As the taxonomic status of the subspecies is still subject to controversy (see above), we defined each of the four putative subspecies as recognized by East S. c. caffer ecophenotype (136 haplotypes). This predominantly South-Eastern lineage also includes seven haplotypes of West-Central (WC) African origin. With the exception of the Namibian samples, haplotypes from Southern Africa  occupied the tip position in the network. About 45% of the Ugandan haplotypes occupied a central position in the network. The second geographic lineage (H2) consisted mainly of West-Central (WC) African samples, including samples from three ecophenotypes: S. c. brachyceros, S. c. nanus and S. c. aequinoctialis. These ecophenotypes were not monophyletic and were distributed throughout the H2 lineage. H2 included 94 distinct haplotypes, including seven of SE African origin.Two lineages that made geographic sense were retrieved with the minimum spanning network . At the population level, low overall sequence divergence among the populations within each lineage was observed, ranging from 0.1% to 1.3% in H1 and from 0.1% to 2.9% in H2. According to the pairwise STF values  can be observed, compared to higher STF values between populations between each of the lineages. Fixation indexes between populations between lineages (STF) were estimated to be 0.52, and among populations within lineages (SCF) to be 0.17 , being on average 1.4 times higher than in the caffer ecophenotype populations. The largest distances, up to 2.9% (higher STF = 0.47 (S. c. nanus). The nanus ecophenotype also displays the lowest haplotype diversity, being on average 1.3 times lower than in the other populations.Nucleotide diversity is lower for H1 (4.6%) than for H2 (6.4%), except for the Campo-Ma’an population  and 2. TT = 0.47 ), were fFs values as well as the raggedness index were significantly negative  which suggests a demographic expansion for both lineages. According to the τ values , demographic expansion for H1 was estimated as starting approximately 48 000 YBP (Year Before Present), and H2 approximately 104 000 YBP.The mismatch distribution analysis showed a predominantly unimodal pattern for both lineages . In addinegative   (p = 0.05). The ancestral effective population size based on the same dataset  was significantly smaller than the current effective size (p<0.00001), indicative of an important population expansion event .The divergence time between the H1 and the H2 lineages under the IM model was estimated at 193 kyr (95% CI: 145 kyr–449 kyr). With the BEAST reconstruction, the divergence time at the root of the tree was estimated at 273 kyr, with a divergence between a Ugandan and a Chad lineage, situated within the confidence interval of the divergence time estimated considering gene flow between both lineages over time. This gene flow was estimated at around five female migrants per generation (95% CI 1–17) with IM. Gene flow did not appear to be directional (p≥0.49) . The effon event . A relatCT = F0.42). Van Hooft S. c. caffer for the SE populations, and S. c. nanus for the WC populations. According to these genetic results, S. c. nanus would include S. c. brachyceros and S. c. aequinoctialis subspecies following standard nomenclature rules . This is further substantiated by the low amount of genetic differentiation between S. c. brachyceros, S. c. aequinoctialis and S. c. nanus. Although inferences based on DNA data are somewhat hampered because of the time required for complete lineage sorting to occur, our data unequivocally show that the separation between S. c. brachyceros, S. c. nanus and S. c. aequinoctialis is not taxonomically equivalent to the split between all of these and S. c. caffer. We note that these are preliminary genetic results as they are based exclusively on single-locus mtDNA. No final conclusion can be drawn concerning the taxonomic status of these subspecies before using complementary nuclear markers.Our study, based on the mtDNA genetic structure points to a genetic discontinuity between the WC (mainly lineage H2) and the SE (mainly lineage H1) populations , with morphological intermediate characteristics, as also proposed by Grubb The absence of genetic structure within H2 based on the analysis of the mitochondrial DNA -contrasting with extreme phenotypic variability- also suggests that buffalo may rapidly adapt (in evolutionary terms) to different ecological conditions, with ecophenotypes not being reproductively isolated. Finer scale resolution of the connectivity between the three ecophenotypes of WC Africa should be investigated using more sensible molecular markers as microsatellites or single nucleotide polymorphism. Besides, based on our results, the fourth species described in Groves and Grubb S. c. caffer and S. c. nanus are very different, the amount of genetic differentiation is typical of that of subspecies, especially when compared to the range of CTF values observed for other large African bovids  Although the morphology, behavior and respective habitats of n bovids . The mosHippotragus equinus) or the bushbuck (Tragelaphus scriptus) The separation into two lineages, distributed in WC and SE Africa respectively, is a pattern observed in many other savanna African mammal species, as for example within the roan antelope (Loxodonta africana), 1.4%; bushbuck (Tragelaphus scriptus), 11.7%; roan antelope (Hippotragus equinus), 1.6%; waterbuck (Kobus ellipsiprymnus), 4.1%; impala (Aepyceros melampus), 2.8%; Grant’s gazelle (Nanger granti), 10.9%) nanus ecophenotype populations are in comparison relatively small and isolated, as indicated by low haplotype diversities and high between-population genetic distances. Pairwise FST values between the nanus ecophenotype populations were about three times as large as those between populations within the other ecophenotypes  confirm our assumption Even if the buffalo would show strong philopatric behavior S. c. caffer subspecies originates from the isolation of a subpopulation of the ancestral population. Fossil records from the late Pleistocene are in agreement with this. Indeed, the close resemblance of the Pleistocene-dated fossils to the actual West-Central African buffalo suggests that the H2 population existed before the H1 population S. c. caffer), which possibly derived from a stock of savanna buffalo originating from West-Central Africa (H2 lineage) According to the IM analysis, although this result was not significant, the split parameter suggests that the The coalescent approach with migration suggested a Middle to Late Pleistocene divergence (150–300 kyr), which is contemporaneous with previous studies of other African species Fs index for both lineages. This is in agreement with two earlier studies based on S. c. caffer, using an alternative coalescent approach with Bayesian skyline plots τ values, was estimated as starting at approximately 48 000 YBP for the H1 lineage, consistent with a previous study on the Cape buffalo by Van Hooft Peloveris antiquus), which dominated the African savanna until the late Pleistocene, as demonstrated by fossil data We also found evidence under the IM model of an important expansion in both lineages since their Pleistocene divergence, which is supported by the star-like topology of the haplotype network of the H1 lineage and the negative Fu’s Syncerus-like species occupied Southern Africa earlier than we could infer based on mtDNA. Our signal of expansion toward Southern Africa could thus be a signal of re-colonization, which supports the hypothesis of the importance of the refugia located in Eastern and Western Africa. Southern Africa could have witnessed multiple colonization-extinction events, following habitat suitability.Nevertheless, recent radiometric studies on fossil records support the presence of buffalo in Southern Africa around 542 kyr (95% CI: 435–682 kyr) Podocarpus forest during favorable periods Lineage H2 on the other hand was inferred to have expanded earlier, at approximately 104 000 YBP. More varied and open habitats prevailed after 1.8 Ma in subtropical Africa S. c. caffer located in Eastern Africa appears to have played an important role in its history, when climatic conditions were unfavorable. Migration from a core Eastern refuge into other parts of SE Africa was found to have happened several times, as these other locations are positioned at multiple separate external branches, dated independently, on internal Ugandan branches, probably related to the climatic oscillation registered during Pleistocene. Further support is found in the network reconstruction, which reflects the same pattern, with Southern African haplotypes forming sub-groups positioned at tip positions, without being monophyletic. Our analysis of the quantification of the strength of connectivity between geographical states in the tree, which attempted to identify the historically most important migration routes, identified strong support for a Uganda-Tanzania and a Tanzania-Kenya link, but not for a Uganda-Kenya link. Thus, a central role of Tanzania in this Southern expansion was also highlighted.The Eastern region around the present-day Uganda appears to have played a prominent role throughout the history of the H1 lineage. Many internal branches had high posterior probabilities for the geographical state of Uganda, and the earliest occurrence of a non-Ugandan branch in H1 was 83–103 kyr , roughlyi.e., Western refuge). The genealogy of H2, with its longer branch lengths, indicates that H2 underwent a demographic expansion earlier than H1, a fact corroborated by the mismatch distribution profiles. For H2, Cameroon and Gabon hosting forest buffalo ecophenotypes form migratory cul-de-sacs connected to CAR, indicating that these populations are the result of a unique migration route from CAR, which is different from the migration route between CAR and the Western savanna buffalo populations. This is consistent with S. c. brachycerosThe H2 lineage showed a less clear geographical pattern with an earlier geographical diversification from a core population in Chad, Cameroon or the Central African Republic (CAR)  was positioned well within H2. This shows an important migration connection between West-Central and West-Southern Africa, which has been proposed before Pelorovis antiquusS. c. caffer is supported by the lack of true Cape buffalo characteristics, i.e., the sweeping horns and pronounced horn boss, in all buffalo fossils from this region In summary, the pattern in i.e., Management Units as defined by Moritz S. c. caffer) and the Western African buffalo (S. c. nanus), which has important implications for the conservation of the species. We found little genetic structure distinguishing the three morphologically distinct ecophenotypes of the Western African buffalo, hence we posit that these ecophenotypes merely represent rapid adaptations to local habitat variations without reproductive isolation. Our results also suggest that the forest buffalo ecophenotype may be an adapted form derived from the West-Central savanna ecophenotype. The more important phenotypic variability observed in West-Central Africa could be the result of an earlier origin of the Western lineage than of the Cape buffalo. More genetic clusters in the Western lineage that very recently diverged may also exist and could be identified using finer genetic markers. We have demonstrated that extensive sampling from the whole distribution of a species makes it possible to infer important aspects of historical migration, refugial areas and taxonomic subdivision.Our main finding is that African buffalo should