Brachyplatystoma platynemum is a catfish species widely distributed in the Amazon basin. Despite being considered of little commercial interest, the decline in other fish populations has contributed to the increase in the catches of this species. The structure, population genetic variability, and evolutionary process that have driven the diversification of this species are presently unknown. Considering that, in order to better understand the genetic structure of this species, we analyzed individuals from seven locations of the Amazon basin using eight molecular markers: control region and cytochrome b mtDNA sequences, and a set of six nuclear microsatellite loci. The results show high levels of haplotype diversity and point to the occurrence of two structured populations (Amazon River and the Madeira River) with high values for FST. Divergence time estimates based on mtDNA indicated that these populations diverged about 1.0 Mya (0.2–2.5 Mya 95% HPD) using cytochrome b and 1.4 Mya (0.2–2.7 Mya 95% HPD) using control region. During that time, the influence of climate changes and hydrological events such as sea level oscillations and drainage isolation as a result of geological processes in the Pleistocene may have contributed to the current structure of B. platynemum populations, as well as of differences in water chemistry in Madeira River. The strong genetic structure and the time of genetic divergence estimated for the groups may indicate the existence of strong structure populations of B. platynemum in the Amazon basin. The Amazon and adjacent river basins is a phenomenal hot spot observed within Neotropical ichthyofauna and has been the focus of numerous studies  have a distinct fish fauna compared with adjacent black-water rivers with low sediment charge draining from lowlands forested   also can deeply affect habitat taxonomical composition and species distribution Roberts . This imThe climatic changes hypotheses are linked to the fluctuation of low and high sea level periods associated with glacial–interglacial periods, respectively. Low sea level events might be related with river interconnections at their lower section, enhancing freshwater taxa dispersal from one basin to another, while high sea level periods would have fragmented populations into newly isolated rivers basins promoting thus lineage diversification. Therefore, not only Pleistocene climatic changes but also earlier orogeny events  occurring during the Miocene and Pliocene likely played an important role in the population divergence of South American freshwater fishes , using sequences of the mtDNA and nuclear markers. We hypothesized that climate oscillation during Pleistocene resulting in marine transgression though low land of the South America continent and differences in water chemistry of the Madeira River, following to isolate local populations, and consequently leading genetic divergence.Therefore, the goal of our study was try to elucidate the genetic variation and the evolutionary process regarding to affect the evolution of B. platynemum individuals were collected in seven location of Amazon Basin, Iquitos (AIQ) (upstream Amazonas River), Purus River (PR), Manaus (AMA) (middle Amazonas River), Belem (ABE) (downstream Amazonas River), upstream Teotônio (UTM), Teotônio Rapids (TRM), and downstream Teotônio (DTM) and the last three in the Madeira River considered the most geographically complex tributary of the Amazon basin  for 216 samples and cytochrome b (Cytb – 1044 bp) for 140 samples. The amplifications of CR were performed with F-TTF primer by Sivasundar et al.  and the μL reaction mixture containing 2 μL DNA template (10–50 ng/μL), 1.0 μL 10× PCR buffer , 0.5 μL forward primer (5 μmol/L), 1.0 μL primer R (5 μmol/L), and 0.5 μL primer M13 labeled at the 5′ with FAM, HEX, or NED fluorescent dyes, 2.1 μL dNTPs (1 mmol/L), 0.30 μL MgCl2 (50 mmol/L), and 0.21 μL of Taq DNA polymerase 1U/μL. The amplification of loci was performed using the following thermocycling conditions: initial denaturation at 92°C for 1 min, followed by 25 cycles at 94°C for 20 sec; 60–62°C for 40 sec; 68°C for 35 sec, followed by annealing of the primer using fluorescence 20 cycles at 93°C for 20 sec; and 53°C for 30 sec an extension to this cycle at 72°C for 35 sec. The final extension for the whole process of amplification was 72°C for 30 min.Heterologous amplifications were performed using the microsatellite loci and PCR conditions developed by Batista . PCR wasμL of this dilution was used for genotyping with a solution containing 7.93 μL Tween-20 (0.1%) and 0.07 μL alleles size standard (TS 500) completing a final volume of 10 L.The amplified products were checked on agarose gels (1.5%). According to the intensity of the bands of amplified products were made dilutions from 1:10 to 1:40. A total of 1 Six loci were selected to show polymorphism . The anaThe overall genetic variability based on mtDNA was estimated using the following parameters: number of haplotypes (H), private haplotypes (PH), haplotype diversity (Hd), number of polymorphic sites (S), total number of mutations (ETA), and nucleotide diversity (Pi) by DnaSP 5.1 software , deviation of Hardy–Weinberg equilibrium (HWE), inbreeding coefficient (FIS), F and R statistics, and allelic richness (Ar) obtained with POP-GEN 1.32 , expected and observed heterozygosity (FST) were conducted with 16,000 permutations to test the significance of pairwise population comparison implemented in ARLEQUIN 3.11 to both mitochondrial and microsatellites markers. The haplotype network was constructed for the mitochondrial markers in NETWORK 4.516  and the fixation index  model implemented in BEAST 1.5.4   were observed registering an overall haplotype diversity h - 0.931 ± 0.000. Considering all base positions, 618 were invariant, 45 were polymorphic sites, and 37 sites were parsimony informative. The total number of haplotypes between sites varied from H - 11 (UTM) to H - 18 (AIP) with absolute frequency of each haplotype between 1 and 21. Shared haplotypes were 19 (28.78%). Haplotypes 4, 5, and 8 were shared among more than one site, mainly of the Amazon River channel. The remainder was shared between two locations except the haplotypes 50, 52, 53, 55, 56, and 58, which were shared only among the Madeira River sites . The highest haplotype diversity was recorded in AMA, h - 0.915 and AB h - 0.905. The Madeira River presented the lowest haplotype diversity (h - 0.635) compared to PR with a haplotype diversity of h - 0.892. All estimates of Tajima's D were positive except for TRM. However, none was significant . Only 21 sites were polymorphic, defining a total of 14 unique parsimony informative haplotypes with absolute frequency of each haplotype between 1 and 16. The total number of haplotypes between locations varied from H - 3 to H - 7 for PR and AMA, respectively. Haplotype sharing was low among only six of the 21 haplotypes (28%) present in more than one site. The haplotype 1 was shared among all locations; haplotype 2, among AIQ, AMA, and ABE; haplotype 4, among AMA, PR, and AIQ; haplotype 16 and 17, among the three sites of the Madeira River ; and haplotype 19, between DTM and TRM. Haplotype diversity ranged from h - 0.545 in PR to h - 0.893 in UTM, and in the same place of the highest haplotype diversity value (UTM) h - 0.893, the second largest number of private haplotypes (H - 3) was observed. Values of Tajima's D were negative, but none was significant (P > 0.05). FigureFor the cytochrome HO - 0.055  to HO - 0.864  and from HE - 0.055  to HE - 0.849 , respectively. Estimates of the inbreeding coefficient (FIS) ranged from 0  to 0.867 . The positive values for FIS index suggested the existence of heterozygote deficiency in 18 of the 36 comparisons in the six locations analyzed, and these values could be derived from inbreeding or from the presence of a substructure within the population. Deviations from HWE were observed in 2 of the 36 estimates performed for the loci analyzed among populations after applying the Bonferroni correction (P < 0.007). The only loci with deviations were BR49 in AMA and BR53 in AIP. The significant values of HWE test were consistent with the estimates of the FIS index and the deviations from HO and HE of each of the assessed locations indicating that low levels of heterozygosity were recorded (HO < HE), and therefore, the values of the FIS index were positive (Table S2). HWE departure is common with microsatellites . A total of 37 alleles were detected in all samples. Private alleles were recorded for the locations of AMA, AIQ, UTM, and TRM, which presented frequencies between 0.029 and 0.083. The number of alleles per locus ranged from 2 to 10 with an average of 6.43 alleles per locus . The obsFST - 0.533 (P < 0.05). The pairwise FST values of the two mitochondrial markers indicated substantial differentiation among the sites sampled in the main channel of the Amazon River and in the Purus River, compared to the sites of the Madeira River, with values ranging from 0.005 to 0.760 (P < 0.05) for the control region and from 0.007 to 0.779 for cytochrome b  and the Madeira River with its three sites . The result obtained revealed that most of the total molecular variance was partitioned between the two identified groups, supporting the FST values for pairwise comparisons. The results indicated that a low percentage of the total variance was attributed within sites (33.60% for control region and 29.21% for cytochrome b) , Purus River (PR), Manaus (AMA), and Belem (ABE) and a second group including the three Madeira River localities .For the genetic structure initially analyzed, all sites were clustered as a unique group. The results of the AMOVA analysis showed a higher percentage of variation among the sites 53.35%) than within the sites 46.65%) with a .35% than6.65% witFST and RST indices displayed moderate values ranging from 0.001 to 0.239 and from 0.004 to 0.339, respectively. Some values were highly significant , showing the existence of genetic differentiation among the samples analyzed   Table. The dif Slatkin . HoweverK - 1–8 populations showed the highest likelihood (ln(P)D) in a population structure of K - 2 (−2113.46 ± 0.171), a result corroborated by the estimation of ΔK  with P - 0.001, respectively, indicating that isolation by distance is unlikely to have generated the observed structure pattern.The Mantel test using the microsatellites data did not show any relationship between the geographic distance and the values for the fixation index (b - −2454.27), relaxed lognormal , and the exponential  molecular clock showed that the exponential model was more adapted to our dataset for both markers. Similarly, the demographic tree prior test indicated that the constant size (BF - −2109.85) hypothesis cannot be rejected as compared with the exponential growth (BF - −2110.13) and expansion growth coalescent priors (−2110.00). Consequently, the relaxed exponential clock and constant population size prior were used to estimate the divergence time. The results show that the groups diverged during