Plasmodium falciparum from different endemic regions especially sub-Saharan Africa. However, few studies have been performed on neglected populations, such as the Pygmy populations. In this study, the population genetic structure of P. falciparum was investigated in the Baka Pygmies of Gabon and compared to that observed in neighboring villages composed mostly of Bantu farmers.There have been many reports on the population genetic structure of Plasmodium infections were characterized by sequencing a portion of the parasite cytochrome b gene. Population genetic structure of P. falciparum in the different villages was analysed using microsatellite markers and genes coding for antigenic proteins .A total of 342 blood samples were collected from 170 Baka Pygmies and 172 Bantus in the north of Gabon (Woleu Ntem Province). P. falciparum was around 57 % and no significant difference of prevalence was observed between Pygmies and Bantus. No significant differences of population genetic structure of P. falciparum was found between Pygmy and Bantu people except for one antigen-coding gene, glurp, for which genetic data suggested the existence of a potentially disruptive selection acting on this gene in the two types of populations. The genetic structure of P. falciparum followed a pattern of isolation by distance at the scale of the study.Overall, prevalence of P. falciparum observed in Baka demonstrates a significant transmission of the parasite in this population, and some exchanges of parasites with Bantu neighbours. Despite that, some antigen-coding genes seem to have had a particular evolutionary trajectory in certain Pygmy populations due to specific local human and/or mosquito characteristics.The prevalence and genetic diversity of The online version of this article (doi:10.1186/s12936-015-0862-5) contains supplementary material, which is available to authorized users. Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi). Among them, P. falciparum is the most virulent.Malaria is the most important parasitic disease based on its impacts on human populations, with 207 million cases and 627,000 deaths recorded in 2012, especially in sub-Saharan Africa [P. falciparum populations has been intensely investigated all over the world because it provides information on the evolutionary potential of the parasite confronted by a new drug or a vaccine, on the risk of diffusion of resistance alleles throughout the world, as well as on its epidemiology and evolutionary history [Plasmodium falciparum populations on the African continent, where most cases of infection occur every year, have largely been studied in this context [During the last 20 years, the genetic structure of  history . Plasmod context –16. HowePygmies are indigenous hunter-gatherers of the Central African forest who diverged from other groups in Africa about 60,000 years ago , 18. TheIn Gabon , there are three distinct Pygmy communities: the Baka, the Babongo and the Bakoya. None of them has conserved a traditional way of life even if some groups live in nomad forest camps for several months for hunting at certain periods of the year. In this country, most population genetic studies on malaria were conducted on the Bantu populations  , 21, 22.P. falciparum was characterized and compared among the Baka Pygmies and their Bantus neighbours in the same region of Gabon using both microsatellite and a set of antigen-coding genes.In this study, the population genetic structure of The study was carried out in Woleu Ntem Province, located in the north of Gabon Fig. . Three cThe study was conducted following two stages: (1) a sensitization stage in May 2013 during which persons were informed of the aim of the study, and, (2) blood sampling from June to July 2013. The sampling was conducted just after the rainy season (occurring from February to May) when malaria transmission is the highest.Participants were recruited from ten villages Fig. ; Table 1In each village, all volunteers were included in the study. Volunteers were recruited with their informed consent and in the case of children the consent of their parents. The translation of consent for participants was done by members of the NGO AGAFI (Gabonese Association of Assistance to Indigenous Women and Indigent). Before blood collection, a medical doctor examined each volunteer. Age and sex were retrieved for each patient. Blood was collected in EDTA tubes using venipuncture. Each sample was aliquoted and stored at −20 °C until used.A total of 342 blood samples were obtained: 170 from Baka Pygmies (121 from MV and 49 from IPV) and 172 from Bantus (38 in MV and 134 in FV). This study was authorized by the Ministry of Health and the agreement of the National Ethics Committee on Research (authorization number: PROT No 0030/2013/SG/CNE).® DNA Blood Mini Kit) according to manufacturer’s instructions.For each blood sample, total DNA was extracted from 200 µl of blood using the DNeasy blood and tissue kit Qiagen (QIAampPlasmodium infections were detected using the method based on the amplification of a portion of the Plasmodium cytochrome b (cyt-b) gene by a nested PCR as described in Additional file P. falciparum prevalence between Pygmies and Bantus (fixed effect) or between village types (fixed effect). In both cases, the factor ‘village of collection’ was considered as a random effect. Tests were realized using the R software [For each sample, software  and the software , 26.Seven microsatellite markers as well as four candidate genes coding for antigenic proteins were genotyped.The microsatellite markers amplified by semi-nested PCR are shown in Additional file msp1—block 2), merozoite surface protein 2 (msp2—block 3), glutamate-rich protein (glurp-region 2), and erythrocyte binding protein-175 (eba-175—region III) genes were genotyped as described in [Msp1 is a single copy gene located on chromosome 9. It encodes a merozoite surface antigen of approximately 190 kDa. The C-terminal part (MSP119) is immunogenic and contains Epidermal Growth Factor (EGF)-like domains. MSP119 is very rich in cysteine and is involved in the invasion of red blood cells by merozoites [msp1 was genotyped in this study. MSP-2 is located on chromosome 2 and encodes a 46–53 kDa antigenic protein located at the surface of the merozoite [msp2 was genotyped in this study. The EBA-175 antigen is a molecule expressed at the surface of the parasite, which binds to red blood cells. It is involved in the invasion of the red blood cells [P. falciparum, as