be partitioned into two MUs , Central , East  and Southern Africa  was analyzed. Details and location of the samples are shown in 2 of the card in 240 µl water and heating this to 90°C for 10 minutes. Following this, 120 µl of the solution was transferred to a new tube together with 480 µl Puregene Cell Lysis solution and 3 µl of a 20 mg/ml proteinase K solution. The remainder of the protocol followed the manufacturer’s instructions.Blood, hair, tissue and dung samples were kept in ethanol solution and stored at ambient temperature, except the dung samples from Ghana, Sierra Leone and Benin, which were collected on Whatman FTA cards D-loop hypervariable region (HVR) and adjacent regions were amplified in the majority of samples using forward L0 (5′- CCCAAAGCTGAAATTCTACTTAAACTA-3′) and reverse S0 (5′-TCTAGGCATTTTCAGTGCCTTGCTTT-3′) primers 5′-AATATAAAGAGCCTCCCCAG-3′) and reverse primer Dsca2 (5′-CGGCCATAGCTGAGTCC-3′) 96 Cleanup kit (Millipore) and sequenced using a DETT sequencing kit according to the manufacturer’s specifications . The resulting sequence PCR products were cleaned with a Montage SEQ96 Sequencing Reaction Cleanup kit (Millipore).The Bubalus bubalis, the Asian water buffalo, the closest relative to Syncerus caffer, were used as an outgroup for our study .Newly sequenced individuals are registered on the National Institutes of Health (NIH) genetic sequence database . Previously sequenced data are available on the same sequence database . Seven sequences of ETAS2 (Extended Termination Associated Sequence) e.g.i.e., missing data) were removed, which resulted in a final dataset of 195 unambiguous characters. The dataset comprised 116 mutations of which 109 were parsimony informative, with a large number of the variability associated to rare variants that differed in just one nucleotide site each. The transition/transversion ratio was estimated at 9.55∶1.The resulting sequences were read and aligned using CLUSTAL_X A haplotype network was constructed using the minimum spanning network method (MINSPNET in ARLEQUIN v.3.1) F-statistics was assessed using 1 000 random permutations. For all populations where the number of included samples exceeded four, we calculated the number of haplotypes, haplotype diversity h and nucleotide diversity πThe hierarchical distribution of genetic variance among and within populations was assessed using an analysis of molecular variance (AMOVA) on the basis of individual nucleotide frequencies. We also performed pairwise comparisons of individual nucleotide frequencies between populations using Wright’s F-statistics, as implemented in ARLEQUIN v.3.1 r computed under a population growth-decline model in ARLEQUIN. The P-value of the test was approximated based on the fraction of times the real data showed a lower value than the simulated data. The timing of demographic expansion can also be roughly estimated by the mode of mismatch distribution τ expressed as τ = 2 µt, where t is the expansion time in number of generations and μ is the mutation rate for the whole sequence D-loop as estimated by Shapiro Fs and Tajima D statistics Past demographic history of each of the buffalo lineages was inferred by a pairwise mismatch distribution analysis between individuals i.e., historical migration patterns) through a proper Bayes factor test rather than by parsimony analysis i.e., above the level of the sampling location)  to incorporate geographical separation of the samples in the analyses. A strict molecular clock was applied with a normally distributed substitution rate prior, with 95% of the probability density between 2.3×10−7 and 4.1×10−7, as estimated by Shapiro Bison priscus. Analyses were conducted assuming a constant population size in order to avoid over-parameterization. The best fitting nucleotide substitution model, according to the Akaike information criterion (AIC) BEAST v1.6.1 ocation) . This yih1 = 0.8 and h2 = 0.9, applying metropolis coupling using a 10 chain geometric heat mode with 45 chain swap attempts per step. The final prior values used for respectively current population size, ancestral population size, migration rate, lower limit divergence time, and upper limit divergence time were currentq = 70, ancestralq = 8, m = 0.12, w = 7 and t = 40, respectively. The splitting parameter s was included, migration rates were estimated in both directions and the HKY substitution model was applied.An isolation-with-migration (IM) model for two closely related populations or ecophenotypes was applied using the IM program Due to computational constraints, 360 samples were randomly selected, 180 from West-Central and 180 from South-Eastern Africa. Three simulations with 360 samples were performed, each using a different random subset of samples and a different random seed number. All populations within a specific group were represented with a random selection of samples, while equalizing sample size per population as much as possible resulting in the following scheme: West Africa (90 samples), Cameroon and Gabon (54 samples), Chad, Central African Republic and Democratic Republic of Congo , Uganda , Kenya and Tanzania (54 samples) and Southern Africa (90 samples). Countries close to the border between two lineages were defined as distinct groups to get maximum sample representation from the presumed center of the divergence.


Maculinea arion) from two distinct and geographically distant parts of its European distribution , which fully represent the ecological and morphological variation occurring across the continent.Climatic oscillations are among the long-term factors shaping the molecular features of animals and plants and it is generally supposed that the rear edges  situated closer to glacial refugia are vital long-term stores of genetic diversity. In the present study, we compared the genetic structure of several populations of an endangered and obligate myrmecophilous butterfly  and the EF-1α nuclear gene and found substantial genetic differentiation among M. arion observed in Italy and in Poland may be explained by the rear edge theory. Although we were not able to pinpoint any specific evolutionarily significant units, we suggest that the Italian peninsula should be considered as a region of special conservation concern and one that is important for maintaining the genetic diversity of M. arion in Europe. The observed pattern of mtDNA differentiation among the populations could not be explained by an endosymbiotic infection.Differences in the genetic structure of  The currently observable molecular biogeography of animals and plants has been historically shaped by the evolutionary history of individual species and recently by their distinctive demographic features. The most important long-term factors that influence European molecular biogeography are related to climatic oscillations . The PleMaculinea arion , which was potentially able to survive glaciations at the base of the Alps and, at the end of the Ice Age, was able to either re-colonise the higher altitudes following shifts of its habitat or adapt to the new biotopes in the lowlands [Hampe and Petit  suggest lowlands .M. arion lives as an obligatorily myrmecophilous species, and its survival depends on the concurrent presence of two types of resources: specific food plants and specific host ants. Its caterpillars, after spending a short period (10-15 days) feeding on Thymus spp. or Origanum vulgare, leave the plant and reach the ground at the beginning of their fourth  instar. There, larval survival is limited by required discovery by a forager ant of the genus Myrmica, which adopts it and takes it to its nest where the larva will spend winter and complete its development by feeding on the ant’s brood [’s brood . Sociall’s brood ,14 and aeriod 10- days fee’s brood .M. arion sensitive to subtle environmental changes; therefore, this species is now threatened in many countries. M. arion became extinct in Britain, and it was later successfully reintroduced after full habitat restoration [M. arion has worsened during the last decade, from near threatened [M. arion is listed in Annex IV of the Habitat Directive and is considered an important indicator of habitat quality and stability as well as an umbrella species for certain types of grassland communities [M. arion inhabits various types of warm and dry grasslands at elevations between 50 and 2500 m [Its complex life cycle makes toration . On a Eureatened  to endanreatened . M. ariomunities ,21 as itmunities . In Eurod 2500 m ,25.M. arion populations from two distinct and distant parts of this species’ European distribution , which fully represent the ecological and morphological variation of the butterfly across the continent. The Polish populations, which inhabit xerothermic meadows, occur on southerly exposed slopes or sandy flat areas, and exploit Thymus spp. as larval host plants (LHP), are usually classified as M. arion arion. In Italy, a more complex pattern is observed with three recognised subspecies. The first subspecies is M. arion obscura , which is characterised by its small size and dark colours, is observed at high altitudes in the Alps and exploits Thymus spp. as LHP. The second, M. arion ligurica , is bright blue in colour, inhabits low altitude grasslands, and exploits Origanum vulgare as its LHP. Finally, M. a. arion encompasses all other populations and Thymus spp. dependences. High variation in host ant use is observed in both countries, as immature butterflies were found with a total number of eight Myrmica species [In the present research, we analysed and compared the genetic differentiation of twenty  species ,21,24,25Maculinea arion and suggested that the lineages originated from different southern refugia during last glaciation. We sequenced the mitochondrial gene cytochrome oxidase subunit I (COI), known as the ‘barcoding gene’ [Wolbachia, whose occurrence has been reported in several lycaenid butterflies including another representative of the genus Maculinea  [Wolbachia infection . Therefore, inspection of M. arion samples for Wolbachia is important for the interpretation of observed patterns because the presence of this endosymbiont can often confound the inference of the evolutionary history based on mtDNA data.Considering the presence of putative subspecies and the fact that Italian populations were potentially able to survive glaciations whereas Poland is a postglacial re-colonisation area, one should expect differences in the populations’ genetic structures. In this regard, we tested two competing hypotheses. The first hypothesis stated that “rear edge” populations  have retained higher genetic variability and are more differentiated from each other than Polish populations occurring in a recently colonised, more continuous, range; the second, the centre-periphery hypothesis ,27, statng gene’ , and theng gene’ ,32. Addia alcon) . This ena alcon) . The hetM. arion adults were captured, and a middle left leg was removed from each individual. After this procedure, the butterflies were immediately released, and the collected legs were stored in 99.9% ethanol. For every population, 5 to 25 samples were obtained.We sampled nine populations that were separated by distances between a minimum of 42 km between VAL and CUN and a maximum of 920 km between VFE and CER Table . In the M. a. ligurica exploits Origanum vulgare as LHP ; mountain pastures in the Alps (1400 - 2200 m a. s. l.) where M. a. obscura is dependent on Thymus spp. ; and clearings or pastures in the Apennines (1000 - 1200 m a. s. l.) where M. a. arion uses Thymus spp.   where M. arion’s distribution in the