early Pleistocene 1.4 Mya (0.2–2.7 Mya 95% HPD) with control region  indicating an excess of low-frequency haplotypes, while in Madeira River group, these values were not significant . The demographic inference using the BSP model is summarized in Fig.eT as a function of time; the uncertainty in the estimated parameters was evaluated using 95% highest probability density (HPD) intervals. The estimated expansion time of individual group was of 0.5–0.25 Myr (95%HPD) for Amazon basin and 0.5–0.45 Myr (95%HPD) for Madeira River. Significantly negative Fs values and Bayesian skyline plots depicting growth provide evidence that group Amazon basin have undergone recent expansion.The difference in likelihood between the strict , with the fixation index showing significant values between Amazon River and Madeira River. The FST values were supported by the statistical network; two weakly supported groups were identified with haplotype network, namely the Amazon River and the Madeira River , the genetic structure was associated with the homing behavior, a process normally related with genetic segregation and association with geographic distribution. However, the Mantel test does not present evidences for isolation by distance among populations in our study. Additionally, we found a high genetic variability in the locality of Belem (ABE)  and the localities of Iquitos (AIQ) and Purus River (APR). These results did not show pattern of segregation genetic, like is normally waited in the homing behavior.Patterns of genetic structure have been identified for other migratory species in the Amazonian basin as in Evolutionary biology and ecology assume that genetic diversity declines and differentiation increases toward the edge of a species' geographic range, where populations tend to be smaller and more isolated. Our results show a different pattern, and we find high genetic diversity between the localities more distant (Iquitos IQ and Belem BE) share most haplotypes mainly with the localities of Manaus and Purus rivers. In contrast, the localities sampled in the Madeira River recorded the lowest genetic diversity and the pairwise significantly higher genetic differentiation with other localities. Our results may indicate that the locality of Madeira River experiences higher population genetic drift and gene flow lower with other localities, possibly due to smaller population sizes, spatial isolation, and a more complex landscape structure.B. platynemum was supported by phylogenetic analyses based on the relationships of haplotypes of both mitochondrial markers. In agreement with the calibration of molecular clock, we found that the groups probably diverged about 1.4 Mya (0.2–2.7 Mya 95% HPD) using control region and 1.0 Mya (0.2–2.5 Mya 95% HPD) using cytochrome b , and also on the pattern of strong genetic structure observed, it is likely that climate changes isolated a population of this species in the Madeira River. According to Hubert et al.  in the upper Madeira River, probably proceed from the Aripuanã refuge. Similar patterns were observed in other fish species, such as Cichla . In contrast, the inferred demographic histories and divergence times of Madeira River group suggest that these populations were able to survive in refuge within colder regions, followed by demographic increases but without evidence of significant range expansion. It is probable that some ecological and chemistry barriers, perhaps between Amazon basin and Madeira River, have maintained the isolations of these populations. Processes of phenotypic differentiation between Amazon and Madeira River populations should not be discarded, but it requires to be confirmed by morphological analysis. However, owing to the small sample sizes for Madeira River group, these results require cautious interpretation, and we suggest that further sampling is needed in these localities before definit-ive statements about the demographic histories of these lineages can be made.Recent studies indicated that the upper Amazon was colonized during the past 4 Mya, and only during the last million years, some of the tributaries were colonized Aleixo , while tt et al. , SerrasaB. platynemum. We identified two distinct groups within this fish species, corresponding to two geographic regions , probably resulted of differences of water chemistry within Amazon basin tributaries and of climate oscillation promoting marine transgression and regression during Pleistocene. Therefore, preservation of genetic diversity and evolutionary potential within this species will rely on ensuring that populations within each of these two lineages are targeted for conservation and be managed independently in fisheries statistics.In addition to other types of molecular studies, the limitations of the techniques , the large geographic ranges of some species, the stochastic effects of sampling, the difficult regarding to understanding species boundaries, and a poor taxonomy of many Neotropical fishes groups conspire to make phylogeographic studies with Amazonian fishes large challenging (Albert and Crampton


Oryctolagus cuniculus) naturally occur and hybridize. In this study we explore the relative roles of historical and contemporary processes in shaping the spatial genetic structure of the rabbit across its native distribution range, and how they differently affect each subspecies and the hybrid zone. For that purpose we obtained multilocus genotypes and mitochondrial DNA data from 771 rabbits across most of the distribution range of the European rabbit in Spain. Based on the nuclear markers we observed a hierarchical genetic structure firstly comprised by two genetic groups, largely congruent with the mitochondrial lineages and subspecies distributions , which were subsequently subdivided into seven genetic groups. Geographic distance alone emerged as an important factor explaining genetic differentiation across the whole range, without the need to invoke for the effect for geographical barriers. Additionally, the significantly positive spatial correlation up to a distance of only 100 km supported the idea that differentiation at a local level is of greater importance when considering the species overall genetic structure. When looking at the subspecies, northern populations of O. c. cuniculus showed more spatial genetic structure and differentiation than O. c. algirus. This could be due to local geographic barriers, limited resources, soil type and/or social behavior limiting dispersal. The hybrid zone showed similar genetic structure to the southern populations but a larger introgression from the northern lineage genome. These differences have been attributed to selection against the hybrids rather than to behavioral differences between subspecies. Ultimately, the genetic structure of the rabbit in its native distribution range is the result of an ensemble of factors, from geographical and ecological, to behavioral and molecular, that hierarchically interact through time and space.The Iberian Peninsula is the only region in the world where the two existing subspecies of the European rabbit ( In most species, populations are genetically structured. This genetic structure may be a consequence of many factors. Foremost among these, geographical factors may lead to vicariant events and divide populations , or ecolThe processes leading to population structure can act at different temporal and spatial scales. Temporarily, historical processes such as isolation in glacial refugia and subsequent expansions can leave detectable signals in the current populations , as wellOryctolagus cuniculus Linnaeus 1758) is a species with worldwide biological and economic importance that has long attracted scientific interest  inhabiting the southwest of the Iberian Peninsula, and lineage B, which predominates in O. c. cuniculus in the northeast of the Peninsula  shaping the spatial genetic structure of the rabbit in Spain, and (2) determining the differences in genetic variability and structure between populations of Samples of 771 European rabbits were obtained from 30 localities covering most of their range in Spain . Samplinn = 144) were a priori assigned to subspecies O. c. cuniculus, 14 localities (n = 410) to subspecies O. c. algirus, and 10 localities (n = 217) to the hybrid zone  following the manufacturer’s instructions.b gene using the primers OcunCB_F: 5′-ATGACCAACATTCGCAAAACC-3′ and OcunCB_R: 5′-TGTCTCAGGGAGAACTATCTCC-3′. The PCR reaction was performed in a final volume of 25 µL containing: 200 µM of dNTPs, 0.2 µM of each primer, 1 U Taq polymerase (Eppendorf), 1× PCR buffer , and 1 µL of DNA extract. The PCR program consisted of 4 min of denaturation at 94 °C, followed by 40 cycles of 1 min at 94 °C, 1 min at 55 °C and 1 min 30 s at 72 °C, plus a final extension of 10 min at 72 °C. PCR products were digested separately with HaeIII and AluI restriction enzymes (Promega) and migrated in 3% agarose gels stained with ethidium bromide for subsequent visualization under UV light. RFLP patterns were identified as described previously , 1× PCR buffer , and 0.5 µL of DNA extract. PCR programs involved 2 min of initial denaturation at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s between 55 °C and 65 °C, 30 s at 72 °C, followed by a final extension step of 7 min at 72 °C. The amplified fragments were analyzed in an ABI3730 automatic sequencer (Applied Biosystems) and allele sizes were assigned using the program GeneMapper v3.7 (Applied Biosystems). The complete data file of microsatellite genotypes and mitochondrial haplotypes was deposited as a All individuals were genotyped according to 10 microsatellite markers: Sat3, Sat4, Sat5, Sat7, Sat8, Sat12, Sat13, Sat16, Sol33 and Sol44 . PCR reaK, from K = 1 to K = 30. Each analysis consisted of 1 × 106 Markov chains with a prior burn-in of 1 × 105 chains. Mean posterior probability values were used to calculate ΔK, a measure of the rate of change of the posterior probabilities between successive K values. Thus, it is possible to detect when the increase in lnP(X|D) is not significant anymore and find the true value of K , allelic richness (RA), observed and expected heterozygosity (oH and eH) and inbreeding coefficient (ISF) for each locus and genetic group using the programs GENETIX 4.02  . AMOVA wmorphism . To avoiijf) (ijd), to give a regression slope lndb and its statistical significance , where ijF is the average kinship coefficient between individuals closer together , and lndb is the slope of the linear regression of the correlation coefficients and the logarithm of the geographical distance. All these tests were conducted in SPAGeDi 1.2 (STF/1−STF) and geographical distances of the localities studied. Also, the presence or absence of putative barriers, such as large rivers and/or mountain ranges, between localities was coded as 1 and 0 in a third data matrix. Using these three distance matrices, a partial Mantel test was performed to determine whether, besides geographical distance, these landforms represented a barrier to gene flow for the rabbit. Both analyses were