well as on the surface of newly released merozoites in human host [Regarding the candidate genes, merozoite surface protein 1  were determined. The two allelic families 3D7 and FC27 of the msp2 gene were considered and the F (FCR3) and C (CAMP) alleles for the gene eba-175. For glurp, alleles were defined based on the amplicon size. PCR products were all visualized on a 1.5 % agarose gel electrophoresis.The specific primers used for genotyping each gene are given in Additional file Allele frequency was computed as the proportion of the total of all alleles detected among the isolates examined  [All population genetic analyses were performed using FSTAT version 2.9.4 . GeneticHe) . CompariF-statistics FST [P. falciparum genetic differentiation while controlling for geographic distance (1000 permutations). For this test, the first matrix was the matrix of FST computed between all pairs of populations. The second matrix was the matrix of pairwise geographic distances computed between populations and the third one was a matrix containing 1 when the populations belonged to the same ethnicity (Pygmy/Pygmy and Bantu/Bantu) and 2 when they belonged to different ethnic groups (Pygmy/Bantu).Population genetic differentiation was estimated using the estimator (θ) of the Wright’s tics FST , 43. ThiP. falciparum populations collected from Pygmies and Bantus within these villages  and 2 when they belonged to two village types (IPV/MV + FV).An additional partial Mantel test was performed with two new groups of populations: one including only the IPVs that are strictly inhabited by Pygmies and the other including the other villages that are mostly inhabited by Bantus (MV + FV). MV isolates were indeed considered more likely to be representative of Bantus because, inside MVs, pygmies represent only a minority of individuals (even if they were sometimes sampled in majority) and also because no genetic differentiation was observed between ges see “”. For thP. falciparum. Prevalence varied from one village to another and is given in Table P-value = 0.154) on the prevalence of infection (while accounting for variations between villages) but differences between village types. Thus, although no significant differences were observed between IPVs and MVs , a significant difference was observed between FVs and IPVs  and between FVs and MVs . Average infection prevalence was significantly higher in the villages from the forest concessions (FV) than in any other village types (IPV + MV).A total of 342 blood samples were analysed: 172 from Bantus and 170 from Pygmies. Distribution and origin of blood samples per village, village types  and ethnic groups are given in Table P. falciparum isolates from two IPVs, five Bantu/Pygmy: MVs, and from three villages of FVs representing 111 isolates from Bantus and 85 from Pygmies. All markers were highly polymorphic with a total number of alleles per locus observed varying from sixto 17. For each village type or ethnic group (Pygmy/Bantu), mean expected heterozygosity (He) is given in Table He) compared either between ethnic groups  or village types .Seven microsatellite markers were genotyped in a total of 196 FST (θ) computed over all loci and populations was low (θ = 0.005) and not significantly different from 0 . Pairwise FST (θ) are given in Table P. falciparum isolates collected from the different ethnic groups . Partial Mantel tests revealed no significant effect of the ethnic group or of the village type  was also performed using 196 P. falciparum isolates. For these genes, alleles were classified according to the size of the amplified fragment. The different alleles and their distribution in the different collection sites are presented in Additional file He) estimated for each gene, each type of village and each population group is presented in Table glurp between IPV and the other villages .The genotyping of the different genes  and significantly different from 0 . Within MVs, no significant genetic differentiation was observed between the isolates collected in Pygmies and Bantus  .In this study, the characterization of the genetic polymorphism of P. falciparum (n = 196). No significant difference was observed in the prevalence of P. falciparum between Pygmies and Bantus. This result is at odds with various studies realized in the middle of the 20th Century in Central Africa, showing a global malaria infection rate lower in Pygmy camps than in surrounding Bantu villages. Explanations for these differences varied from one study to another. Schwetz attributed the differences to ethnic characteristics because they considered that the different human communities were submitted to a similar malaria risk [Anopheles gambiae s.s, the main vector of Plasmodium in Equatorial regions, was mostly absent from the undergrowth and so the Pygmy camps because of the ecological preferences of their larvae for temporarily sunned breeding sites [Over all samples (n = 342), around 57 % of the individuals were infected by ria risk . Howeverria risk , 46. Thung sites .An. gambiae, the main vector of P. falciparum and to an efficient malaria transmission. All these factors may explain the increase of malaria prevalence in present Pygmy communities and the similarity in the intensity of transmission with the neighbouring Bantu communities.The absence of differences observed in this study between the IPVs and the neighbouring Bantu villages  can mainly be explained by the drastic cultural changes Pygmies have experienced in the last 50 years. Most Pygmies are no longer nomadic and now live in settlements very similar to those of the Bantus. Pygmy communities spend more time outside the forest in fixed settlements, within-village population densities have increased as well as their contact with the Bantus . As a coP. falciparum population genetic diversity and its distribution, no substantial difference between village types or ethnic groups (Pygmies vs. Bantus) was observed. Genetic diversity as measured by expected heterozygosity was high in the different population groups and equivalent to levels of diversity measured in other African areas with a hyper endemic transmission [Because microsatellite markers are expected to be neutral, population genetic structure estimated using these markers is considered to be mainly the consequence of demographic events and not selection acting on these populations. Regarding smission , 48  or ethnic groups. No significant genetic differentiation was observed between Pygmies and