country and the sampling included eleven Polish populations. The distances between the Polish populations ranged from 8 km at a minimum (KLU and SRO) to a maximum of 460 km (GUG and SRO). The localities can be divided into two types: dry grasslands (often clearings in pine forests) on sandy flat areas where Thymus serpyllum was used as LHP and xerothermic grasslands on southerly exposed slopes with T. pulegioides . Both primers were used for sequencing both strands. The sequencing products were run on an ABI PRISM 3130 capillary automated sequencer (Applied Biosystems).Genomic DNA was extracted using a Genomic Mini Kit . The Ron and Hobbes primers were used for the COI mtDNA fragment amplification, and ef51.9 and efrcM4 primers were used for the EF-1α gene . PCR wasWolbachia in M. arion was performed with the same amplification protocols using the W-Specf and W-Specr primers [In addition, PCR to amplify the 16S rDNA to test for the presence of  primers . We screNh), haplotype diversity (h), nucleotide diversity (π), and the number of segregating sites were calculated using ARLEQUIN 3.11 [FST values in ARLEQUIN 3.11 [FST values for the COI and the EF-1α genes was ascertained by 1000 permutations. The isolation by distance (IBD) pattern was tested by comparing the genetic differentiation between populations, as measured by pairwise FST/(1- FST), to the logarithm of geographical distance using IBD software [ΦST). To explore patterns of genetic divergence in more detail, we applied the spatial AMOVA procedure using SAMOVA ver. 1.0 [FCT value based upon 10000 simulated annealing steps.DNA sequences were aligned in BioEdit v 7.0.4  and reviUIN 3.11 . RelatioUIN 3.11 . PopulatUIN 3.11 . The sigsoftware . Analysisoftware  was perfsoftware   to NST (based on haplotype frequencies and distance between haplotypes), as described by Pons and Petit [To test the phylogeographic structuring of haplotype distributions, we compared the average nd Petit , using Dnd Petit .M. arion were fixed for the same haplotype (H1). Moreover, H1 was the only haplotype shared between the two countries  were obtained for the 804-bp fragment in 225 individuals. We found twelve (eight singletons) haplotypes (GenBank accession nos. KC316050 - KC316061), which were defined by thirteen variable sites (eleven transitions and two transversions). The haplotypes differed by one to five substitutions. Possible relationships between the COI mtDNA haplotypes were estimated as a network, as shown in Figure π) and haplotype (h) diversity values were estimated at 0.12% and 0.480, respectively. In the Italian populations, the average π and h were 0.17% and 0.651, respectively. For M. arion in Poland, the corresponding values were much smaller  because the polymorphism was found only in the SRO population that had only two haplotypes (one being a singleton)  differed by a single substitution from the Wolbachia sequence found in the chrysomelid beetle Diabrotica virgifera[All samples, regardless of the population origin, were positive for virgifera and therNuclear DNA sequences EF-1α) were obtained by comparing a 460-bp fragment from 263 individuals. Altogether, 26 segregating sites  comprised the 30 haplotypes (17 singletons) (GenBank accession nos. KC316062 - KC316091) in the combined samples. Of these 30 haplotypes, the Italian populations contained 18 (4.56 haplotypes per population on average) and the Polish contained 17 (average per population 3.73) Table α were obπ for the pooled sample was 0.21% (range: 0.02% - 0.47) and haplotype diversity was 0.632 .Only five out of the 30 EF-1α haplotypes (17%) were shared between the two countries. For all populations studied, the haplotype numbers ranged from two in the Mt. Cervati (CER), Sowlany (SOW) and Piaski (PIA) populations, up to seven in the Colle delle Finestre (CDF) populations and eight in the Hutki Kanki (HUT) population . The pairwise FST values between the populations were highly variable and ranged from 0.000 to 0.955 . However, in Poland, the average genetic differentiation at the COI gene among all the populations was very low and not statistically significant .Based upon the COI genetic data, there was a statistically significant and high degree of genetic differentiation among the 20 M. arion for the EF-1α gene was also high (FST = 0.179) and statistically significant (P < 0.001). The pairwise FST values ranged from 0.000 to 0.678. The average pairwise FST value between Italian populations was high and statistically significant . Moderate and significant, genetic differentiation was found among the Polish populations   or for the EF-1α gene  nor among Polish populations with respect to the EF-1α gene . The IBD analysis was not performed on the Polish samples for the COI gene because the majority of pairwise FST values were zero.The average genetic differentiation among all 20 populations of M. arion haplotypes was highly supported by AMOVA results, for which all sampling sites were treated as a single group . When the Italian and Polish samples were treated as two separate groups, the AMOVA was not significant for the COI gene . However, significant structuring between Italy and Poland was found for the EF-1α gene . In Italy, no significant structuring was observed with respect to the LHP for either marker . The same result was obtained when the Italian populations were regrouped as lowland (up to 600 m) and highland (over 600 m).The geographical structuring among the M. arion for the COI and EF-1α genes. For the COI gene in Italy, the data were best explained by assuming four groups of populations. The first group consisted of the AUR and CET samples; group two included populations from the BDR, LOA, CUN, VAL and VFE; and groups three and four were represented by the CER and the CDF populations, respectively. The percentage of variation among these four groups was high at 48.43% (P < 0.001); among the populations within the groups, the variation was 3.26% (P < 0.001), and within the populations, the variation was 48.31% (P < 0.001). The maximum percentage of variation for the EF-1α gene in Italy  occurred under the assumption of two population groups , while the variation among populations within groups was 5.37% (P < 0.001) and within populations was 66.29% (P < 0.001). The analysis of the Polish samples revealed no grouping of populations, and the EF-1α gene data were best explained by assuming a single population group . No such analysis was performed for the mtDNA on Polish samples because of the almost complete lack of polymorphism.SAMOVA was used separately on the Italian and Polish populations to identify which subdivision was most likely to explain the genetic structure observed in NST value (0.156) was significantly (P < 0.001) higher than the average GST value (0.076) among 20 populations studied, indicating a phylogeographic structuring of M. arion haplotype distributions. The same pattern was also found when the Italian samples  and the Polish samples  were analysed separately. Interestingly, both the NST and GST values for the EF-1α gene were significantly higher among Italian samples than among Polish ones (P < 0.001). No phylogeographically significant structuring was found for the mtDNA in Italy .For the EF-1α gene, the average M. arion Italian populations, both at the COI mtDNA and the nuclear EF-1α genes. This is in contrast to the almost total absence of mtDNA polymorphism and the low to moderate genetic differentiation at the nuclear gene found for M. arion populations inhabiting Poland, a recently deglaciated area. Moreover, the rear edge populations  possessed as many as 11 out of the 12 mtDNA haplotypes recorded in this study; thereby, emphasising their relevance in the context of biodiversity conservation and, most importantly, from the evolutionary perspective.Perfectly matching rear edge theory , this stThe mtDNA diversity in Italy may be thus an example of 'southern richness’ as opposed to ‘northern purity' as defined by Hewitt ,3,48, e.Bombina pachypus[NST and GST values emphasize the role of the genetic drift as the main force responsible for the differentiation among Italian populations. In Italy, we found more structuring and greater genetic differentiation for the COI gene than for the EF-1α gene. Mitochondrial markers are characterised by much faster lineage sorting rates and more frequent haplotype extinctions than the bi-parental nuclear markers. Consequently, as is widely recognised for many Lepidoptera e.g., [Thus, our data do not support the alternative scenario that suggests lower genetic diversity in populations at the edge of a species’ distribution compared to that in the centre ,27. The  pachypus. The idera e.g., ,53, diffM. arion populations. In Poland, we almost exclusively found a single haplotype, which was also the most frequent in northern Italy (H1). Of course, a “European” origin of north Italian populations cannot be ruled out either. This would mean that the dispersion from the East has reached northern Italy. Our results have indeed shown that the ancestral haplotype (H1) is dominant in northern Italy, but is rare (AUR) or absent  in central and southern Italy. Ugelvig [M. arion, Italy was colonised from Iberia. It should be noted, however, that only two samples from Italy were analysed, while our extensive study showed that Italy was rather colonised from two different routes.Although our data on mitochondrial genetic material suggested a significant population genetic structure, they did not support the subdivision of the populations into macrogeographic groups . The most common mtDNA haplotype in our samples (H1) is indeed the most widespread throughout the species’ range because it was recorded also from Catalonia, Germany, Denmark, Sweden, Estonia, Finland, the Czech Republic, Slovakia, Romania, Hungary, Ukraine, Russia and Kazakhstan. Moreover, the second most common haplotype (H5), which we found in five Italian sites , is known to occur in Catalonia, the Czech Republic and Sweden . As a re Ugelvig  suggestsEuscorpio carpathicus complex, where Salomone et al. [Melanargia galathea[Polyommatus coridon[We detected three unique and closely related mtDNA haplotypes  in southern Italy. A similar pattern was found in Italy for the e et al.  concludee et al. , and the galathea. Howevers coridon. This coM. arion phylogeographical pattern further studies should be based on a much higher number of specimens, especially coming from other South European areas as well as central populations.On the other hand, the presence of unique haplotypes in southern Italy concurs with the hypothesis that, contrary to common belief, most relic rear edge populations may not have been the source of major postglacial re-colonisations, thereby preserving their high genetic distinctiveness . Rear edM. arion showed moderate to high levels of diversity in the nuclear gene EF-1α. Altogether, 30 haplotypes were found, 18 occurring in Italy and 17 in Poland. Interestingly, a study carried out on M. alcon in a closely overlapping area found only six haplotypes among the 179 individuals sampled [M. arion exhibits a rather high nuclear genetic variability when compared to other members of the genus Maculinea[Maculinea butterflies makes them prone to genetic drift and therefore to a loss in genetic variation [M. arion is considered a less advanced social parasite because it preys on ant larvae and shows a lower level of host ant specificity. In this case, it is possible that the effective population size of M. arion is higher than in other members of the genus, especially M. alcon and M. ‘rebeli’. In this regard, it