performed in ARLEQUIN 3.1.The effect of geographical distance on the genetic differentiation between individuals was also tested for all the samples in peninsular Spain  and for each lineage and the hybrid zone separately. Regression was performed for the kinship coefficient between pairs of individuals (fij)  and theiificance . Also, sificance . Geograpsampling , so to cling . In only five localities with over 10 individuals analyzed, all individuals belonged to one lineage. Rabbits from Mallorca, Lérida and Galicia belonged to lineage B, while those from Jaén3 and Sevilla1 belonged to lineage A. Although all the other localities showed a mixture of both lineages, there was a clear predominance of B haplotypes in the northeast of the Iberian Peninsula and Balearic islands, A haplotypes in the southwest, and a mixture of both in the center of the Peninsula .AN per locus = 26.29 ± 9.07). In all localities heterozygosity values were larger than oH = 0.6 (average oH = 0.7 ± 0.15). The localities showing greatest diversity, measured as allelic richness, were Madrid1 and Sevilla2 (RA = 7.70 and 7.62), whereas the least diverse were Mallorca (RA = 5.15) and La Rioja (RA = 6.20)  .Deviations from Hardy–Weinberg equilibrium were detected in 17 of the 26 locations analyzed . In all P(X|D) increased substantially from K = 1 to K = 2 and then was attenuated as the number of K increased but without reaching a clear asymptote. Notwithstanding, calculation of ΔK clearly revealed the existence of 2 genetic populations or groups (K1 and K2). Taken together, these results suggest that the sampled rabbits belong to two large and distinct genetic groups, but do not completely exclude the possibility that more gene pools exist. The distribution of the two genetic groups exhibited high geographical correlation such that the localities to the south of the Iberian Peninsula were assigned with a greater likelihood to group K1 and the northern localities to K2 group .Both the assignment probabilities to the genetic groups based on the nuclear DNA and the frequencies of mitochondrial lineages closely conformed to sigmoidal functions  for 7 genetic clusters. Most of the inferred genetic clusters showed a well-defined geographical distribution . The firGenetic diversity parameters estimated for the inferred genetic clusters in BAPS, indicated a greater diversity for K-ZH and K-S1, which were also the only clusters in Hardy–Weinberg disequilibrium, due to a significant deficit of heterozygotes . All genP < 0.001, P < 0.001, respectively). Conversely, STR was much higher , indicating that the effect of mutation is of greater importance than drift in the differentiation of rabbit genetic clusters . Relationships between individuals decreased rapidly as geographical distances increased and this autocorrelation was significantly positive up to a distance of approximately 100 km  than for O. c. algirus and the hybrid zone, which showed similar values , indicating a greater genetic structure of rabbit populations in the northeast of the Iberian Peninsula   , therefoe rabbit  could leOverall, the European rabbit in Spain showed considerable genetic structure, which was similar to that described for rabbit populations in the northeast and southwest of the Iberian Peninsula  and sligSTF and STR statistics, indicate that the rabbit has a hierarchical genetic structure. First, the oldest and largest differences are mainly reflected by the two genetic groups, based on nuclear markers, and their high STR values. Within these, there are other genetic groups identified in BAPS that are determined by other factors that could be either environmental or ecological. In turn, these inferred populations consist of even smaller groups conditioned by the social behavior of the rabbit, and are reflected by the significant values of STF between localities and the significantly positive spatial autocorrelation  showed greater relatedness among close individuals and more spatial genetic structure and differentiation than the southern populations or those in the hybrid zone (Lasiorhinus latifrons), that where soft soils occur the construction of burrows is facilitated so animals do not need to share their shelter with other groups of individuals , it is surprising that the hybrid zone shows so many exclusive alleles, when we would initially expect it to only hold the sum of the parental alleles. Unique alleles have been previously described in the rabbit hybrid zone for the HBA haemoglobin alpha chain gene, which probably originated by recombination of alleles from the parental lineages , showing a greater genetic introgression of lineage B, characteristic of the northern O. c. cuniculus, into lineage A, O. c. algirus, than vice versa. This is consistent with recent findings related to autosomal loci , but significantly greater proportions of individuals had been assigned to K2 (K1 = 0.37 and K2 = 0.63) (mal loci , yet conmal loci . The facmal loci . In thismal loci . In thismal loci .Ultimately, multiple factors ranging from geographical and ecological, to behavioral and molecular, are interacting and shaping the overall genetic structure of the rabbit subspecies and their hybrid zone. Future studies using genomic data coupled with behavioral and ecological information could further clarify how these issues are related to the differences in genetic variation and structure of the rabbit subspecies.10.7717/peerj.582/supp-1Table S1Rabbit localities sampled in this work, number of individuals analyzed, geographical coordinates and subspecies occurring in each locality according to it its natural distribution range. Numbers correspond to those indicated in Click here for additional data file.10.7717/peerj.582/supp-2Table S2N, number of samples; AN, number of alleles; oH, observed heterozygosity; eH, expected heteroygosity; ISF, inbreeding coefficient.Click here for additional data file.10.7717/peerj.582/supp-3Table S3N, number of samples; AN, number of alleles; oH, observed heterozygosity; eH, expected heteroygosity; ISF, inbreeding coefficient.Click here for additional data file.10.7717/peerj.582/supp-4Data S1Full data set of 10 microsatellite genotypes, including locality, coordinates, and mitochondrial haplotype for each individual analyzed in this study.Click here for additional data file.


Triatoma sordida is widely distributed throughout the Chaco and the Eastern Region of Paraguay. It is associated to palm trees and artificial ecotopes located in peridomestic environments. The aim of this work was to determine genetic and morphometric variability and feeding behavior among population of T. sordida captured in domicile and peridomicile areas of Paraguay. Feeding contents and levels of genetic and morphometric variation were determined in 124 T. sordida from domicile and peridomicile populations of San Pedro and Paraguarí departments of the Eastern Region and Boquerón and Presidente Hayes departments of the Western region using Double Diffusion Gel, random amplified polymorphic DNA (RAPD), and head and wings morphometry. Morphometric analysis revealed isolation of populations by geographic region and larger size in triatomine populations from the Western Region. RAPD showed no specific patterns for domicile and peridomicile populations. The estimator of diversity  and high gene flow obtained  did not allow the establishment of genetic differentiation within the same region. The blood meal source showed that poultry feeding was 38% of host preferences, and human blood was the second feeding preference (24%) in the insects from the Eastern Region while poultry feeding was predominant in those from the Western Region (30%). This work showed homogeneity between T. sordida populations of the same region and between domicile and peridomicile. The genetic diversity was determined among T. sordida populations of both geographical regions suggesting differentiation associated to eco-geographical isolation by distance. It is important to notice that pattern feedings were different between the two regions. Further studies should be focused on how phenetic and genetic variations could be related to the adaptation capacity of these triatomine populations to domicile, increasing their vector potentiality in the transmission of Chagas disease. Trypanosoma cruzi among mammals. However, not all of them are epidemiologically important (Triatoma infestans (T. sordida (T. cruzi (The subfamily Triatominae (Hemiptera: Reduviidae) includes over 144 species of strictly hematophagous insects, considered potential vectors of nfestans  and T. s sordida  have beeT. cruzi , 8.T. infestans. T. sordida of wild origin seems to have been disseminated from Brazilian plateaus toward the south, and now is found in Argentina, Bolivia, Paraguay, and Uruguay where it occupies extensive geographical areas but generally in small populations of individuals  in ulations . Similar species . In relazygosity . Similarsomatids .T. sordida, the polymorphism levels and its implication in the infestation of dwellings in endemic areas for Chagas disease in Paraguay are still unknown. The objective of the present work was to determine the feeding behavior and genetic and morphometric variability among population of T. sordida captured in domicile and peridomicile of the two geographical regions of Paraguay. Finally, this study contributes to improve surveillance strategies embracing this potential vector.Thus, triatomines studies based on diverse molecular markers have been used to clarify phylogenetic and evolutionary relationships between species, apart from inferring divergences and population structure, as it has been demonstrated previously in others triatomines, where wide polymorphism between discrete populations suggest the existence of a species complex , 26. TheThe Eastern Region is humid, sub-tropical, composed by valleys, small hills, and wooded areas. The average annual temperature is 24.3°C and the average annual rainfall is between 1000 and 1600 mm. . The WesT. sordida according to Lent and Wygodzinsky  and Paraguarí (PA) departments of the Eastern Region of Paraguay; Boquerón (BO) and Presidente Hayes (PH) departments of the Western Region Figure . All triodzinsky .T. cruzi were identified as described by Hoare . All parameters were calculated using the JMP 4.0.0  was extracted; 34 of them were from the Eastern Region (PA) and 28 from the Western Region (Pte. Hayes). In order to do this, a section was made in the front third of the abdomen of the specimens. When the content volume was insufficient, the complete promesenteron was transferred to a vial . Each viThe Gel Double Diffusion method was carried out in a glass slide (7.5 cm × 5 cm) using 3.5 mL of 1.3% agar (I.D. Oxoid Agar) diluted in veronal hydrochloride buffer (pH 8.6). This preparation was maintained in a humid chamber for 24 h , 42. In The antisera used for the identification of the feeding source was against human, poultry (chicken), dog, cat, goat, mice, and guinea pig blood and were also put in contact with the intestinal content of the triatomines searching