Bantus within MVs. Over all the studied area, a pattern of isolation by distance was observed  thus suggesting that exchanges of parasites among villages occur mostly at small geographic distances and that no particular barrier to migration exists between Pygmy and Bantu communities Fig. .The distribution of genetic diversity at candidate genes, if under selection, is the consequence of the demographic history of the populations (as for microsatellites) but also of the action of selection. Comparing their population structure to that of microsatellites is thus a way to detect phenomenon of selection and in particular phenomenon of local adaptation for certain genes.Overall, similar patterns of population structure were observed at the candidate genes, namely no differences in the levels of genetic diversity among village types or ethnic groups and a global isolation by distance at the scale of the study . The only substantial difference was observed for the gene GLURP. Not only the level of genetic diversity of this gene was significantly lower in the IPVs Table  than in GLURP is expressed at different stages in the parasite including mosquito and vertebrate stages . If disrP. falciparum populations collected in different regions of the world at genes encoding ten leading vaccine antigens [glurp gene. Authors attributed this observation to several possible factors, including variations in human genetic resistance to malaria or adaptation to different anopheline species transmitting P. falciparum worldwide [The findings of this are compatible with results of another study comparing the level of population structure among antigens , includiorldwide .P. falciparum in Pygmies. In this study, the prevalence and genetic diversity of P. falciparum observed in Baka demonstrates a significant transmission of the parasite in this population, and some exchanges of parasites with the Bantu neighbours. Despite that, some antigen-coding genes seem to have had a particular evolutionary trajectory in certain Pygmy populations due to specific local human and/or mosquito characteristics. It is necessary to extend this research to other sites, including the Bakoya Pygmies in the northeast and Babongo Pygmies in southern Gabon, as well as to other sites in Central Africa. It would in particular be interesting to analyse the prevalence and population genetic structure in the last Pygmy groups that have conserved a traditional nomadic and forest-dependent way of life, such as in the Central African Republic.Very few studies have been conducted on the population genetic structure of


Pulmonaria officinalis. Because in disjunct populations of this species the heteromorphic self-incompatibility system showed relaxation in the long-styled morph, but not in the short-styled morph, we also hypothesized that the extent of fine-scale SGS and clustering differed between morphs.Geographical ranges of plants and their pollinators do not always entirely overlap and it has been suggested that the absence of specialized pollinators at range margins may induce changes in mating systems. Because a species’ mating system is known to have a considerable effect on within-population pollen movement, the extent of fine-scale spatial genetic structure (SGS) can be expected to differ between populations located at different parts of their geographical range. To test this prediction, we compared the fine-scale SGS between two core and two disjunct populations of the distylous forest herb Sp = 0.0014). No evidence of stronger SGS in the long-styled morph was found in the center of the range whereas one disjunct population showed a significantly (P = 0.029) higher SGS in the long-styled morph (SpL = 0.0070) than in the short-styled morph (SpS = 0.0044).Spatial autocorrelation analyses showed a significant decrease in genetic relatedness with spatial distance for both core and disjunct populations with the weakest SGS found in one of the core populations  may be as important in determining variation in SGS. In natural plant populations, genetic diversity and the extent of spatial genetic structure are determined by a variety of population characteristics, ecological conditions and historical events that affect natural selection, gene flow and genetic drift -3. PopulLupinus perennis, Clarkia xantiana, Embothrium coccineum) . As. AsP. ofP. officinalis is deposited in significantly larger amounts on both the proboscises and heads of its principal pollinators compared to S-morph pollen. This was attributed to the limited length of the proboscis of these principal pollinators (mostly short-tongued bumblebees) in combination with the occurrence of floral hairs at the entrance of the corolla tube of L-morph flowers, which easily collect pollen as the proboscis moves past  reduces the proximity of potential mates and may decrease local female reproductive success ,69. Howeast (see ). If shoast , whereas no such difference was observed in the center of the range   genotype and the S-morph the heterozygous (Ss) genotype [Z-values and thus positive spatial autocorrelation at distances smaller than 5 meters except for the core population 'Bertsdorf’, which also had a significantly lower SGS compared to the other three populations (Figure Interestingly, the asymmetry in mating between L- and S-morphs found by Brys et al.  in the sd Triest  found hih morphs . As in mgenotype . Mating genotype . HoweverP. officinalis, due to lower levels of gene flow between the partially selfing L-morphs of distant populations [In this study, we documented substantial variation in the fine-scale SGS of a heterostylous species across a broad geographical scale. Although the extent of fine-scale SGS tended to be low, we showed that the extent of SGS was more than four times stronger in one of the disjunct populations than in one of the core populations. This difference in SGS between these populations can be most likely explained by differences in mating between regions in combination with population-specific characteristics. The mixed mating system of the L-morph as opposed to the outcrossing system of the S-morph was earlier found to influence the genetic structure of the metapopulation in the disjunct range of ulations . These rSM carried out the sampling in the field, the genetic analyses and drafted the manuscript. HJ directed the research, participated in its design and assisted together with OH, with the drafting of the manuscript. All authors read and approved the final manuscript.