is interesting to observe that recent genetic data obtained on Danish and Swedish populations suggest that M. arion is more dispersive and less sensitive to genetic erosion than was previously assumed [ sampled . Our res sampled ,57 by shMaculinea,35,58. I assumed ,60.The AMOVA analysis of nuclear genes, through EF-1α, showed significant genetic differentiation between the Italian and Polish populations. This may indicate a different population origin occurred in both countries and some indications of this can be found among 24 sequences available in Genbank. A total of twelve EF-1α haplotypes in addition to our samples (30 haplotypes) were deposited by Als et al. . In the M. arion was extensive, yielded the highest level of polymorphism in both EF-1α and microsatellite markers [The low to moderate population differentiation observed in Poland for the EF-1α gene concurs with results from a previous study involving microsatellite markers that was conducted on the same DNA material; in which, rather low levels of differentiation for the species were determined, although some populations were highly isolated and the genetic differentiation was indicated to be mainly shaped by habitat fragmentation . Interes markers .Bombina pachypus by Canestrelli et al. [We did not find any evidence for IBD for any population grouping or for molecular marker. The lack of any correlation between geographic and genetic distances can be explained by historical demographic processes or cycles of colonisation and extinction. A similar pattern was identified for some Apennine populations of i et al. .M. arion. Among the Italian samples there were representatives of both M. a. ligurica  and M. a. obscura , while the remaining Italian populations , as well as all Polish populations belonged to M. a. arion. The analysis of the bar code sequence showed that two of the three populations belonging to M. arion ligurica were characterised by fixed or almost fixed haplotype H1, which was also fixed in 10 of 11 Polish populations and almost fixed in the remaining population. No species or subspecies-level differentiation was detected also as far as EF-1α gene is concerned. Hence, the results of our study are in agreement with the allozyme analysis of Hungarian populations indicating no differences between Origanum and Thymus dependent populations [M. arion is a species with an absence of deep historic population isolation. Sielezniew & Dziekańska [Our genetic data do not support any subspecies divisions in ulations . This suiekańska  hypothetMaculinea taxa , molecular analysis revealed no differences supporting division into separate species [Homona mermerodes.It is worth noting that in the case of two other  species ,62 and p species . A lack  species  from New species  in the mM. arion, has not been a mean to promote speciation, but can be ascribed to local adaptations in the species’ phenology that have evolved recently. For instance, when one of the glacial refugia was localised at the southern base of the Alps, after deglaciation, some populations could expand their range to higher altitudes tracking hosts re-colonisation, while others were able to survive in the lowlands by shifting to a novel host plant [M. a. ligurica populations may represent a relic of M. arion populations that survived at the base of the Alps and were a source for the re-colonisation of the Alps.We can speculate that use of different LHPs by a locally monophagous butterfly species, such as st plant . If thisWolbachia, and screening of all our samples suggested 100% prevalence of a single strain of the bacterium (supergroup A). The most common effect of a Wolbachia infection is cytoplasmic incompatibility (CI), which results in infected males that are unable to successfully reproduce with either uninfected females or females infected by a different strain. As a consequence, the distribution of mtDNA variation in Wolbachia infected populations may not conform to the expectations of the neutral theory [Wolbachia, as hypothesised for Polygonia c-album[All the populations in this study were infected by l theory ,65, evenl theory . The stal theory , but coua c-album.Maculinea alcon (including the xerothermophilous ecotype ‘M. rebeli’) is also reported to have Wolbachia infection [. However, a different strain (supergroup B) was detected in samples of this species collected across Poland and Lithuania. Sielezniew et al. [Wolbachia is not responsible for the observed pattern of genetic differentiation among the studied M. arion populations. It is probable that the Wolbachia infection in M. arion populations preceded both the expansion from glacial refugia and ecological specialisation.The congeneric nfection . Howeverw et al.  hypothesM. arion between the two distant parts of the European species range. Although we were unable to demonstrate any evolutionarily significant units, we suggest that Italy is an example of a rear edge area and should be considered as a region of special conservation concern, and therefore important for retaining genetic diversity in M. arion. However, the lack of correlation between the genetic differentiation and any subspecies divisions or ecological variation indicate that the observed specialisations are relatively recent in origin. We also demonstrate that the patterns of mtDNA diversity found in Poland and Italy cannot be explained by endosymbiotic infection and results from the colonisation patterns as well as genetic drift.Our study shows that the rear edge theory may explain the observed differences in the genetic differentiation pattern in The authors declare that they have no competing interests.DP, MS, MR and EB designed the study. DP, MS, SB, FB and MW carried out field work. DPT, MR, DP, MB and RR performed laboratory work, genetic and statistical analyses. MS, MR, DP and RR drafted the manuscript. SB, FB, MW and EB critically reviewed the manuscripts. All authors read and approved the final manuscript.Table S1. Haplotype frequencies of the COI mtDNA gene in nine populations of M. arion from Italy and eleven populations from Poland. S2. Haplotype frequencies of the EF-1α nuclear gene in nine populations of M. arion from Italy and eleven populations from Poland. S3. Genetic differentiation (pairwise FST main text and supplementary table s3) at the COI gene  and for EF-1α  among nine populations of M. arion in Italy and eleven populations of M. arion in Poland (for full names of the locations see Tab. 1). Any significant FST main text and supplementary table s3 values are given in bold.Click here for file


Cornu aspersum aspersum. Deliberately or accidentally imported, the species has become recently a notorious pest outside its native Mediterranean range. We compared the spatial structure and genetic variability of invasive  versus native populations using five microsatellite loci and mitochondrial (Cyt b and 16S rRNA) genes as a first step towards (i) the detection of potential source populations, and (ii) a better understanding of mechanisms governing evolutionary changes involved in the invasion process. Results based on multivariate analysis , Bayesian statistical inference  and demographic tests allowed a construction of the introduction pathways of the species over recent centuries. While emigrants originated from only one of the two native lineages, the West one, the most likely scenario involved several introduction events and “source switching” comprising (i) an early stage (around 1660) of simultaneous introductions from Europe  towards Oceania  and California, (ii) from the early 18th century, a second colonization wave from bridgehead populations successfully established in California, (iii) genetic admixture in invasive areas where highly divergent populations came into contact as in New Zealand. Although these man-made pathways are consistent with historical data, introduction time estimates suggest that the two putative waves of invasion would have occurred long before the first field observations recorded, both in America and in Oceania. A prolonged lag period as the use of an incorrect generation time could explain such 100–150 years discrepancy. Lastly, the contrasting patterns of neutral genetic signal left in invasive populations are discussed in light of possible ways of facing novel environments (standing genetic variation versus new mutation).This study is the first on the genetics of invasive populations of one of the most widely spread land mollusc species known in the world, the “Brown Snail”  However, to avoid attributing present genetic changes to post-invasion genetic or ecological events, whilst they must simply reflect genetic differences in source populations, the original variability might be assessed with as much precision as possible Elucidation of the mechanisms driving the success of invasive species requires detailed knowledge of the phylogeography and genetic characteristics of both source and invading populations. A large number of studies devoted to invasive taxa have increasingly demonstrated the importance of identifying the geographic origin and lineage sources of introduced populations and the mode and pathways of their introduction in order to determine factors influencing successful invasions, to design management strategies and to prevent further spread of species which sometimes turn into agricultural pests th century, the Mediterranean mollusc Cornu aspersum is one of the most widely spread land snails in the world C. a. maximum whose origin is still doubtful since its range has not been investigated, and the common form ‘petit-gris’ or C. a. aspersum. Most probably native to north Africa aspersum subspecies shows strong discontinuities between two well-defined geographical groups indicative of independently evolving lineages aspersum form (western Mediterranean and European coastlines) strongly indicates a deep genetic split into an Eastern and a Western lineage. The East one (lineage C) includes especially samples from East north Africa (from East Kabylia to Tunisia), and the West one (lineage B) consists of populations from western north Africa (from western Kabylia to Morocco) and Europe. Signatures left in the geographical distribution of the genetic variation suggest that Pliocene and Pleistocene events involving vicariant and dispersal processes through tectonic changes and climatic fluctuations would explain such an East-West genetic split, also described in other co-distributed taxa  If introduced populations originated from one or two lineages? and if both lineages were involved, did the East and West lineage supply roughly equal numbers of migrants? (ii) Whatever the source lineage, which introduction scenario can be inferred from the genetic signature left in introduced populations? Did migrants originate from a single or multiple introduction events? In the case of multiple introductions, which are often thought to account for the invasive success of genetically variable populations The aim of this study, devoted for the first time to invasive populations of 1, Ca2, Ca4; 1 sample from Texas, Tx1; 1 sample from Utah, Ut1), Hawaï , South America , Africa  and Oceania  (maximum coming from a snail farm in Brittany (France) were also analyzed and used as an outgroup in phylogenetic and network-based analyses. Sample site characteristics and haplotype codes are given in C. a. aspersum individuals required no relevant permission.Specimens of the native area were either previously investigated  . All speb versus 16S) consisted of two steps. First we performed a preliminary analysis using all native individuals (390 and 