for the corresponding antigen. All the antisera were prepared and tested previously in the Laboratory of General Ecology of the University of Buenos Aires, Argentina.The extraction of DNA was carried out according to the protocol of Promega Wizard Genomic Purification Kit, USA  in five 1 (5′-TCACGATGCA-3′), A2 (5′-GAAACGGGTG-3′), L4 (5′-GTGGATGCGA-3′), and L5 (5′-AAGAGCCCGT-3′). The PCR was set up as follows: a final volume of 25 μL PCR mixture that contained 0.25 mM dNTPs , 10 pmol of primers, 1.0 unit of Taq polymerase (Gibco Life Technology), 5 ng of DNA template in a buffer with 2 mM MgCl; 50 mM KCl, 10 mM Tris–HCl, pH 8.8 was used; each amplification included a DNA-free negative control. The visualization of the products was obtained using 2.5% ultra-pure agarose gel electrophoresis stained with ethidium bromide. The bands obtained were digitalized by KODAK 1D  software.The amplification reaction was performed according to the protocol of Williams et al.  modifiedT. sordida populations were in Hardy–Weinberg equilibrium and that there were no selection processes favoring any particular genotype. All loci were entered in a binary matrix and a similarity index was obtained from this matrix (FST) was determined according to Nei and Chakraborty (Nm). These parameters were analyzed using the software POPGENE (version 1.31)  .T. cruzi. None of them showed natural T. cruzi infection, confirming in this occasion the low infection rates of T. sordida specimens in both regions (data not shown).Feces of 124 insects were analyzed looking for 2, R1, EO) was significant (p = 0.01–0.0001), excepting the post-ocular (PO) distance of the head (p = 0.08). The Bonferroni correction showed that the characters of the wings differentiated the sexual dimorphism better than those of the head.The Guillaumin profile allowed, in general, determining that individuals from the Western Region were larger than those from the Eastern Region. The sexual dimorphism of the analyzed structures  for head and (p = 0.001) for wings.The size analysis using the principal components showed that, although females were consistently larger than males, the populations from BO department had significantly larger wings and heads than those from the other studied localities Figure . The sigp = 0.0003 (head) and Wilks lambda = 042, p = 0.0001 (wings). With the elimination of the allometric size, only the head variables were compatible with the CPC  model . Through the DA, the specimens were correctly classified into their respective groups with considerable concordance (Kappa between 0.66 and 0.83). This canonical variation analysis also showed the isolation of PA population for both sexes : 30% for Gral; Bruguez community of Western Region and 38% for Cerro Guy community of Eastern Region. In the latter, the most frequent second blood source was human (24%) followed by cat and dog blood. In the Western Region, the second frequent blood source was multiple blood  where the most common blood mixtures were poultry-human, poultry-rat, and poultry-dog-cat.Figure Nm) was 5.7 individuals per generation.The RAPD profiles were complex Figure A, and thT. sordida invading and trying to colonize houses in both Paraguayan regions. These areas have been intensively sprayed and a tendency of invasion has been observed when residual insecticide activity ends. Previous reports demonstrated very low rates of domiciliary colonization of T. sordida in Argentina and Brazil , this fact seems logical as they are insects of the same species. However, we suppose that the separation between regions and the morphobiometric differences of PA populations could be related to the genetic changes caused by local selective pressures. The grouping observed in the dendrogram could be associated with epidemiological differences in their respective origin focuses, considering that the Western Region is an area with high pressure of triatomine infestation (T. sordida populations in Paraguay and the use of more sensitive molecular markers to compare these findings with cryptic speciation groups previously described (The similarity analysis shown by the estation , 58. Howescribed , 34.T. infestans populations that demonstrated allelic differences among neighboring localities, which increase among populations more distant from each other (T. sordida between neighboring populations and the results presented suggest a genetic homogeneity between T. sordida from the same region, which is due to the permanent genetic flow between neighboring populations. However, the observed heterogeneity between specimens from Western and Eastern Regions could be associated with the big distances and even with the presence of the Paraguay River as a geographical barrier, which would be in agreement with the separation obtained with the morphometric analyses, i.e., that the differences between PA and BO involves a differentiation process, possibly associated with the eco-geographic isolation by distance and absence of genetic flow. Feeding behavior also confirm differences in these two populations, while triatomines from the Western Regions showed a poultry feeding profile, patterns of the insects from Eastern regions were associated with poultry feeding and human blood feeding profiles in triatomines captured in domicile. Mixed feeding showed an intense mobilization behavior of these triatomines between peridomicile and domicile areas.The dendrogram is similar to the result previously obtained for ch other . It has ch other . The migT. infestans in Paraguay as other secondary vector species such as T. sordida are more frequently detected in the studied region.It is important to notice that the genetic analysis has shown intraspecific divergences that allow us to think of possible gene variations involved in the shape expression, although we could not discard the possibility that the separation reflected in the dendrogram is influenced by the variation of allelic frequencies of the individuals. In this case, new questions arise and further studies will be required with models of population genetics to obtain better markers related to the infestation risk of potentials triatomines, mainly as a consequence of the control of Further studies should be focused on how phenetic and genetic variations could be related to the adaptation capacity of these triatomine populations to domicile, increasing their vector potentiality in the transmission of Chagas disease.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Many reintroduction projects for conservation fail, and there are a large number of factors that may contribute to failure. Genetic analysis can be used to help stack the odds of a reintroduction in favour of success, by conducting assessment of source populations to evaluate the possibility of inbreeding and outbreeding depression and by conducting postrelease monitoring. In this study, we use a panel of 306 SNP (single nucleotide polymorphism) markers and 487–489 base pairs of mitochondrial DNA control region sequence data to examine 321 individuals from possible source populations of the Eurasian beaver for a reintroduction to Scotland. We use this information to reassess the phylogenetic history of the Eurasian beavers, to examine the genetic legacy of past reintroductions on the Eurasian landmass and to assess the future power of the genetic markers to conduct ongoing monitoring via parentage analysis and individual identification. We demonstrate the capacity of medium density genetic data (hundreds of SNPs) to provide information suitable for applied conservation and discuss the difficulty of balancing the need for high genetic diversity against phylogenetic best fit when choosing source population(s) for reintroduction. Castor fiber, to Scotland, we demonstrate the capacity of medium -density SNP (hundreds of single nucleotide polymorphisms) genotyping derived from RAD sequencing data , Telemark  and Elbe  fur trade (henceforth FT) refugia in the west, and the Voronezh , Konda , Azas  and Bulgan  FT refugia in Eastern Europe and central Eurasia  and it is listed under the Bern Convention (Appendix III) and the EU Habitats and Species Directive . Population estimates for Eurasia are around 1 million individuals  were available but few cross-amplify successfully to the Eurasian beaver . The C. canadensis samples were included in order to assess cross-amplification and polymorphisms in this closely related species. Samples came from blood stored in EDTA or a variety of tissue types  and were extracted using the DNeasy blood and tissue kit . DNA was quantified using a Nanodrop 1000  and was normalized to between 35 and 50 ng/μl concentration.Samples of beaver were collected from throughout Europe and Asia . DNA samples were placed in a randomized order, and negative controls and positive controls were placed on each plate as standard. Genotype scoring was conducted using Genome Studio (Illumina). After initial clustering of the SNP data, individuals that showed a low call rate (<95% of the SNPs) were rejected and the data were clustered excluding these individuals. All clusters called were manually inspected and adjusted by eye if necessary.SNP genotyping was conducted by ARK Genomics  on a BeadXpress System .Analyses were performed on two data sets: all loci, that is, the entire data set of 306 SNPs which remained once monomorphic and failed loci were excluded; and the reduced set of 104 randomly selected SNPs. This reduced set of SNPs was used for the SBasic population genetic statistics, such as Hardy–Weinberg, heterozygosity, allelic richness, Fst, and the assessment of the markers’ power for individual ID and parentage testing were conducted in Genalex version 6  in Genalex 6 to gain a general overview of the structure of the data set. Principle component analysis has been shown to have a high power of population assignment  and the model of correlated allele frequency (λ = 1). Ten independent replicates of K = 1–20 were conducted. How to choose which value of K is biologically meaningful remains a subject to debate  was not significantly lower than the K + 1'th value  is integ et al. ). As thes et al. ). Using albicus type’.To examine the evolutionary relationship between the FT refugial beaver populations, a phylogenetic network of the SNP data was assembled using the method neighbour-net (Bryant and Moulton http://www.biomatters.com), with final correction done by eye. Sequences were aligned to the GenBank sequence NC_015108.1 .The alignment was visualized using the median-joining network  SNPs scored successfully and were polymorphic (Data S1). Thirty-three SNPs were discarded because they either failed outright (22) or gave profiles that could not be scored (11). A further 45 SNPs were discarded because they amplified, but gave profiles that were monomorphic in the samples screened.C. canadensis can be found in Of the 306 polymorphic SNPs for Eurasian beaver, 250 (79%) cross-amplified successfully in the Canadian beaver (scored in ≥3 beaver) and 8 (2.5%) were polymorphic. No markers were fixed for alternate alleles between