Salix caprea is a cold-tolerant pioneer species that is ecologically important in Europe and western and central Asia. However, little data is available on its population genetic structure and molecular ecology. We describe the levels of geographic population genetic structure in natural Irish populations of S. caprea and determine the extent of gene flow and sexual reproduction using both chloroplast and nuclear simple sequence repeats (SSRs).HT = 0.21-0.58) and 79 different haplotypes were discovered, among them 48% were unique to a single individual. Genetic differentiation of populations was found to be between moderate and high (mean GST = 0.38). For the nuclear SSRs, GST was low at 0.07 and observed heterozygosity across populations was high (HO = 0.32-0.51); only 9.8% of the genotypes discovered were present in two or more individuals. For both types of markers, AMOVA showed that most of the variation was within populations. Minor geographic pattern was confirmed by a Bayesian clustering analysis. Gene flow via pollen was found to be approximately 7 times more important than via seeds.A total of 183 individuals from 21 semi-natural woodlands were collected and genotyped. Gene diversity across populations was high for the chloroplast SSRs (The data are consistent with outbreeding and indicate that there are no significant barriers for gene flow within Ireland over large geographic distances. Both pollen-mediated and seed-mediated gene flow were found to be high, with some of the populations being more than 200 km apart from each other. These findings could simply be due to human intervention through seed trade or accidental transportation of both seeds and pollen. These results are of value to breeders wishing to exploit natural genetic variation and foresters having to choose planting material. Salix L.  belongs to a family of catkin-bearing trees, the Salicaceae. The basic chromosome number of Salix is 19, and most species are diploid (2x = 38), but ploidy levels up to dodecaploid (12x = 228) have been reported [S. caprea L. and S. scouleriana Barratt [Salix flowers are predominantly insect-pollinated, but wind-pollination also occurs [The genus  diploid x = 38, bo occurs .Salix burjatica Nasarow [S. reinii Franch. & L. Sav. [S. lanata L., S. lapponum L. and S. herbacea L. [S. hukaoana Kimura [S. arbutifolia Pall. [Microsatellite markers have been developed successfully for some species of willows. They have been characterized in  Nasarow , S. rein L. Sav. , subarctbacea L. , S. hukaa Kimura , S. arbuia Pall.  and up tia Pall. . A subseia Pall. .Salix caprea is a cold-tolerant pioneer species native to Ireland which occurs in a broad range of habitats and is one of the few willow species able to grow in forest understories [S. caprea [S. caprea.rstories . It is frstories . It is srstories . Only on. caprea , which sSalix has been studied throughout the world. Lian et al. (2003) [S. reinii, a creeping shrub which is a pioneer colonist of volcanic substrates on Mount Fuji, Japan. Evidence of clonal growth and seedling recruitment were detected in this polyploid species.However, the molecular ecology of several other species of . (2003)  used nucS. lanata and S. lapponum [S. herbacea) and found evidence of clonal growth in individuals growing up to seven metres apart.A study has been conducted in the UK for conservation and restoration of lapponum . They foS. eriocephala Michx.) and a naturalized one (S. purpurea L.) to compare the genetic diversity and structure of their populations [S. purpurea contained plants with identical multilocus genotypes (inferred to be clones), while clonal individuals were rare among S. eriocephala populations. They suggest that vegetative propagation in combination with sexual reproduction has contributed to the naturalization of S. purpurea in the USA and has resulted in higher levels of genetic differentiation among S. purpurea populations than among native S. eriocephala populations [Another study in the USA focused on a native willow  traditional population-genetic methods that often require a priori population designation such as diversity statistics, allele frequencies across Ireland, unique genotypes, analysis of variance, and tests of isolation by distance, and 2) Bayesian algorithms that cluster individual samples into populations without a priori population designation. Results were compared to those presented in previous studies on other woody species with a particular focus on Salicaceae.In order to investigate the genetic diversity, the extent of gene flow and the population genetic structure of natural Irish populations of S. caprea, describe nuclear and cpDNA allelic and haplotypic diversity in natural Irish populations of S. caprea, determine the level of geographic population genetic structure in natural Irish populations of S. caprea, and determine the extent of gene flow and sexual reproduction in this species.The specific aims were to test existing chloroplast and nuclear SSR markers for their ability to detect and describe genetic diversity and differentiation of populations in Salix caprea was sampled in semi-natural woodlands, defined hereafter as “woodlands which resemble the natural woodland cover, dominated by native trees but altered by human activity. Stands originating from previous planting may be termed semi-natural if they are now regenerating naturally, as may stands which were formerly coppiced” [oppiced” . Irelandoppiced” . In ordeoppiced”  was alsoSalix caprea were collected across Ireland during the summers of 2010 and 2011. 183 individuals from 21 sites in counties Cavan, Clare, Fermanagh, Galway, Laois, Leitrim, Longford, Mayo, Meath, Offaly, Roscommon, Tipperary, Waterford, Westmeath and Wicklow were sampled  [Salix spp. from an enriched library of Salix burjatica [DNA was extracted from dried leaf tissue with a DNeasy Plant Extraction kit . The markers used included eight chloroplast and six nuclear SSR loci. The chloroplast SSR markers were produced using a set of primers designed for universal application for dicotyledonous angiosperms and were developed on tobacco (acum L.) . CCMP2, urjatica . SB24, 32, 0.25 units of GoTaq® DNA Polymerase. Every primer was used at 0.2 μM except forward and reverse primers of CCMP5 which were both used at 0.4 μM. PCR parameters included 95°C for 4 min, then 35 cycles at 95°C for 30 s, 50°C for 45 s and 72°C for 1 min 15 s, following a final extension at 72°C for 8 min. Amplification using the nuclear SB primers  used 10 ng DNA, 1× colorless GoTaq® Flexi Buffer, 0.2 mM of each dNTP, 0.32 μM of each primer, 1.5 mM MgCl2, 0.25 units of GoTaq® DNA Polymerase. SB38 and SB85 forward and reverse primers were both at 0.4 μM whereas the others were at 0.32 μM. The PCR parameters were 94°C for 2 min, then 35 cycles at 94°C for 40 s, 54°C for 1 min and 72°C for 2 min, following a final