405 sequences for 16S and cyt b genes respectively) and then, we discarded groups of populations or lineages that could not be the source of the invasive individuals. The resulting reduced data set comprised 262 sequences for cyt b (192 native and 70 invasive) and 263 sequences for 16S (188 native an 75 invasive). For nuclear variation, 587 individuals were genotyped for polymorphism in five microsatellite loci but only 424 (252 native and 172 invasive) were used after removing individuals with missing data . For the 16S region, they were manually adjusted, based on the secondary structure of the large ribosomal subunit of C. nemoralisnaSP v4.10.9 rlequin v3.1 hymlv2.4.4 rBayes v3.1.1-p1 etwork v4.2.0.1 Mitochondrial sequences were aligned using the built-in assembly algorithm of the Cenepop v4.0 enepop. Tests for difference among groups of native versus introduced populations for several statistics  were carried out using comparisons among groups of samples as implemented in Fstat v2.9.3.2 initab version 15) with 10 native and 10 invasive populations to examine the effects of population origin  and locus on allele number (Na), allelic richness (A) and heterozygosity (observed Ho and expected He) estimates. Non-normal data were first transformed by applying the Box-Cox transformation.Standard population parameters were estimated to describe genetic variability within and between samples. Allele number, allelic richness, allele frequencies, observed heterozygosities and unbiased estimates of expected heterozygosities under Hardy-Weinberg expectations were calculated for each population and locus using Gdegenet v1.2–3. k) or homogeneous groups of genotypes was assessed using the K-means clustering approach. An increasing number of clusters (k) were tested and the optimal k value was chosen on the basis of the lowest associated Bayesian information criterion (BIC) provided for each k tested. Misclassified individuals or individuals not assigned in the population where they were sampled could then be potential migrants. In the same way, homogeneous groups clustering individuals originating from different populations could suggest multiple introductions.To detect population genetic structure and explore the relationships between the introduced populations and possible source populations, we used multivariate , we carried out 20 independent Markov Chain Monte Carlo (MCMC) runs with 7500 iterations discarded as burn-in followed by an additional 20 000 generations. The optimal number of clusters k was verified by estimating the ad hoc statistic ΔK suggested by k value inferred were aligned using Clumpp v1.1.2 software k ≈ 15, we used the LargeKGreedy algorithm with G’ and 1000 random input orders. The Distruct v1.1 software lumpp.Clustering of individuals was investigated using Bayesian algorithms implemented in S2, see iy ABC th century. Based on the geographic distribution of the mtDNA lineages and the genetic admixture at microsatellite loci found in NZ2, samples were pooled into one “native” and three introduced populations: “NZ2” for admixed invasive NZ2 population, “Cal” for invasive Californian populations, “inva2” for all other invasive populations. A fifth population considered as a ghost population (“ghost NZ”) was also included in both scenarios tested. This unsampled population acts as a parent in the admixture event occurring in New Zealand. The admixture of genetically diverse source populations in NZ2 could then be explained by  and New Zealand gives birth at that time to the admixed population “NZ2”.scenario 1: two independent introductions occurred simultaneously from native range (“native”) to New Zealand (“ghost NZ”) and California  at time t3 and then served as source of colonists for California  at t2. A second wave of introductions occurred from California to other regions (“inva2”) leading to admixed populations in New Zealand (“NZ2”) at t1.scenario 2: New Zealand (“ghost NZ”) was first colonized by native migrants (“native”) at Ne = 5000 for “native” and Ns = 1000 for all introduced populations. However, new established populations started by small numbers of migrants  so that Ns was reached in introduced areas after a few generations (db = 5). Each individual was genotyped at five microsatellite loci and two mitochondrial genes. Microsatellite loci are assumed to follow the SMM mutation model, each locus has a mutation rate drawn from a Gamma distribution (mean = mean mutation rate and shape = 2). The two mtDNA genes are assumed to be of 600 and 400 bp and to follow the HKY model . Approximate date of first introductions recorded in California were used to define the prior distribution of the coalescence time in all scenarios . A number of 106 data sets were simulated per scenario following the parameter values drawn from the prior distribution. To compare scenarios, we computed the posterior probability of each of them by performing a logistic regression based on 1% of the simulated data closest to observed data.We first simulated genetic data set under scenario 1 and then used this simulated data set as a test data set to compare both scenarios and evaluate the one that fits better the observed data. The effective population size is set to FsR2 statistics which were estimated using DnaSP v4.10.9 rlequin v3.1 The dynamics of demographic expansion before and after introduction was inferred using tests of population growth. As described in b and 16S respectively) eastern haplotypes excluded from data sets. For both genes, all invasive haplotypes linked to the West lineage were scattered throughout the main sub-lineage W1 except some haplotypes exclusive to the New Zealand population NZ2  which were part of the haplogroup W2  distributed in a star-like pattern belong to the sub-lineage W2. The putative source populations of NZ2 haplotypes varied between the two sub-lineages but also between genes; 16S and cyt b individuals of W2 could only have originated from native-range source populations (Algiers area and Europe) while individuals of W1 would have diverged after secondary introductions from previous ones that occurred earlier in America or closer geographically in the South Island of the country. All remaining exotic 16S haplotypes of the W1 haplogroup were specific to introduced populations and diverged greatly from native ones except s54 arranged around the widely distributed common haplotype s1 shared by Californian (Ca4), Chile, French and Sicilian individuals , they were all specific to invasive samples . Subsequent analyses were made with most , locus  and population . Thus, allelic richness (A) and gene diversity (Hs) were significantly lower in the invasive populations than in the native ones. However, univariate tests (ANOVA) showed that population effect was only significant for Na. Matrix of pairwise correlations showed a relative decrease of the partial r only between He and Na but it remained highly significant . Results from mtDNA mirrored the lower nuclear diversity in invasive populations (b respectively. The within-population divergence in NZ2 was high compared to estimates of other invasive populations and the relatively low haplotype diversity found (Estimates of genetic diversity based on microsatellite loci are shown in ulations . Nucleotty found .1), whilst the second axis differentiated individuals from continental Spain . Such strong differentiation between invasive samples may explain the strongest FST value found for invasive populations (versus native sets of samples respectively (39 and Ba4) and similarly, four native individuals out of 23 were assigned to invasive samples  clustering mainly native individuals from Spain and France since only two individuals from NZ2 and Ca2 joined groups 1 and 9 respectively. With a different “status-ratio”, all seven other groups defined clustered native and invasive individuals and were all weakly genetically differentiated from each other.A first DAPC on genotypic data of 424 individuals  was made using all 20 populations as clusters . The firulations . The aveectively . Amongsttructure clustering indicated that the most parsimonious estimate of the number of clusters was k = 2. Whatever the model tested, the structure inferred mirrored results of the first DAPC with multilocus genotypes of the three Spanish samples assigned predominantly into one of the two clusters  and α (0.53±0.10) suggested that sampling location was roughly informative and that each individual would have originated mostly from a single population, the LOCPRIOR model ignored the sample group information to cluster NZ2 individuals since NZ2 genotypes split into two main distinct clusters, with most individuals having ancestries both in NZ2 and NZ1. The ancestry of individuals seemed also uncorrelated with the sampling location of Chilean populations with fractions of their genome having ancestries in the French F39 (green) and F41 (red) populations.Results of the Sclusters  and genoinferred . At k = 2. Contemporary invasions would occur from California to other places (“inva2”). The medians of the posterior distribution of t3 and t2 were 221 (95% CI = 83–296) and 156 (95% CI = 59–197) generations respectively. Assuming a generation time of two years, estimates  implied events which would have occurred over times much longer than the first introductions recorded around 1850 in California.Posterior probabilities of scenarios estimated through a logistic regression showed better support for scenario 1 than for scenario 2 , meaningversus non-native groups of populations  and without NZ2 (“invasive-NZ2”). For the 16S gene, one or both Fu’s Fs test of selective neutrality and R2 statistics yielded significant results for “invasive”, “invasive-NZ2”, “native” and “native West” sets of populations. For cyt b, demographic expansion characterized “native”, “native West” and “invasive-NZ2”. Locally, tests performed on partial range of invasive populations indicated that the null hypothesis of constant size is not rejected for North-American populations either for cyt b as for 16S genes. These results were confirmed by the observed mismatch distributions which closely matched those expected under a model of a sudden expansion for these data sets  a French or a Spanish origin of founders, (ii) an immigration pattern in several steps involving bridgehead populations located in California. The New Zealand population of Auckland (NZ2) is differentiated from all other invasive samples since some snails of this population fall into the distinct W2 sub-lineage without sharing haplotypes with native populations. As derived haplotypes of French and Spanish populations found in W2, the position of specific haplotypes of NZ2 would suggest that invaders for that New Zealand sample would have partially originated from Western Europe that has been originally colonized by migrants from the Algiers area in north Africa. Although they were described only in vague terms, historical data reinforce this assumption with a likely introduction of the species from Europe for producing food Although all founding snails originated from the western lineage only, both mtDNA and microsatellite markers indicate that highly distinct sources of western populations would have supplied migrants. The two evolutionary sub-lineages W1 and W2 identified in the native range participated in the colonization of North and South America, South Africa and Oceania although with unequal contributions. The haplotype distribution of cyt iy ABC analysis suggest a chronology of colonization events which occurred in New Zealand and indicate the direction of