the two species. A list of eight polymorphic SNP markers for Of the 321 animals screened, 279 had profiles which amplified for 95% or more of the 306 SNPs; these were taken forward for further analysis. A table of raw genotype data can be found in Polymorphism and heterozygosity at the 306 markers varied widely across populations Table  in the dUsing principle component analysis , clustering of the genotype data was analysed using four different overlapping SNP (sub)sets: all 306 SNPs, the ‘random’ panel (104 SNPs) and the panels that were chosen to be variable in Bavaria (84 SNPs) and Norway (81 SNPs) . The effK , so K = 5 was chosen as the lowest value of K that captured the majority of the data structure. The five clusters which fell out consistently across all ten replicates in the following order were as follows:In STRUCTURE, posterior likelihood (LnPD) showed a pattern of increase towards an asymptote with increasing K . Between1. Norway, 2. Central Eurasian populations  3. France, 4. Germany: Hesse and 5. Eastern European populations Fig. . These fThese population structure analyses of nuclear data reveal a number of factors about the Eurasian beaver which are briefly summarized here:C. f. fiber) population represents a distinct population that appears not to have introgressed with other European populations.The Norwegian  population also represents a distinct population that appears not to have introgressed with other European populations.The French  clustering entirely with other populations .Despite widespread reintroductions in Germany Table , there dC. f. belorussicus) and Voronezh (C. f. orientoeuropaeus) in Russia appear to belong to a common genetic cluster and group with the populations from Lithuania and Poland and Russia Kirov, both reputedly mixed belorussicus and orientoeuropaeus  falling out at K = 6 and a Lithuania/Poland cluster at K = 7. At K = 8, a clear split within the Belarus (belorussicus) populations becomes apparent , or showing introgression between Norwegian, Eastern European and French clusters. The six animals that belonged entirely to the French cluster came from the Rhône watershed area in the Swiss cantons of Geneva, Vallais and Vaud and are presumably immigrants from France. Hardy–Weinberg disequilibrium in the Swiss ‘population’ is high for the reason that it actually spans a number of disconnected populations. Fine-level population structure was not the purpose of this study, but see Frosch et al.  and the reintroduced populations of Germany: Bavaria (50%), BWB (80%) and Hesse (10%), grouped within the western clade  was broadly concordant. Under both analyses ‘eastern’  and ‘western’  haplotypes original to the Durka et al.  papers cA network of the haplotypes reveals that there were 42 mutational steps needed to move across extremes of the network from haplotype nh3 to bi1 . In all of the populations except Mongolia and Russia: Azas, a panel of 20 or fewer markers was sufficient to have a probability of identity (PID) of <1/10 000. This increases to a panel of 30 or fewer assuming a population of full sibs (PID_SIB) (Sup.Mat 4). Due to the low number of polymorphic markers in Mongolia and Russia: Azas, the maximum achievable PID in these populations was 0.00712 (7 markers) and 0.09879 (four markers), respectively. In all but Mongolia, Russia: Azas, Russia: Konda and France, 30 or fewer markers was sufficient for parentage assignment . Tables of probabilities and markers suitable for addressing parentage and individual ID questions in each population are available in There are two predominant reasons why it is desirable to conduct genetic analysis in support of population reintroduction and augmentation work:To aid with the selection of founder population(s) and individuals from within these population(s) to be released.To conduct ongoing management and postrelease monitoring of population.−1μl), but uniplex SNP assays or approaches using rapid parallel SNP genotyping based on nanofluidic dynamic arrays  . VariatiThe first consideration, to aid with the selection of source populations and individuals from within these populations for reintroduction, is more complex. There are three primary genetic considerations when choosing animals for a reintroduction, which may not always be synergistic:To select individuals with low levels of inbreeding and high combined genetic diversity.Conversely, to ascertain that the introduced combination of animals is not likely to suffer outbreeding depression.To select the most similar individuals to those historically present IUCN .These issues will now be examined in turn with respect to the results of this study.Arguably, the most important consideration is choosing individuals with low inbreeding coefficient and a high combined genetic diversity. Most animals of conservation concern either currently exist in small populations or have come from populations that have previously undergone a bottleneck (as in this example of the Eurasian beaver) and so are subject to high levels of inbreeding. Although the possibility of inbreeding is often skimmed over by conservation practitioners [as illustrated by the fact that 37% of published reintroduction studies report fewer than twenty founding animals or fail to report the number of founders  pedigree data, not measures of genetic diversity (e.g. heterozygosity) alone, especially if only few genetic markers are available  and albicus (Germany: Hesse) samples  is higher than in the FT refugial populations  Table . This fies Table   and C. f. orientoeuropaeus (Voronezh). While the phylogenetic tree of the nuclear data , which should group geographically within the eastern populations according to Durka et al. (putative C. f. belorussicus) exhibits a mixture of haplotypes that span the putative east/west division , Russia (Voronezh) and French stock, show more recent signs of admixture between the expected population clusters  as indicated by the more variable contribution from each cluster within admixed individuals  may not share the same adaptive traits , and distantly related populations may evolve similarly adaptive traits in parallel. In the case of the British population of the Eurasian beaver, which went extinct in the 16th century  is the least desirable in view of the low genetic diversity in FT refugia populations, although we underline that there is no experimental evidence available for inbreeding depression in beaver and highly bottlenecked, single source populations exist apparently successfully. We also conclude that there is no genetic evidence to preclude either of the two remaining scenarios proposed by Halley  as we seGenetic considerations are not the only consideration when choosing founders for a reintroduction. Decisions must be made based on availability of source animals, the impact of removing of the founding animals from the source population, animal welfare, veterinary and ecological consideration IUCN , 2013. R


Aedes aegypti mosquito. Ae. aegypti is prevalent in the tropics and sub-tropics and is closely associated with human habitats outside its native range of Africa. While long established in the southeastern United States of America where dengue is re-emerging, breeding populations have never been reported from California until the summer of 2013. Using 12 highly variable microsatellite loci and a database of reference populations, we have determined that the likely source of the California introduction is the southeastern United States, ruling out introductions from abroad, from the geographically closer Arizona or northern Mexico populations, or an accidental release from a research laboratory. The power to identify the origin of new introductions of invasive vectors of human disease relies heavily on the availability of a panel of reference populations. Our work demonstrates the importance of generating extensive reference databases of genetically fingerprinted human-disease vector populations to aid public health efforts to prevent the introduction and spread of vector-borne diseases.Dengue fever is among the most widespread vector-borne infectious diseases. The primary vector of dengue is the  Aedes aegypti. Breeding populations of this mosquito were reported for the first time in California in summer 2013, and the origin of these populations was unknown. We genotyped Ae. aegypti from California and compared their genetic composition and diversity against reference populations from Asia and the Americas, and with laboratory strains, to identify the source of this introduction. We identified the southeastern United States of America as the likeliest origin of California Ae. aegypti. This information will aid the design of containment, control, and elimination strategies to prevent the establishment of a permanent breeding vector population in the state. Note that the resolution achieved in tracking the origin of new disease-vector introductions is proportional to the completeness of the reference dataset. Our analysis was delayed to gather further data on regions close to California and would otherwise have been completed within a week. This work highlights the importance of having an extended dataset of human disease-vector genotypes in place prior to the occurrence of invasions that compromise public health.Dengue fever is a vector-borne disease that has dramatically increased in frequency and severity in recent decades, affecting over half of the world's population. The primary vector of dengue is  Aedes aegypti is the mosquito species responsible for DENV transmission. Ae. aegypti is a domestic species in the sense that, outside Africa, it is closely associated with human habitats and is often transported by humans and their commerce Ae. aegypti is currently found in the southern states, Arizona eastward, with year-round breeding confined to latitudes below 33°N Ae. aegypti near airports Ae. aegypti in California were never reported until the summer of 2013, when they were found in the Central Valley counties of Fresno and Madera and the coastal county of San Mateo  after many years of absence an Mateo . When exAedes aegypti adults and larvae for this study came from collections made between 2004 and 2013 from 33 locations across 11 countries in the Americas and Asia (and Asia . MosquitAedes aegypti mosquitoes with the DNeasy Blood and Tissue kit (Qiagen), according to manufacturer instructions. Samples were treated with 4 µl of RNAse A (Qiagen) and stored at −20°C until further analysis. Our reference dataset included eleven populations of Ae. aegypti from around the world that have been screened for allele frequencies at 12 highly polymorphic microsatellite loci in previous studies Total nucleic acids were extracted from 1205 individual is, as well as for linkage disequilibrium (LD) among loci pairs, using the online version of the GENEPOP software is, analogue to Fis, was also calculated in GenoDive v.2.0b25 All microsatellite loci were analyzed for within-population deviations from Hardy-Weinberg equilibrium (HWE) by Fa priori information of sample location. The most