extension at 72°C for 20 min. The annealing temperature was different depending on the primers, it was 48°C for SB38, 50°C for SB85, 52°C for SB194 and 54°C for SB24, SB93 and SB199.Prior to amplification by PCR, the quantity of DNA of each sample was checked using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Amplification using the CCMP primers was as follows : 10 ng DNA, 1× colorless GoTaq® Flexi Buffer, 0.2 mM of each dNTP, 0.2 μM of each primer, 1.5 mM MgClBetween 1:5 and 1:80 dilutions were performed according to the brightness of the band after checking the quantity of DNA on an agarose gel. PCR products were multiplexed and 1 μL of the diluted mix was added to 8.75 μL Hi-Di formamide and 0.25 μL of an internal lane size standard  and run on an ABI 3130xl Genetic Analyzer (Applied Biosystems), following the manufacturer’s protocol. After genotyping, the fragments were sized using GeneMapper v4.1 (Applied Biosystems).pi2)), Shannon's information index (−1*Σ(pi*ln(pi))), gene diversity/expected heterozygosity [pi2), , observed heterozygosity (number of heterozygotes/n) and Jost’s estimate of differentiation [For both the chloroplast and nuclear markers, the frequency distribution of each marker was graphed in Microsoft Excel and mapped into ArcGIS 10.1 (ESRI) for each population. Diversity indicators were calculated in total, per population and per locus in GenAlEx 6.5 , POPGENEzygosity  (1-Σpi2)ntiation ,31 were ntiation .HT), diversity within populations (HS), genetic differentiation (GST = 1-HS/HT), inbreeding coefficient within individuals in each subpopulation (FIS), inbreeding coefficient of an individual relative to the total population (FIT), inbreeding coefficient within subpopulations, relative to total /HT). The values of GST were taken to calculate the ratio of pollen mediated/seed mediated gene flow [POPGENE was used to calculate overall diversity in collections  based on the infinite allele model and the sum of squared size difference (RST) based on the stepwise mutation model. Gene flow was estimated from FST obtained from the AMOVAs (Nm = (1-FST)/FST for cpSSR data or Nm = 0.25*(1-FST)/FST for the nuclear SSR data) [The average gene diversity over loci was calculated in Arlequin. Exact tests of Hardy-Weinberg (HW) equilibrium using a Markov chain were performed in Arlequin for the nuclear loci. Analyses of molecular variance (AMOVA) were carried out in Arlequin with two different analyses of distance, the number of different alleles (SR data) . Unique SR data) .FST for each population obtained from Arlequin [Isolation by distance (IBD) estimation was carried out using a Mantel test. Two types of test were made: 1) with all the individuals against the haploid genetic distances matrix (for the cpSSRs) or the codominant genotypic distances matrix (for the nuclear markers) obtained from GenAlEx, or 2) with the matrix of Slatkin linearized Arlequin . Both teK value was set from one to ten, and ten runs were performed for each value of K. The length of the burn-in period was set to 50,000, and the MCMC chains after burn-in were run for an additional 100,000 times. The optimal value of K was determined by examination of the ΔK statistic [Genetic structure was investigated using STRUCTURE v2.3.4. ,38, whictatistic  using Sttatistic .A maximum of one allele per locus per individual was detected. They were the predicted length suggesting there was no contamination and the target region was amplified. 31 alleles were discovered in total for the 8 cpSSRs. Microsatellites CCMP4 and CCMP7 were found to be monomorphic (respectively 113 and 133 bp) but the other microsatellites were variable, with mononucleotide repeats in every case (1 bp difference). Between 2 and 7 alleles per locus were found  (Table NE) ranged from 1.15 for CCMP10 to 3.66 for CCMP6. HT ranged from 0.13 for CCMP10 to 0.73 for CCMP6. HS was lower than the overall diversity for all markers, as not all the alleles were present in every population. Nm was equal to 2.29, Nm > 1.0, which shows little differentiation among populations.Indicators of genetic diversity are provided in Table HE) were variable across loci reflecting the different number and frequencies of the alleles found. For three loci, HW tests were significant. It was especially visible for SB93 and SB199 where a small HO was observed, this was confirmed by both FIS and FIT where these indicators were found to be high, indicating a dearth of heterozygotes at these two loci. No excess of heterozygotes were detected even for the SB194 loci. Nm was equal to 2.76, Nm > 1.0 again, which indicates a constant gene flow among populations.The number of effective nuclear alleles was lower than the total number of alleles, showing that few alleles contributed to the variation Table . AverageHO) were less than the expected heterozygosities (HE) except for CORR and KILC where an excess of heterozygotes was observed.A mean number of 2.2 alleles and 3.9 alleles were found per locus and per population for the chloroplast and nuclear SSRs respectively Table . The ave79 haplotypes were discovered from the analysis of the cpSSRs when every allele from each individual were combined. Among them, 38 were unique and 41 were shared among two or more individuals . In contrast, 165 unique multilocus genotypes were found for the nuclear markers . Individuals WEST6, 7, 8 on the one hand and CORR2, 4, 6, 7, 8 on the other hand have the same genotypes. When the results are combined with the cpSSRs, CORR2 and 6, CORR4 and 7, and WEST7 and 8 have the same genotype, demonstrating that these individuals might be clonal.GST was moderate for the cpSSR data  suggesting low differentiation. The ratio of pollen mediated/seed mediated gene flow was calculated according to Petit et al., 2005 [GST from Table l., 2005 . The meaD (Dest) [GST or its analogues should not be used because when diversity is high this measure will approach zero (no differentiation). These data support this, as SB85 was the least variable and SB38 was the most variable even though their GST is similar (0.04 against 0.06). Dest was very low for SB85 (0.002) and considerably higher for SB38 and SB194 (0.33 and 0.27). It was moderate for SB24 and SB93.A third measure of differentiation was calculated in GenAlEx, Jost’s D (Dest) . Jost suFST and RST (Table FST based AMOVA and 63% for the RST based AMOVA), the rest of the variation being among populations . However, the 2nd test with Slatkin linearized FST showed no IBD . For the nuclear SSR data, similar results to the cpSSR analysis were obtained (data not shown). The first test among all individuals was significant  but the Mantel test based on population FST showed no significance .For the cpSSR data, the first Mantel test among all individuals showed a slight pattern of isolation by distance, although the slope was nearly equal to zero , as this hybrid is frequent