colonization. Since about 1600, two independent pools of immigrants would have dispersed simultaneously from native range towards New Zealand and California. More than one century later (around 1770), there is evidence for a second wave of invasion from successful populations established in California and introduced at varying distances throughout the world. The high level of genetic diversity observed in the population located in the North Island of New Zealand (NZ2) would then result from genetic admixture between highly divergent source populations, the ghost one (first invasion) supplanted by the arrival of Californian snails (second invasion). Haplotype relationships would suggest that the South Island (NZ1) first received Californian founders that subsequently expanded until they came into contact with snails of the first wave of introduction located in the North Island. Strong nuclear and mitochondrial affinities between South African and NZ1snails indicate that exchanges between the two continents could also have existed before and/or after the colonization of New Zealand by Californian invaders. Although the geographical situation of South Africa suggests that all scenarios are possible, the alternate derived or ancestral position of NZ1 and South African haplotypes both diverging from Californian ones seem to be unclear. Since accidental introduction of the species from the 17th century would prevail over the intentional one recorded in 1855 , a colonization event from South Africa prior to the arrival of Californian snails cannot be ignored. Further analyses involving novel New Zealand and South African samples need to be conducted to shed light on exact source populations and routes of introduction of the species in Oceania. This would indicate whether early explorers used South Africa as a “port of call”. By contrast, the intermediate position of Californian snails specifically from Monterrey Bay (Ca2) provides evidence for the existence of a bridgehead effect 2 population may have served as the source of colonists for secondarily invaded territories either closely located as other American states , or more distant geographically as Haiti, South America, South Africa and New Zealand. The notorious spread of this pest in North America and the earliest record of the species (around 1950) in the Hawaiian Islands support this bridgehead scenario 1) also diverging from Ca2 reflects a strong genetic bottleneck in the founding population. The higher frequency of new alleles or alleles rarely found in other samples suggests that this population probably experienced a rapid recovery preventing the loss of genetic diversity while promoting rapid local adaption of invaders.Results of the DC. a. aspersum in non-native areas are consistent with historical data, time estimates for introduction suggest that the two putative waves of invasion would have really occurred long before the first field observations were recorded, both in America and in Oceania. Two reasons might help to explain such a 100–150-year discrepancy. First, differences between estimates and historical records could correspond to the lag period between initial colonization and subsequent population expansion 1). Unlike those drawn directly on standing genetic variation, novel alleles are indeed not immediately available C. a. aspersum could be reduced in introduced populations compared to native ones from which this time has been inferred. Due to a reduced density in pioneer populations, the rapid adaptation of some invasive species to face novel environmental conditions has been partially attributed to their capacity to change some of their life-history traits C. a. aspersum.Although invasion pathways of C. a. aspersum provides preliminary evidence for reconstructing the introduction pathways of the species over recent centuries. Not surprisingly, only immigrants of the western lineage would have been successful at all stages of the invasion process. However, the single origin of invasive lineage does not imply a simple colonization pathway. Strong genetic divergence among and within introduced populations suggests a scenario comprising (i) an early stage of two simultaneous eastward and westward introductions, (ii) a second colonization wave from bridgehead populations successfully established in California, leading to genetic admixture in invasive areas where divergent populations came into contact, such as in New Zealand. The contrasting patterns of neutral genetic signal left in introduced populations mirrored two different ways of managing novel environments, through (i) a high level of genetic diversity pre-existing in native populations and retained in bridgehead ones, (ii) the emergence of genetic novelties resulting either from new mutations or from admixture of divergent source populations. Although the relationship between genetic diversity and invasiveness seems to be not straightforward C. a. aspersum into a large range of habitats.This first study on the invasive populations of Table S1Cornu aspersum aspersum populations analyzed in this study and in n), name of individuals sequenced  and their corresponding cyt b and 16S RNA haplotypes and GenBank accession numbers. Two kinds of haplotype codes are provided: (i) the sequence name for unique haplotype, (e.g. A1.1), (ii) hk (k up to 71 distinct types) and sk (k up to 56 distinct types) for cyt b and 16S respectively for haplotypes shared by distinct individuals  of (XLSX)Click here for additional data file.Table S2C. a. aspersum. He, expected heterozygosity per locus and per population; Na, number of alleles per locus and per population, Ae: allelic richness per locus and per population . Population code: (i) Native: Ba, Balearic islands; E, Spain; F, France mainland, Si, Sicily; (ii): Invasive: Ca, California; Ch, Chile; Hw, Hawaiian islands; NZ, New Zealand; SAf, South Africa. n: number of individuals analyzed per population; n*, number of individuals used for analyses after discarding individuals with too much missing data.Diversity measures from microsatellite loci comparing 10 native and 10 invasive populations of (XLSX)Click here for additional data file.


Rinelocaria were compared through the allozyme electrophoresis technique: one morphotype, R. pentamaculata, from the Keller River in the middle stretch of the Ivaí River basin and the other, R. aff. pentamaculata, from the São João River in the upper portion of the Ivaí River basin. The morphotype from the São João River was collected upstream from the São João waterfall, which is about 80 m deep. Twelve enzymatic systems  were analyzed, which allowed to score 22 loci. Only lociAat-2, Est-3 and Mdh-C showed polymorphism. The two samples differed in allele frequencies at the three polymorphic loci. The average expected heterozygosity for all loci was 0.0806 ± 0.0447 in the Keller River sample. For the São João River morphotype, this value was 0.0489 ± 0.0350. Nei' s genetic identity and distance between the two populations were respectively 0.9789 and 0.0213. Wright's FIS , FIT and FST over all loci were estimated as 0.3121, 0.4021 and 0.1309, respectively. We consider that the two morphotypes represent species in statunascendi.Two allopatric morphotypes of the genus  The Neotropical region harbors the most diversified ichthyofauna of the planet, corresponding to approximately 6,000 of the 13,000 species of freshwater fish. Of these, 4,475 have been described and there are probably 1,550 yet to be described  and the The species concept has been a subject of debate for a long time and, according to Genetic difference is one of the ways to verify whether or not two populations belong to the same species. Studies on isozyme electrophoresis have supplied important information about the genetic variability of natural populations, estimation of gene flow, elucidation of interspecific limits and the establishment of evolutionary relationships among different taxa . When thRineloricaria pentamaculata was described by Langeani and Araújo (1994), based on nine specimens collected from several locations in the upper Paraná River basin. Collections from the Ivaí River basin showed the presence of two morphologically different populations of R. pentamaculata. The main characteristic of R. pentamaculata is the presence of five dark dorsal stripes over the dorsum. It differs from congeners by having a naked snout tip, without platelets or odontodes, and an upper caudal-fin ray with an extension usually equal in length to the orbital diameter.Considering that a waterfall can represent a geographical barrier to gene flow and that the two populations present morphological differences as regards the form of the snout, form of the lip, disposition of the dermal plates and form of the caudal fin, this work was done to verify whether or not these two populations belong to the same species.Rineloricaria from the Ivaí River basin  a rounded vs. a pointed , more triangular snout tip; II) a lower lip distal border that is smooth or weakly serrate, vs. strongly serrate; III) each abdominal platelet being surrounded by skin, vs. abdominal platelets that are attached to each other and not surrounded by skin and; IV) an upper caudal-fin spine that usually does not extend beyond the branched ones, vs. an upper caudal-fin spine that is prolonged beyond the branched ones (the extension being similar in length to the eye diameter).The Keller River sample was identified as g at 4 °C for 30 min. Due to the presence of a great amount of fat in the liver, 100 μL of carbon tetrachloride (CCl4) was added to the tubes  containing 100 μL of Tris-HCl buffer 0.02 M, pH 7.5 and centrifuged at 44,720 Gels were prepared with 15% corn starch , Tris 0.He and Ho) of The genetic variability was estimated according to the average heterozygosity  were analyzed, which allowed to score 22 loci. Aat-2, Est-3 and Mdh-C  showed allele variation, with four, three and two alleles, respectively. In the sample from the São João River, only Aat-2 and Est-3  presented variation, with three alleles each.Voucher specimens were deposited in the ichthyological collection of the Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura, Universidade Estadual de Maringá (NUP): locus for both samples. The average observed heterozygosity for all loci was 0.0475 ± 0.031 in the sample from the Keller River and 0.0411 ± 0.03 in the sample from the São João River, values that are not statistically different . The average expected heterozygosity for the three polymorphic loci was estimated to be 0.0806 ± 0.0447 for the Keller River sample and 0.0489 ± 0.0350 for the São João River sample, values that are not statistically different . The sample from the Keller River was not in Hardy-Weinberg equilibrium for Est-3 , while the R. pentamaculata sample from the São João River was in Hardy-Weinberg equilibrium for the three polymorphic loci. The two populations differed in the allele frequencies of Est-3 , Aat-2  and Mdh-C .loci of four populations of Hypostomus of the Keller River to be in Hardy-Weinberg equilibrium. Hypostomus of the Maringá Stream. A great number of factors can deviate a population from Hardy-Weinberg equilibrium for a given locus, e.g. inbreeding, assortative mating, natural selection and gene flow  similar to the obtained number  is smaller than the average estimated by loci, while the population of the São João River presented only two. The high heterozygosity of Keller River population seems paradoxical, because that population was polymorphic at only 13.64% of the loci. loci for the population and Hypostomus strigaticeps