likely number of clusters (K) was determined by conducting 20 independent runs from K = 1 to 29 on individuals of all American and Asian populations, and from K = 1 to 21 on individuals of the North American cluster. Each run assumed an admixture model and correlated allele frequencies using a burn-in value of 100,000 iterations followed by 500,000 repetitions. The optimal number of K clusters was determined following the guidelines of Prichard et al. Population structure and assignments of individuals from the three California populations to their genetic cluster of origin was performed via the Bayesian clustering method implemented by the software STRUCTURE v. 2.3 N = 10,000 and α = 0.05GENECLASS2 To test for possible sample size effects on STRUCTURE, we evaluated the possibility that uneven sample size might have an effect on the STRUCTURE analysis and our conclusions. We performed resampling of the data from all 20 North American samples, with and without replacement, with all samples equal to 15 (without replacement) and 30 (with replacement) genotypes. When the genotypes were subsampled without replacement, 6 out of 10 runs resulted in K = 3 being the optimal number of clusters, while the remaining 4 had an optimal K = 2. When the genotypes were resampled with replacement, the optimal number of clusters was K = 3 for 7 out of 10 runs, K = 5 for 2 out of 10 runs, and K = 2 for 1 out of 10 runs. In all simulations, the California samples had greatest affinity to the Southeast US group samples at K = 3 (data not shown).Raw data  can be found in Supplementary Materials, Ae. aegypti, to address whether the California introduction was a consequence of a laboratory release (o) for natural populations was significantly higher than that of laboratory strains . Allelic richness across loci within natural strains was 3.6206±0.5681 and 2.1633±0.6104 within lab strains, consistent with the genetic diversity pattern observed from the heterozygosity values. When the California populations were analyzed independently, the Ho values of all three California collections fell within the values of the natural populations  and a set of reference populations from the Americas and Asia . Becauselifornia . GeograpLikelihood estimation methods In addition, we ran a Principal Components Analysis (PCA) on the allele frequencies of the 20 North American samples. The California samples are indistinguishable from samples from the Southeast US, but distinct from all other samples .Ae. aegypti was introduced to California from the southeastern USA by multiple founder mosquitoes. Populations of recently introduced species are often relatively low in genetic variation due to the new populations having been started by a few founders. In comparing the level of genetic variation of California Ae. aegypti populations to other populations there is no indication of a decrease in genetic diversity . This suggests that commerce via air, railroad, or trucks from the New Orleans/Houston areas may have been responsible, though the mode of introduction remains unknown.Our data indicates that the origin of the California invasion can be tracked to the region of New Orleans and Houston, cities ∼300 km apart, and likely did not originate from spillover from geographically adjacent regions that have long had Ae. aegypti invasion into temperate Netherlands The extensive mosquito surveillance program in California allowed for a rapid response to the initial detections, emphasizing the importance of enhanced surveillance and personnel training, especially in regions susceptible to invasion of exotic disease vectors. In the USA, dengue became a nationally notifiable infectious disease in 2010, and for 2013, California has reported 124 imported cases Ae. aegypti is proportional to the completeness of the reference population panel. Prior to this study, we had very sparse sampling from Arizona and northern Mexico; this delayed our analyses by several months while additional samples from this region were obtained and genotyped. Had our genetic panel been more complete, the study could have been carried out within a week. This highlights the importance of having in place extensive population genetic databases for invasive vectors prior to new invasions, which occur on a regular basis and are expected to increase as the climate patterns change. While spread of invasive vectors usually occurs inadvertently, purposeful introductions by bioterror activities could be tracked in a similar manner. An analogy can be made with the notorious 2001 anthrax event, whereby the origin of the strain was quickly traced thanks to an extensive database of DNA sequences of hundreds of anthrax genotypes The degree of accuracy in the identification of the source population of the California Figure S1Aedes aegypti. STRUCTURE bar plots indicating relatedness of Aedes aegypti populations worldwide, based on 12 microsatellite loci. Each vertical bar represents an individual. The height of each bar represents the probability of assignment to K = 2 optimal clusters (different colors) determined using the Delta K method. Pantropical cluster is shown in pink and the African cluster in blue. Populations from the African cluster were originally analyzed in Brown et al. 2011 California populations belong to the pantropical cluster of (EPS)Click here for additional data file.Figure S2Aedes aegypti using 12 microsatellite loci. Note that California samples (*) are indistinguishable from samples from the Southeast US.Principal Component Analysis in 20 North American samples of (EPS)Click here for additional data file.Table S1Aedes aegypti populations analyzed for 12 microsatellite loci.Collection information for (DOCX)Click here for additional data file.Table S2Number of alleles per microsatellite locus.(DOCX)Click here for additional data file.Table S3Aedes aegypti populations.Genetic diversity of (DOCX)Click here for additional data file.Table S4Raw allele frequencies at 12 microsatellite loci.(DOCX)Click here for additional data file.Text S1Marker validation.(DOCX)Click here for additional data file.


In the present study we have assessed whether the Carpathian Mountains represent a genetic barrier in East Europe. Therefore, we have analyzed the mtDNA of 128 native individuals of Romania: 62 of them from the North of Romania, and 66 from South Romania.outside the Carpathian range (South Romania) displayed some degree of genetic differentiation compared to those living within the Carpahian range (North Romania).We have analyzed their mtDNA variability in the context of other European and Near Eastern populations through multivariate analyses. The results show that regarding the mtDNA haplogroup and haplotype distributions the Romanian groups living The main differentiation between the mtDNA variability of the groups from North and South Romania can be attributed to the demographic movements from East to West (prehistoric or historic) that differently affected in these regions, suggesting that the Carpathian mountain range represents a weak genetic barrier in South-East Europe. Assessing the genetic structure of different populations is important for understanding the population history of a certain geographic area from a genetic point of view. It is also important from the epidemiological point of view to avoid spurious association between genetic markers and certain diseases ,2. The gWhile most of the genetic studies of European populations have focused on the Western part of the continent, few studies have assessed the genetic structure of Eastern Europe. Among these, one study assessing the variability of the Y-chromosome has proposed that, although the population structure of the Carpathian basin is relatively homogenous, the Carpathian range from the present territory of Romania represents a genetic barrier between populations living in the East and West of this barrier and mountain populations encompassed within the Carpathian arch . In thisThe present population of Romania is homogenous from a cultural and linguistic point of view, with an absence of language dialects within the borders of the country despite the relatively large and diverse geographic areas inhabited. Several historical events impacted the demography of Romanian populations inside and outside the Carpathian range. In this regard, although relatively little is known about potential differences in the Neolithic and the Bronze age, an impact of Celtic migration has been reported mainly in Transylvania (inside the Carpathian range), rather than in the planes East and South of the mountains . During The mitochondrial variability obtained in the population from the North of Romania  N = 62) was classified in 51 different sequences or haplotypes, which indicates a high degree of sequence diversity (0.9905 ± 0.0072) , was classified in 43 different sequences or haplotypes, which indicates also a high sequence diversity (0.9804 ± 0.0059), but lower than that obtained in the sample from the North of Romania Table . ThirteeST test . This could be due to the fact that North and South Romanian samples share only eight mitochondrial haplotypes. On the other hand, the analysis of the mitochondrial haplotype variability obtained in Romanian samples in the context of the Near Eastern and North East European populations, showed that 30 out of 51 haplotypes (58%) obtained in the North Romanian sample have been found only within the populations of North East Europe, and 21 out of 51 haplotypes (41%) have been found in both North East Europe and the Near Eastern populations. Regarding the Southern Romanian sample, 16 out of 43 haplotypes obtained (37%) have been found only in the Near Eastern populations, and 10 out of the 43 haplotypes obtained (23%) have been found in both the Near East and North East European populations.In the First Component (43% of the variance) of the Principal Component Analysis (PCA)   Figure . Thus, tThe Bulgarian, Hungarian, Russian and Czech populations are located between the North and South Romanian populations in this First Component. Bulgaria shows more similarity with South Romania than with North Romania regarding the frequency distribution of haplogroups K (7.5%), HV (6%) and H (43%) . HoweverThe Second Component of the PCA, which explains 14% of the variance, did not show a clear clustering of populations. The haplogroup with a higher correlation with this Second Component is haplogroup M . The populations from Northern Romania, Czech Republic, Russia and Bulgaria differ from the rest in their high frequency for haplogroup M (4.3-0.9%) -14, wherFST distance matrix. The analysis showed an RSQ of 0.98037 and a stress value of 0.07343, which indicates that the representation of the MDS obtained showed a good description of the real mitochondrial variability. In the MDS analysis, the Near Eastern and the European populations presented the greatest distance  analysis, where all mitochondrial variability was taken into account, was also carried out to provide a two-dimensional view of the e Figure .The analNext, we decided to obtain