in Ireland [All ispersal . PropaguRepublic  that founeration , it is p Ireland  and it wHO = 0.41 for the nuclear SSRs) and gene diversity (H = 0.39 for the cpSSRs and H = 0.53 for the nuclear SSRs) which are comparable to other Salicaceae like Populus tremuloides, S. purpurea, S. viminalis or S. daphnoides [All populations showed relatively high values of observed heterozygosity ; therefore populations are expected to remain genetically stable over time [Nm decreases with increasing FST because greater differentiation between populations corresponds to lower levels of gene flow [S. caprea which is partly wind pollinated, gene flow is expected to be high between and within populations [S. caprea appears to account for a large (roughly 13%) component of total gene flow.ver time . Nm decrene flow . For an ulations . From ouulations , which wGST) of populations was pronounced for the cpSSRs (0.38 on average) but low for the nuclear microsatellites (0.07 on average). It is not completely unexpected as cpDNA is generally maternally inherited in angiosperms [GST among populations is generally more pronounced for cpDNA than nuclear DNA. GST was found to be much lower for populations of S. caprea sampled across Europe for chloroplast DNA [GST of 0.090 for PCR-RFLP markers and a GST of −0.017 for cpSSRs.Genetic differentiation . These values are higher than those estimated for natural populations of S. viminalis in the Czech Republic (FST of 0.05) based on 38 nuclear SSRs [Fraxinus excelsior trees (FST of 0.09) based on six nuclear SSRs [From the nuclear SSR analysis, low to moderate genetic differentiation between populations was discovered depending on the method used . For the nuclear SSRs study, however, the among population genetic variation estimations were substantially lower and differed depending on the method used. Most of the variation was estimated to occur within individuals for the  species  and withiminalis ,50. In aata used . The resFST among the sampled sites. This is in accordance with a study on S. viminalis in the Czech Republic [S. caprea across Europe where an absence of geographical structure was found from the analysis of three cpSSRs and four PCR-RFLPs [The tests for isolation by distance gave similar results for both the cpSSR and nuclear markers. A slight IBD was identified for the tests among all individuals on pairwise distances but no IBD was detected for the tests with the linearized Republic . The IBDCR-RFLPs .S. caprea within the sampling area and this is expected as S. caprea is recalcitrant to natural vegetative regeneration except for a few genotypes [GST/FST/Dest values were low especially for the nuclear SSRs, are consistent with outbreeding and indicate that there are no significant barriers for sexual reproduction and gene flow within Ireland over large geographic distances.Sexual reproduction is inferred to be high for enotypes . Our datPopulus trichocarpa [2 (radius 10 km) in western Oregon, the mean pollination distance was 7.6 km, with many recorded matings over 10 km [P. trichocarpa, as the maximum within stand pollination distance simply reflects the maximum potential distances between trees within the study site, which was verified for other studies [Both pollen-mediated and seed-mediated gene flow are high, some of the populations being 230 km apart from each other. Such a finding could simply be due to human intervention through seed trade, extensive planting or accidental transportation of both seeds and pollen. The absence of population structure could indicate the existence of one largely continuous population throughout Ireland. A parentage study has not been undertaken in our work but the results could also be consistent with long distance pollination. Parentage analyses were undertaken in chocarpa . In a ster 10 km . They co studies .S. caprea across Ireland. New markers were tested and shown to be suitable for genetic characterization of S. caprea. High levels of allelic and genetic diversity were found with these markers, with within population variation accounting for the majority of the variation, and a high number of unique genotypes detected. Population structure and differentiation analyses, as well as IBD tests confirmed low levels of geographical structuring of variation but moderate differentiation was detected with the cpSSRs. However, gene flow through seeds and pollen was shown to be large. These results are of value for breeders wishing to exploit natural populations and foresters having to choose planting material. Further analysis should analyse the variation of Irish populations in relation to those found in Europe.This paper used chloroplast and nuclear microsatellites to examine the genetic diversity, the geographic population genetic structure and the extent of gene flow in natural populations of


Although resistance can occur among separate populations of a species across the landscape, the spatial scale of resistance in many weeds is often left unexamined. We assessed the potential that resistance to the herbicide glyphosate in the agricultural weed Ipomoea purpurea has evolved independently multiple times across its North American range. We examined both adaptive and neutral genetic variations in 44 populations of I. purpurea by pairing a replicated dose–response greenhouse experiment with SSR genotyping of experimental individuals. We uncovered a mosaic pattern of resistance across the landscape, with some populations exhibiting high-survival postherbicide and other populations showing high death. SSR genotyping revealed little evidence of isolation by distance and very little neutral genetic structure associated with geography. An approximate Bayesian computation (ABC) analysis uncovered evidence for migration and admixture among populations before the widespread use of glyphosate rather than the very recent contemporary gene flow. The pattern of adaptive and neutral genetic variations indicates that resistance in this mixed-mating weed species appears to have evolved in independent hotspots rather than through transmission of resistance alleles across the landscape.Strong human-mediated selection  The evolution of herbicide resistance in weedy plants is an excellent example of adaptation to strong human-mediated selection , then it is inferred that populations are independently evolving resistance across the landscape. In this case, a pattern of high neutral genetic structure across populations would suggest rather limited gene exchange among populations, supporting the possibility that populations are independent evolutionary units. Hence, pairing an assessment of the level of resistance across the landscape with investigation of the genetic structure of a weed can allow us to identify the evolutionary units of herbicide resistance  Is there a geographic mosaic pattern of glyphosate resistance in I. purpurea, indicating