of the Maringá Stream; however, the expected heterozygosity was only 0.028. A similar fact occurred with a Hypostomus sp. 3 population collected in the Itaipu Reservoir analyzed by loci, despite an expected heterozygosity was 0.048. The relatively high heterozygosity estimated in our work, when compared to the above studies, can be explained by the similarity among the allele frequencies in the analyzed polymorphic loci. The value of expected heterozygosity is maximal when the allele frequencies are similar and indeed, at Mdh-C we observed similar frequencies for the alleles Mdh-C (a) and Mdh-C (b) . In loci were quite divergent, which explains the lower He value.The value of He values estimated for species of Loricariidae have high variation, as found in several Hypostomus populations. The currently available results show that the family Loricariidae contains species with larger genetic variability than others, i.e. 0.011 and 0.017 for two populations of the Iguaçu River basin .The FIS, FIT and FST per polymorphic locus were statistically different from zero, which indicates a significant excess of homozygotes. The averages over all loci were estimated as FIS = 0.3121, FIT = 0.4021 and FST = 0.1309, respectively. Nei's genetic identity and genetic distance were estimated as 0.9789 and 0.0213, respectively.Wright's FIS value represents the level of gene fixation within subpopulations. FIT represents the level of gene fixation present in the total population (ignoring the subdivision). The FST value indicates the genetic differentiation between two populations . These values show that the two populations are genetically different at these three loci; however, the average for the 22 loci turns the differentiation between the two populations moderate.Values of ulations . Followilocus represents a barrier to the gene flow for sympatric populations and may result in, at least partial reproductive isolation. In organisms with sexual reproduction, this variation indicates that two populations should be considered different species.According to Rineloricaria pentamaculata of the Keller River and R. aff pentamaculata of the São João River of the Ivaí River basin are isolated geographically. As they differ morphologically, as well as in allele frequencies at the polymorphic loci, it is probable that they represent species in statu nascendi (i.e. in the process of genetic divergence to become two distinct species.nascendi , i.e. in


Neosporacaninum is one of the main causes of bovine abortion worldwide and is of great economic importance in the cattle industry. Recent studies have revealed extensive genetic variation among Ncaninum.  isolates based on microsatellite sequences (MSs). MSs may be suitable molecular markers for inferring the diversity of parasite populations, molecular epidemiology and the basis for phenotypic variations in Ncaninum. , which have been poorly defined. In this study, we evaluated nine MS markers using a panel of 11 Ncaninum. -derived reference isolates from around the world and 96 Ncaninum.  bovine clinical samples and one ovine clinical sample collected from four countries on two continents, including Spain, Argentina, Germany and Scotland, over a 10-year period. These markers were used as molecular tools to investigate the genetic diversity, geographic distribution and population structure of Ncaninum. . Multilocus microsatellite genotyping based on 7 loci demonstrated high levels of genetic diversity in the samples from all of the different countries, with 96 microsatellite multilocus genotypes (MLGs) identified from 108 Ncaninum.  samples. Geographic sub-structuring was present in the country populations according to pairwise FST. Principal component analysis (PCA) and Neighbor Joining tree topologies also suggested MLG segregation partially associated with geographical origin. An analysis of the MLG relationships, using eBURST, confirmed that the close genetic relationship observed between the Spanish and Argentinean populations may be the result of parasite migration  due to cattle movement. The eBURST relationships also revealed genetically different clusters associated with the abortion. The presence of linkage disequilibrium, the co-existence of specific MLGs to individual farms and eBURST MLG relationships suggest a predominant clonal propagation for Spanish Ncaninum.  MLGs in cattle.The cyst-forming protozoan parasite  Neosporacaninum is a cyst-forming obligate intracellular parasite that is phylogenetically related to Toxoplasma gondii and has been recognised as a worldwide cause of neuromuscular disease in dogs and a major cause of abortion in cattle [Ncaninum.  life cycle have confirmed that canids  are intermediate and definitive hosts and that cattle and other ungulates, including sheep, goat, water buffalo, bison and deer, are natural intermediate hosts [Ncaninum. -life cycle can be maintained by domestic dogs and cattle. Dogs can become infected by ingesting Ncaninum. -infected bovine tissues, and oocysts are shed in the faeces after sexual reproduction presumably in intestinal cells, which in turn can orally infect cattle [T. gondii, sexual crosses of Ncaninum.  in canids could produce new isolates with distinct biological characteristics by recombination and reassortment of genetic material between two different parental genomes [Ncaninum.  can also be transmitted transplacentally to the foetus by a primary infection of a dam with oocysts during pregnancy (exogenous transmission) or the recrudescence of a chronic infection in the dam (endogenous transmission), causing fetopathy [Ncaninum.  infection is not likely to persist in the absence of horizontal transmission [Ncaninum.  has been described involving white-tailed deer (Odocoileusvirginianus) and coyote (Canislatrans) as intermediate and definitive hosts, respectively [n cattle -3. Advante hosts -3. Theret cattle -6. Based genomes . N. etopathy ,9. Endogetopathy ,3,9, butsmission  and recesmission . The extsmission . In addiectively ,13.Ncaninum.  isolates with specific host preferences and virulence. These topics have been poorly investigated in Ncaninum. , likely due to a lack of suitable molecular tools. Several different studies have recently shown the discriminatory power of microsatellite (MS) markers to genetically identify Ncaninum.  isolates from diverse hosts and geographic origins [Ncaninum.  in clinical samples was facilitated by the development of nested-PCR [T. gondii [Ncaninum. -derived reference isolates from around the world, 96 Ncaninum.  bovine clinical samples and one ovine clinical sample collected from four countries, including Spain, Argentina, Germany and Scotland, on two continents over a 10-year period. These markers were used to investigate the genetic diversity of Ncaninum.  at global and country level, geographic distribution of Ncaninum.  genotypes and the genetic relatedness between country populations and population structure of Ncaninum.  in cattle.Suitable genetic discriminatory methods for differentiating parasites at the strain or isolate levels are necessary to investigate the intra-species diversity and epidemiological and clinical aspects associated with neosporosis. Molecular genotyping techniques have been used to study the taxonomy, evolutionary mechanisms and phylogenetic relationships, population genetic structure and epidemiology of many organisms, including protozoan parasites -19. Thes origins -25. The sted-PCR ,26. Mini. gondii -30. In tNeospora DNA samples included in this study were obtained from isolates maintained in a laboratory and clinical samples from contaminated tissues collected during the necropsy of ruminant abortions, resulting in a total of 108 samples from various regions of the world , oocysts shed from a naturally infected dog , bovine abortions, stillbirths or calves with neurological signs , a clinically healthy calf (Nc-Goiás 1) and a naturally infected sheep (named as Nc-Sheep) were used in this study. The majority of these isolates have been previously genotyped [The he world . The DNAenotyped ,31. ThisNcaninum.  samples from asymptomatic-congenitally infected calves from different provinces in Spain (Nc-Spain1H-Nc-Spain10) collected from 2003–2006 were included in this study [Ncaninum. -specific ITS-1 PCR [Microsatellite multilocus genotype (MLG)-data of 10 is study ,32. The TS-1 PCR . The PCR2 in Galicia (north-west Spain), which is one of the main cattle-producing regions in Spain; 11 bovine samples were collected from the Madrid, Navarra and Zaragoza provinces  covering a geographic area of 343 Km2 and 7,097 Km2 for the central and north-east regions, respectively, and 1 ovine sample from Jaén province (southern Spain). These areas were located 285 to 556 Km apart in an area of 385,106 Km2. Of these, a total of 18 samples were genotyped in a previous study [A total of 53 DNA samples from the infected tissues of the aborted bovine foetuses collected primarily from Holstein-Friesian cattle between 2001 and 2010 were included in this study . A totalus study   and dairy (Holando Argentino and Jersey) breeds.A total of 16 DNA samples obtained from bovine foetuses between 2004 and 2008 were included in this study . The foe2 and from farms located approximately 10 to 556 Km apart  and a 3730 DNA analyser (Applied Biosystems) at the Unidad Genómica del Parque Científico de Madrid. The sequences were analysed using BioEdit Sequence Alignment Editor v.7.0.1 .Neospora by ITS-1 PCR, were included in each round of PCR. In each round of amplifications, a Ncaninum.  (Nc-Liv) reference isolate for which the sizes of the nine MS loci had been determined by sequencing was included as an internal control [Negative controls, including reactions without a template and extraction of bovine DNA negative for  control .Allele assignment was performed according to the sizes determined by capillary electrophoresis and sequencing of the MS10 and MS7 markers. Allele designation in this study was performed according to the length of the variable repeat motifs identified in the MS sequences based on previous analyses ,23,26 T.Ncaninum.  were used for subsequent analyses.The seven-loci MS profiles  obtained from 108 samples infected with We measured the allele number (A), allele frequency and allele richness (Ar) per locus in whole and separate parasite country populations using FSTAT v.2.9.3.2  between each group of Ncaninum.  samples from different geographic locations [FST index based on Weir and Cockerham’s θ estimator [The GDA package was used to calculate Nei’s genetic distance , implemented in the LIAN v 3.5 web interface . Because LIAN cannot analyse MLGs with missing data, only MLGs with complete allelic data for the seven markers were used for analyses. The presence of LD was assessed for whole and separate populations in: the complete dataset and a reduced dataset of unique MLGs, where duplicate MLGs (found in more than one sample) were only included once.Multilocus linkage disequilibrium (LD) amongst seven-loci genotypes was assessed using a standardised index of association  and Cavalli-Sforza distances among the sample MLGs was calculated from the complete dataset by applying POPULATIONS v. 1.2.3.2 software (http://bioinformatics.org/~tryphon/populations/). An unrooted Neighbor-Joining (NJ) tree was constructed with the DAS matrix using POPULATIONS software. This analysis was performed with bootstrap values for 1,000 replicates. The NJ tree was visualised with FigTree v.1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/).Ncaninum.  