Median Joining Networks (MJN) for haplogroups J Figure  and K K because In the present study we carried out an assessment of the mtDNA variability of Romanian populations from outside (South Romanians) and within (North Romanians) the Carpathian mountain range, in order to assess its influence as a potential genetic barrier on the genetic variability of Romanians.The North Romanian population exhibited several differences in the frequency distribution of certain mitochondrial haplogroups comparing with other populations. Haplogroup H showed a very high frequency 59.7%) Table , while i9.7% TablHomo sapiens out of Africa, and it can be found mainly in Southern Asia, having a high frequency in Indian subcontinent [The frequency of the remaining mitochondrial haplogroups in the North Romanian sample  were within the European mitochondrial haplogroup frequency variation, except for haplogroups M, X and A, which are very rare in Europe. Haplogroups X and A are found mostly in Eastern Asia and America, and haplogroup M is the root of many of the haplogroups derived from the dispersion of ontinent . The preThe mitochondrial haplogroup variability  of the sample from Southern Romania is, in general terms, within the range of variation of other European populations, except for haplogroups HV and K. The frequencies of haplogroup HV 10.6%) and K (7.6%) are closer to the range of variation described in the Near East  compared with other European populations  [.6% and KST analysis based on haplogroup frequencies did not indicate statistically significant differences between North and South of Romania, the pairwise FST analysis based on mitochondrial sequence frequencies showed statistically significant differences between the two populations. The Northern Romanian haplogroup distribution is statistically significant different from that observed in the Near Eastern populations, while the haplogroup distribution from Southern Romania does not present such differences  or historic (Eastern Asian migrations) events that differentiate the region of Transylvania compared to the rest of Romania.outside the Carpathian range (South of Romania) displayed some degree of genetic differentiation compared to those living within the Carpahian range (North of Romania). However, this differentiation can be mostly attributed to the demographic movements from East to West (prehistoric or historic) that differently affected in North and South Romania.In the present study we evaluated whether Carpathian Mountain represent a genetic barrier in East Europe. Regarding the mtDNA haplogroup and haplotype distributions the populations living In the present work, we analyze the mtDNA variability of 128 individuals from two different Romanian regions separated by the Carpathian mountains, including 62 samples from Cluj-Napoca (North of Romania) and 66 from Dolj and Mehedinti (South of Romania) . Likewi Andrews .2, 0.1 μM of each dNTP, 0.4 μM of each primer, 5 units of Taq (Bioline) and 10 μl of diluted DNA (1 μl of DNA extract in 10 μl of distilled water). Cycling parameters were 95°C for 5 min, followed by 35 cycles of 95°C for 10 sec, 58°C for 30 sec and 72°C for 30 sec, and a final cicle of 72°C for 10 min. The sequence of the primers to amplify the HVS-I and HVS-II, were those in Hervella et al.[Life Technologies). The results obtained were edited with BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and the sequences were manually aligned.PCRs were performed in 25 μl of reaction mixture containing 10 mM Tris–HCl pH 8.3, 2 mM of MgClla et al.. In the Finally, in order to classify the mitochondrial variability of the individuals analyzed in this study, we proceeded to amplify 11 markers to define the 10 Western Eurasian haplogroups . The proST distances) were calculated on the basis of haplogroup and haplotypes frequencies using Arlequin software v3.11 [http://www.fluxus-engineering.com/). Given the high mutation rate of HVS-I, the substitution rates obtained by Meyer et al.[Intrapopulation genetic diversity parameters such as the number of different sequences (K), sequence diversity (Ĥ) , number re v3.11 . In addire v3.11 -14,27-50re v3.11 . This dier et al.,52 have The genetic barriers associated with each geographical location including in Figure The authors would like to declare no competing financial or personal interests in the preparation of this manuscript.Conceived and designed the experiments: CR, MGN, MI, SA, NI. Performed the experiments: MH, SA, NI, CR. Analyzed the data: MH, NI, SA, CR, MGN. Contributed reagents/materials/analysis tools: CR. Wrote the paper: MH, CR, MGN. Revised the manuscript critically for important intellectual content: SA, NI, MI. Approved final version to be published: MH, SA, NI, CR, MGN, MI. All authors read and approved the final manuscript.The different mitochondrial sequences (nps 16000–16399) obtained in this study as FASTA file.Click here for fileFST analysis: FST values  and p-values with standard deviation (p ± sd)  based on mitochondrial haplogroup frequencies .Click here for file


Revealing long-term effects of contaminants on the genetic structure of organisms inhabiting polluted environments should encompass analyses at the population, molecular, and cellular level. Following this concept, we studied the genetic constitution of zebra mussel populations from a polluted (Dp) and reference sites (Cl) at the river Drava, Croatia, and applied microsatellite and DNA damage analyses ). Additionally, mussels from both populations were exposed to polluted wastewater in the laboratory for three days, and DNA damage was analyzed to evaluate acclimatization and genetic adaptation of the investigated populations to the polluted environment. The two populations differed in their genetic constitution. Microsatellite analysis suggested that Dp had undergone a genetic bottleneck. Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that Dp had an increased percentage of micronuclei in hemocytes in comparison to Cl. The laboratory experiment revealed that Dp had a lower percentage of tail DNA and a higher percentage of micronuclei than Cl. These differences between populations were possibly caused by an overall decreased fitness of Dp due to genetic drift and by an enhanced DNA repair mechanism due to acclimatization to pollution in the source habitat. The fast growing human population has altered freshwater ecosystems profoundly . HazardoTo obtain an integrated assessment of the impact of pollution on populations, the use of multiple biomarkers at different levels of biological organization has been strongly suggested , 7, 11.  Dreissena polymorpha has been applied as bioindicator for passive as well as active biomonitoring [The zebra musselnitoring –14, for nitoring .In particular, we present a case study of two mussel populations from the river Drava (Croatia). These two populations are considered to have the same genetic background but are exposed to different environmental conditions. The first population (Dp) was collected at a polluted site where mussels were constantly exposed to the effluent of a municipal WWTP, whereas the second (Cl) was collected at a reference site.We first studied the genetic constitution of both populations applying microsatellite analysis at the population level and DNA damage analysis at the molecular and cellular level. For the population level we hypothesized to detect a genetic bottleneck in population Dp, caused by increased mortality resulting from the exposure of the population to the WWTP effluent (hypothesis 1.1). This bottleneck could be a result of selection or of random genetic drift due to a decrease in population size. At the molecular and cellular level, we expected increased genotoxic effects in Dp compared to Cl (hypothesis 1.2).Second, in a laboratory experiment, we exposed randomly selected individuals from both populations to polluted municipal wastewater. For this experiment, we expected that the two populations would react differently to these altered environmental conditions. (i) Under the assumption that selective processes have changed the genetic constitution of population Dp in the source habitat, Dp could be more resistant to the wastewater and show less DNA damage than Cl (hypothesis 2.1). (ii) If alternatively, Dp had a decreased genetic diversity due to random genetic drift leading to a decreased fitness under altered environmental conditions , 16, we The zebra mussels were sampled in July and August 2009 in northern Croatia . The refAll chemical analyses of water and sediment were performed by the Institute of Public Health “Dr. Andrija Štampar,” Zagreb, Croatia, except for pH and oxygen, which were directly measured in the field.b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, benzoperylene, and indenopyrene) were analyzed according to the EPA 550 [63Ni Electron capture detector  according to the HRN EN ISO 6468 [Water samples were collected at the Dp site, at the WWTP effluent channel, approximately 20 m upstream from the outlet into the river Drava, and at a reference site Cl2 . For the EPA 550  standardISO 6468  standardOnly one sediment sample could be collected in the WWTP effluent channel W.C., , since t63Ni Electron capture detector  according to EPA 8082 [The methods used for determining heavy metal concentrations , arsenic, mineral oil hydrocarbons, and PAHs have been previously described in detail . The conEPA 8082  standardFor population genetic analyses, the sample size was 48 individuals for each of the populations. Genomic DNA was extracted from each of the individuals from the posterior adductor muscle of ethanol-preserved samples using the High Pure PCR Template Preparation Kit . The six primers used for microsatellite analysis and the respective PCR protocols are based on Naish and Boulding , AstaneiThe microsatellite data set was checked for scoring errors due to stuttering, large allele dropouts, and null alleles with the program Microchecker . We testMussels collected from the two sites Cl and Dp were transported within 90 min to the Department of Zoology, Faculty of Science in Zagreb. Hemolymph was withdrawn from the posterior adductor muscle sinus with a hypodermic syringe for subsequent Comet assay and MNT. The Comet assay was conducted as described in detail by Štambuk et al. . For eacAliquots of 0.1 mL hemolymph mixed with 0.1 mL phosphate-buffered saline (PBS) in 10 mM ethylenediaminetetraacetic acid (EDTA) were placed on slides and left for 15 min in a humidified chamber at room temperature allowing hemocytes to settle down. The subsequent procedure was conducted as described previously . MicronuHE > HEQ). In a bottlenecked population, the number of alleles is reduced faster than the allele frequencies by a strong reduction in population density. As HE is calculated from allele frequencies and HEQ is calculated from allele numbers, a significant excess of heterozygosity (HE > HEQ) is evidence of a recent bottleneck in a population [We checked both field populations for possible bottlenecks using the program BOTTLENECK, version 1.2.02 . With