that resistance has evolved independently in separate populations across the landscape, or is there a pattern of isolation by distance suggesting a single origin? (ii) Does the pattern of neutral genetic structure across this species' range provide evidence that populations are genetically isolated or that gene flow, whether historical or contemporary, has occurred or is occurring? and (iii) Is there evidence for migration between populations of the southeastern USA after glyphosate was put into widespread use across the landscape, indicating that contemporary gene flow is responsible for the spread of resistance? While the majority of studies that assess neutral genetic variation in herbicide-resistant weeds have investigated either predominantly outcrossing, wind-pollinated species, or alternatively highly selfing species, the work presented herein considers a species that exhibits a mixed-mating system, and one that, by all indications, is in the early stages of glyphosate-resistant evolution across the landscape.Defelice . This spDefelice , which iDefelice . AdditioDefelice . AlthougDefelice , we currI. purpurea located within soya, cotton, corn or alfalfa fields selected at random from six states across the Midwestern and Southeastern USA . Ten seeds from each population  were scarified and then planted in pine bark soil in SC10 super containers  in each of six experimental treatments, described below. Individual plants were randomly assigned to racks that were then randomly assigned to flow trays (four racks per flow tray). Pots were watered daily, and flow trays were filled with water to prevent desiccation. Only plants that germinated prior to June 26, 2013, were included in the experiment—however, germination was high  in both experiments and ranged from 50 to 100% among populations. A total of 4614 plants were used in our resistance assays, with 1995 and 2619 individuals planted in each experiment. The number of individuals per population and treatment combination used in the experiment can be found in 2-pressurized sprayer . The same applicator treated each of the two replicate experiments. We sprayed plants at a rate of 187 liters/ha at 30 psi with a stride pace of 90 paces per minute at 1.5 meters above the plants. Three weeks after glyphosate application, we scored survival and the height of the living stem of each plant.We measured the height of the stem and number of leaves of each individual 3 weeks after planting, when the majority of individuals were at the 5-leaf stage. Average height of the plants prior to herbicide application was 13.1 ± 0.3 cm. Plants were then sprayed with RoundUp PowerMax  at rates around the recommended field rate (1.54 kg ai/ha) of 0, 0.21, 0.42, 0.84, 1.70, and 3.40 kg a.i./ha (the 0 kg a.i./ha treatment was used as a water control) using a handheld, COμl for subsequent PCR. We identified 15 SSR loci that showed compatibility for multiplexing and were easily scorable of 20 that were previously described for I. purpurea . All DNA samples were quantified by spectrophotometry and diluted to 20 ng/purpurea . Forwardμm of IP8, IP2, IP27, 0.20 μm of IP31, and 0.25 μm of IP34, IP18, and IP1. The second multiplex consisted of 0.05 μm IP36, 0.10 μm of IP47, 0.15 μm of IP6, and 0.25 μm of IP12, IP21, IP45, IP26, and IP42. Ten microlitre PCRs were run with Qiagen Master Mix . Thermocycler conditions consisted of 95°C for 3 min, 35 cycles of 94°C for 30 s, 50°C for 90 s, 72°C for 60 s, and a final extension at 72°C for 10 min on an Eppendorf  MasterCycler Pro thermacycler. One microliter of PCR product was used for fragment detection using an Applied Biosystems 3730 DNA Analyzer  at the Cornell Life Sciences Core Facility . An ABI GS500 for multiplex 1 and GS600 for multiplex 2 size standards were used for fragment length comparison. All sample genotypes were analyzed using Applied Biosystems PeakScanner 1.0 analytical software . A PP (Primer Peaks adjustment) sizing default was used for the analysis.Because fragment size and dye incompatibilities precluded running all 15 loci in one multiplex, we split loci into two multiplex PCRs. One multiplex consisted of 0.15 We included populations with at least 10 germinants per experiment  in the analyses of spatial variation of herbicide resistance. We first report a species dose–response curve and then follow with patterns of survival across populations at doses above the recommended field rate.R = 0.480, P = 0.004), showing that although we identify differences in the survival of plants in separate greenhouse experiments, the populations performed similarly relative to one another in the different experiments. To estimate the dose–response curve, we first fit the residuals by regressing a general linear model of survivorship on experiment with a binomial distribution to account for the effect of replicate. The residual data were then fit to a Weibull 2.4 parameter model (Weibull 50) was expressed as:Preliminary analysis showed a significant effect of replicate experiment on the proportion that died. Thus, we used residual values after accounting for the effect of greenhouse experiment to estimate our dose–response curves. The use of residual is recommended by Kelly and Rice  to smoot Weibull  using th Weibull  in R (R  Weibull . The WeiY is the response (survivorship), c is the upper limit of the curve, d is the lower limit of the curve, e is the ED50, and b is the relative slope around e. We first estimate a species-level ED50 using all individuals, followed by a regional (Southeastern and Midwestern USA) ED50 value, and ED50 values per state. We included state in the models because we hypothesized that management policies and herbicide procedures might vary at the state level, and this could influence the level of resistance among states.where We assessed survival at the 1.7 and 3.4 kg a.i./ha treatment levels (a rate that is similar to the suggested field rate of 1.54 kg a.i./ha and a dose that is twice that) to determine whether there was a significant effect of population, state, and/or region of origin on survival postherbicide. To do this, survival was modeled as a binary character (1/0) in a generalized linear model with a binomial distribution using the lmer option of the lme4 package Bates  in R R . Fixed eWe calculated Moran's I to determine whether there was a correlation between survival following herbicide application and geographic distance. Specifically, we calculated the correlation between survivorship and its spatial lag using the Spatial Autocorrelation Analysis in Macroecology . We used MicroChecker .We assessed patterns of neutral genetic variation across this species' range using a variety of metrics to determine whether genetic diversity and population structure were influenced by recent selection kerham's  θ using kerham's . A confiamova to test the level of genetic differentiation