genotyping were located on six chromosomes  based on a BLAST search in the ToxoDB database (http://toxodb.org/toxo/)  . Three Mg/toxo/) . Most ofg/toxo/) .r2= 0.9288, p<0.0001 by linear regression analysis in GraphPad Prism v.5 software).The number and size of the alleles at each MS marker are also displayed in in vitro passages of the parasite on the stability of the repetitive motif for each MS was evaluated in a stock of Ncaninum.  DNA from the NC-1 isolates routinely maintained in 7 different research laboratories. The unique MS variation among NC-1 samples was detected on the MS6B marker from a unique NC-1 sample lacking one AT repeat  to investigate the genetic diversity in bovine quencies . Predomiquencies . The numectively . These re values , likely Genotypic diversity was similarly high across all of the populations from different countries . All of D) and pairwise FST analysis . A lower level of genetic differentiation was observed between the Scottish and German populations . However, the pairwise FST value estimated between these populations was not significant, which is likely due to the limited sample size from both populations. Notably, when the Spanish and Argentinean populations were compared, minimal genetic differentiation was also observed . Geographical association of Ncaninum.  MLGs was examined by overall structure PCA analysis . LD was slightly reduced in the Spanish population when the analysis was performed with unique MLGs, but with a higher level of statistical significance . These results suggest a non-random association between the MS loci in these parasite populations and support the possibility of genetic isolation. By contrast, significant LD was not detected among the German Ncaninum.  MLGs. However, established LD estimations should be considered with caution due to the limited number of complete MLGs for some of the populations included in the analyses (<10). LD could be a consequence of the physical linkage of the polymorphic loci used for genotyping, such as MS4 and MS5. Complementary analyses of LD between all pairs of loci ruled out the influence of this MS pair in multilocus LD (data not shown).LD estimations were determined for each country population . MultiloHh-Berlin (German) and Nc-Spain 5H (Spanish) MLGs  MLGs , Group 2sh) MLGs , Group 3sh) MLGs . These rse herds . Clonal MS amplification of the clinical samples in this study was performed using nested-PCR developed by Pedraza-Díaz et al. , with slNcaninum. . Novel alleles were found in most of the MS markers after analysis of the complete dataset, particularly for the most polymorphic marker MS10. This finding confirmed and increased the high discriminatory power for these markers established in previous studies [Genotyping of a substantial number of samples also allowed us to define the suitability of these markers for the population genetic analysis of  studies ,21,23,26 studies . Several studies ,41, whic studies -42. MostNcaninum. , as suggested in previous studies [Ncaninum. , with 96 MLGs identified in 108 Ncaninum.  samples typed even when the multilocus analysis was limited to seven loci. High levels of genotypic diversity were also determined for the phylogenetically related T. gondii isolates when five MS markers were applied to French and French Guiana parasite populations or seven MSs were used to genotype 271 T. gondii isolates with different geographic origins [Ncaninum.  at global and local scale.Multilocus analysis of MSs confirmed the great discriminatory power of these markers for the large-scale genotyping of  studies ,21,23,26 origins ,43. ThesHe values were found in populations originating from different countries, indicating high genetic diversity in Ncaninum.  within each population. Similarly, high genotypic diversity was also observed for each Ncaninum.  country population. Indeed, detection of identical MLGs was basically restricted to samples from individual herds confirming the high diversity of Ncaninum.  on a smaller regional level.Similar average Ar and FST and D values demonstrated a clear genetic separation between the Spanish and Argentinean populations, on one side, and the German and Scottish populations, on the other side. However, less genetic divergence was observed between the Spanish and Argentinean populations. Population segregation by NJ analysis was partially associated with the geographic origin by country, although dendrogram analysis based on these data was unstable. NJ analysis based on MLGs may failure in represent the complex population structure of Ncaninum.  and detect the association between genetic divergence and geographical origin with accuracy [Ncaninum.  was also explored by PCA analysis, results revealed similar relationships for these country populations, showing a closer relationship between the Spanish and Argentinean populations and the German and Scottish populations. These results confirmed that there is a level of geographical sub-structuring between these populations. Considering the large distances and physical geographic barriers between these country populations - the Atlantic Ocean and Pyrenees Mountains - geographic differentiation is expected. However, genetic isolation by geographic distance cannot explain the minor genetic differences observed between the Spanish and Argentinean populations, and likely between the German and Scottish populations. When the relationships between individual MLGs were visualized by eBURST analysis, we observed two main clonal groups, clustering mainly Spanish and Argentinean MLGs . Additional investigations using a higher number of Ncaninum.  samples, representing different European and American locations and a higher range of intermediate and definitive hosts, are needed to confirm this hypothesis. Moreover, new polymorphic markers based on genes and intron sequences must be evaluated in order to establish the history and geographic dissemination events for Ncaninum. .Similar to the Spanish cattle breeds in South-America, the Holstein-Friesian breed originating from Holland-Germany was introduced worldwide into dairy herds during the second half of the 19th century and the first decades of the 20th century, including Spain . As a reNcaninum.  MLGs co-exist and are maintained by clonal propagation. This hypothesis is consistent with the results obtained from eBURST showing that different clonal clusters, some co-existing in individual herds, were described in the Spanish MLG population. Thus, the eBURST analysis suggests the clonal as the predominant form of propagation of Ncaninum.  MLGs in the Spanish herd.The level of LD estimated for each population in this study also support parasite migration events. The observed LD implies low genetic exchange between parasite populations in the following scenarios: 1) “epidemic-ephemeral clonality”, 2) “cryptic biological speciation”, and 3) “clonal population structure”. LD might also reflect physical separation in either space or time or both, or the recent introduction of genetically different populations due to immigration and insufficient time for random mating or prolonged maintenance by clonal propagation ,49. ThisNcaninum.  in domestic hosts, where the parasite is predominantly transmitted in cattle via vertical transmission routes after the reactivation of infection during the pregnancy or oral consumption of oocysts, likely after the inbreeding in the dog [AS values, the extensive genetic diversity showed by Ncaninum.  with different clusters and the presence of singletons that are not associated to main cluster through eBURST analysis could be interpreted as the result of sexual recombination and genetic exchange. The life cycle of T. gondii and Ncaninum.  allows clonal and sexual expansion in populations. Recent molecular studies in phylogenetically related T. gondii species have demonstrated different population patterns depending on the level of sexual recombination [T. gondii has been associated with disease outbreaks due to the rapid amplification of a single clone into millions of oocysts and out-breeding as a potent mechanism to generate offspring with a range of phenotypic traits, including virulence [Ncaninum.  in cattle herds [Ncaninum.  .This clonal model is also compatible with the life cycle of  the dog ,50. Nevebination ,43. Receirulence . The horNcaninum.  MLGs for some populations and representative values of the Ncaninum.  samples from this parasite population. Despite increased efforts to obtain a representative number of Ncaninum.  samples suitable for genotyping, the final parasite dataset was primarily limited to bovine abortion samples and Ncaninum.  MLGs could be biased towards more virulent isolates in cattle. Samples from other domestic and wild intermediate and definitive hosts from different geographic regions should be included to establish the genetic diversity of Ncaninum.  in these hosts and to investigate if there is host preference across Ncaninum.  isolates and the global population structure of Ncaninum. . Additionally, the MS markers used in this study were physically distributed on 6 out of the 14 chromosomes of Ncaninum. . The seven MSs used in multilocus genotyping represent a suitable molecular tool for genetic studies in Ncaninum.  according to the results obtained in this study. Future approaches should also be directed towards the identification of a new set of markers that represent the remaining 8 chromosomes and apicoplast DNA to improve this integrated method of Ncaninum.  genotyping.A limitation of this study was the low number of Ncaninum.  from cattle using a set of seven MSs. The results demonstrate the suitability of these MSs as molecular tools to characterize the population structure of Ncaninum. . Multilocus MS analysis confirmed an extensive genetic diversity across Ncaninum.  isolates from different countries and continents that is maintained at local level. Genetic differentiation between Ncaninum.  geographic populations demonstrates that there is a degree of geographic sub-structuring by genetic isolation, although it could be influenced by parasite migration and host movement between countries. The results also support a predominant clonal propagation of Ncaninum.  MLGs, at least in Spanish herds, but maybe under the presence of some degree of genetic exchange. Nevertheless, it has to be kept in mind that conclusions derived from the molecular analysis directly depend on the resolution of the markers and sampling used in the study. Extended analyses with an increased number of markers and representative samples from different hosts and geographic origins are needed to confirm the conclusions of this study.In summary, this work is the first extended study to infer the genetic diversity, geographic distribution and population structure in Figure S1Ncaninum.  samples included in this study.Geographic distribution of the  The expanded window shows the detailed geographic origins of the European Ncaninum.  samples. Circle sizes represent the number of samples included for each country population as indicated by the number inside.(TIF)Click here for additional data file.Figure S2Allele frequency distribution of the seven microsatellite loci used for genotyping each country population. Fragment size and number of repetitive motifs were determined using Genescan software and sequencing. Frequencies were assessed based on the total number of samples included for each population. Allele lengths as number of repetitive motifs Click here for additional data file.Table S1(DOCX)Click here for additional data file.Table S2(DOCX)Click here for additional data file.Table S3(DOCX)Click here for additional data file.