thpulation . We chospulation , 36. TheU test) differentiating between heterozygote deficiency (HO < HE) and excess (HO > HE) [The overall observed and expected heterozygosity (genetic diversity) of the two populations and respective heterozygosities for each of the loci were estimated with the program GENEPOP, version 4.0.10 . The samO > HE) . In caseFST) and the inbreeding coefficients (FIS) using the F-statistics by Weir and Cockerham [FST and FIS estimates were calculated over all loci, as well as separately for each of the loci.To assess overall differences in gene pools of the populations Cl and Dp, we calculated the differentiation between the two populations  were estimated and tested for significant deviation from zero, using a nonparametric permutation approach described by Excoffier et al. [FIS) were estimated and also tested for significance.The r et al. . FurtherFIS), variances among age classes within populations (FSC), variances among age classes among populations (FCT), and the overall fixation indices (FIT) were calculated and tested for significant deviation from zero. All AMOVA analyses were computed with the program Arlequin, version 3.5.1.2 [The distance method applied in the locus-by-locus AMOVA was the number of different alleles. With the help of this method, the locus-specific inbreeding coefficients , mean values of DNA damage were calculated based on the mean of each individual within a group. The data are presented as mean ± SEM (standard error), for both Comet assay and MNT. To assess differences between groups, we performed the Mann-Whitney  Chlorella sp.Approximately 200 mussels per site were acclimatized to laboratory conditions for two weeks in glass aquaria. The aquaria contained dechlorinated, well-aerated water, which was renewed every other day, but not any sediment. Every other day the mussels were fed with algaeAfter this acclimatization period, 50 mussels of each population were exposed to the polluted municipal wastewater of Zagreb in aquaria for three days. The wastewater in the aquaria was renewed every day. At the same time, other samples of 50 mussels of each population were exposed to dechlorinated tap water and were used as a control. At the end of the experiment, seven to twelve individuals were taken from each treatment to assess genotoxicity by the Comet assay and MNT.Results of the water and sediment chemical analysis are presented in The sediment analysis of the WWTP effluent channel clearly showed that the water running through this channel has been heavily polluted, at least from time to time.The analysis of the genetic data set with the program Microchecker  did not HE > HEQ) in Dp (P = 0.023), but not in Cl (P = 0.219). This result suggests that the Dp population has undergone a recent genetic bottleneck [The Wilcoxon test revealed a significant excess of heterozygosity  while no difference was observed for this locus in Cl   .PCl = 0.443,  PDp = 0.237). The score tests of heterozygote excess also corroborated HWE , whereas the score tests of heterozygote deficiency revealed a significant deficit of heterozygotes in Dp (P = 0.033), but not in Cl (P = 0.414). Score tests for heterozygote deficiency individually carried out for each of the six loci and each of the two populations revealed that all loci conformed to HWE, except for locus C5 in Dp, which had a significant deficit of heterozygotes  of the two populations did not differ from zero and thus indicated no genetic differentiation between the two populations  and the overall fixation index  both deviated significantly from zero. In contrast, the variances among age classes within the populations  and among the populations  were not significant  and third age class  of Dp had an increased proportion of homozygotes in comparison to the first age class of Dp and to all three age classes of Cl.AMOVA indicated that the major proportion of genetic variation was due to genetic differences within individuals (96.96%). The overall inbreeding coefficient  for locus C5 . The genotoxic response measured by the Comet assay in the individuals from the Cl population exposed to the wastewater was significantly higher in comparison to the response of mussels from the Dp population . The frequency of micronuclei in mussels from the Cl population exposed to the wastewater was not significantly different from the Cl mussels in tap water (P = 0.089).A significant increase in frequency of micronucleated hemocytes was detected in the mussels from the Dp population after the exposure to wastewater in comparison to the mussels in dechlorinated tap water  and the control field population Cl (Comet assay: Ensis siliqua) after an oil spillage [Consistent with hypothesis 1.1 we found that Dp had undergone a significant genetic bottleneck which reflects a strong reduction in number of individuals in the past. The genetic bottleneck in Dp was accompanied by a loss of rare alleles in four out of six of the analyzed microsatellite loci. This suggests that nonselective random genetic drift had affected population Dp , 45. Basspillage .The water analysis at the Dp site and the effluent channel revealed only a decrease in oxygen concentration. However, in high flow systems like rivers, assessment of pollution status by water analysis is greatly confounded by sampling strategy and frequency and provides very little information on pollution pressure occurring through the extended periods of time. D. polymorpha in the literature [ Dreissena polymorpha larvae remain in the plankton up to three weeks or even longer and consequently can be drifted over 300 km before settlement [ Orchomenella pinguis [ Mytilus galloprovincialis inhabiting polluted marine ecosystems [The populations Cl and Dp had nearly identically high levels of genetic diversity . Similarly high levels of genetic diversity (0.79–0.94) assessed by microsatellite analysis have been reported for other populations ofterature , 48. Theterature . He founttlement , 51. Thi pinguis  and the osystems . The lat D. polymorpha mussels could only result from the downstream swamping of free-swimming larvae in high densities [There was no significant genetic differentiation between the two populations Cl and Dp. A high gene flow between the two populations is consistent with their high genetic similarity. In this part of the river Drava, gene flow in theensities . This paensities . As the  D. polymorpha populations based on microsatellite loci and concluded that it was most likely caused by null alleles, other studies did not find deviations from HWE [ D. polymorpha population studied by us which inhabits a heavily polluted site in the river Sio, Hungary. This population also showed a deviation from HWE at locus C5 (unpublished data). Other population genetic studies also found a selective effect on a single microsatellite locus that was explained by the genetic hitchhiking model [ D. polymorpha could clarify which coding gene is linked to C5.We found a significant deviation from Hardy-Weinberg equilibrium (HWE) for the Dp population, whereas population Cl was in HWE. This deviation observed in Dp was caused by locus C5, which only deviated significantly from HWE and showed a deficit of heterozygotes. While Astanei et al.  also repfrom HWE , 56. Ourfrom HWE , 58. A lng model . A genetFIS) increased with the age of the mussels and significantly differed from zero for the age classes 2 and 3. This suggests that the proportion of homozygotes increased with the age of the mussels indicating ongoing selection. The increase in homozygosity is reflected by the overall deficit of heterozygotes observed in Dp.The results obtained for age classes also indicated an indirect selection process affecting locus C5. For population Dp, the inbreeding coefficients .Based on the results of the population genetic analysis, the Comet assay, and the MNT carried out for the field populations, we have hypothesized that the mussels of the contaminated Dp site had been acclimatized to the local WWTP effluent discharge by an enhanced DNA repair mechanism and/or enhanced antioxidative mechanism. This possible acclimatization was apparently reflected only in the successful repair of DNA damage revealed by the Comet assay and not in MNT. A more efficient DNA repair system and/or antioxidative mechanisms in the Dp population resulting from acclimatization processes could also explain the differences in responses of the two populations seen in the pollution treatment in the Comet assay. Acclimatization through the DNA repair system and/or antioxidative mechanisms could be concordant with the low level of tail DNA found in the field population Dp, and the significantly lower percentage of tail DNA found in the laboratory Dp population in comparison to the laboratory Cl population in the pollution treatment. This explanation is consistent with our hypothesis 2.1 for the laboratory experiment, where we assumed that population Dp was more resistant to wastewater due to previously acting selection processes in the source habitat. It is also possible that the DNA repair mechanism and/or antioxidative defense were facilitated by differential gene expression, but the relation between these processes is not clear. So far, the role of specific DNA sequences in the repair of DNA damage has not yet been studied in ecotoxicology and should be the focus of future study [Our alternative hypothesis for the laboratory experiment (hypothesis 2.2) was that if the impacted population (Dp) had a decreased genetic diversity due to random genetic drift, it would be affected by wastewater more severely than population Cl. The microsatellite analysis, contrary to hypothesis 2.2, detected no decreased genetic diversity in Dp in comparison to Cl, but we could show that random genetic drift had affected the Dp population. Possibly, genetic drift and resulting decreased population fitness were responsible for the high rate of micronuclei of the Dp population in the laboratory experiment. D. polymorpha [The mussels of the Cl and Dp populations reacted differently to the exposure to wastewater in our laboratory experiment, which was probably due to their differing genetic constitution. This differing constitution was apparently a consequence of the different environmental conditions, to which the mussels had been exposed to in their natural habitat. The combination of the responses of the three biomarkers gave comprehensive information about the impact of both treated and nontreated wastewater on the genetics of zebra mussels at different levels of biological organization. A limitation of our approach is that it is not possible to directly prove the selection of one or several specific genes by the analysis of neutral microsatellite markers. Therefore, future research should answer which genetic process  enabled the adaptation or acclimatization to contamination. A challenging perspective could be the widening of our approach to gene expression studies or genetic linkage mapping of the microsatellite loci to coding genes of the model organismlymorpha . The mailymorpha , 7, 16.