across regions , states within regions , and populations nested within states using GenAlEx v. 6.1 ), were assessed from five runs using a range of k values from 1 through 35. We used the delta-k method , and Cavalli-Sforza distance over the natural log of geographic distance using ISOLDE, Option 6 in GenePop  and a third scenario assumed admixture after the use of glyphosate (time t5) within NC and TN regions.We next used a Bayesian coalescent approach , which is the year that RoundUp was approved for chemical weed control (Duke and Powles t3), from an initial effective population (Ne) size ranging between 250 and 1000 individuals, which encompasses the range of previously estimated Ne for I. purpurea  had previously been described in Defelice , which is similar to the manufacturer's recommended field dose of 1.54 kg a.i./ha. Twelve populations, all of which were from VA, SC, NC, and TN, exhibited a proportion survival that was significantly higher than the species average, that is, resistance values that were greater than the species 95% CI  and state on survival , but we observed no isolation by geographic distance across all sampled populations in survival . The Southeastern USA exhibits greater diversity in the proportion survival per population than the Midwestern USA; although the most resistant populations do not, in general, cluster together in a PCoA  and observed (Ho) heterozygosity ranged between 0.230–0.372 (mean = 0.304) and 0.191–0.400 (mean = 0.294), respectively , and allelic richness per multilocus genotype × population combination ranged between 1.23 and 1.37 (mean = 1.30). Expected , and populations within states . The majority of genetic variation in I. purpurea is found within populations .The FST across the species' range to be 0.127, (95% CI: 0.071–0.183), which is lower than a previous estimate using floral color . One hundred and eight (21%) of 595 pairwise-FST values between populations were significantly greater than 0, and ranged from 0.035 . Of the significant FST values among states, the majority were observed between populations in TN and NC (15%) and SC (15%)—interestingly, these were states in which we observed highest levels of resistance. There was, however, no indication that resistant populations exhibited more or less differentiation compared to other populations, as the majority (64%) of the significant pairwise-FSTs were among populations that exhibited resistance values within the species' 95% CI, and, less than 2% of the significant pairwise-FSTs were between resistant and susceptible populations.We estimated Weir and Cockerham's  FST acroI. purpurea was k = 3 (ln(P(D) = −8265.7). All 3 genotypic clusters were found within individuals sampled from North Carolina suggesting that populations within this state are either the source of introduction for other weedy populations or this state has had multiple introductions of different seed lots.Further, although we found a moderate level of genetic differentiation across populations sampled from North America, our STRUCTURE analysis uncovered a pattern of widespread migration and admixture among individuals within populations Fig. . The mosamova result for regional genetic differentiation . However, these populations were contained within the range of variation across the Southeastern US populations  or ‘susceptible’  in PC1 or PC2 mean scores . Hence, there was no indication that the neutral genetic variation of this species clustered according to resistance status rather than geography, as would be expected if propagules from, for example, resistant TN populations had migrated to the resistant Carolina populations and established and/or admixed. We did not uncover evidence of isolation by distance using linearized FST over geographic distance , nor did we uncover significant isolation by distance measured as the Cavalli-Sforza Edwards chord distance . Pairwise estimates of Nei's genetic distance similarly did not correlate with geographical distance , reinforcing our finding of either widespread gene flow across populations or colonization following a recent bottleneck.Our PCoA of neutral genetic variation revealed a slight clustering of Midwestern US populations, which had similarly been found in the ons Fig. , and theWe found overwhelming support for admixture prior to glyphosate use  and that it is unlikely increased resistance is due to contemporary gene flow between populations, but rather, results from independent regimes of selection via the herbicide. We discuss each of these main points below.Our comprehensive analysis of herbicide resistance and neutral genetic variation in the weed that is, resistant populations were located near susceptible populations—suggesting that resistance has independently evolved across disparate areas of this species' distribution. This pattern of potentially independent resistance hotspots has been shown in other resistant weed species  , we uncovered populations that exhibited very high or very low survivorship postherbicide application. Populations that exhibited high survival and thus high resistance did not appear to cluster in one region of the landscape—50 estimates and survivorship compared to Midwestern US populations. Because management practices are often regulated at the state level, it is possible that the difference in resistance between regions may result from differences in the recommended dose across areas. For example, TN has the highest recommended application rate in corn  as does survival postherbicide , we find little evidence for a geographic pattern to that structure beyond the slight clustering of Midwestern US populations identified in the PCoA. In particular, we found no isolation by distance within the species, suggesting a scenario of either widespread gene flow between populations or their relatively recent colonization.With some notable exceptions , but still historical gene flow is responsible for the low genetic differentiation. Populations then went through rapid adaptation of increased resistance across separate areas within the past ∼20 years due to the prevalent use of Roundup herbicide. To conclusively rule out the possibility that rare gene flow events may have introduced resistance alleles across disparate areas, however, we will need to determine whether the genetic basis of resistance across populations differs, and perform an analysis of the phylogeographic history of resistance alleles.An interesting and remaining question is whether or not the potential independent evolution of resistance is due to selection on pre-existing and hence similar genetic variation, or due to novel mutations in the same or different genomic architecture. Previous work in this species has identified genetic variation for glyphosate defense in accessions of


Alternaria alternata isolates sampled from the main potato production areas in China to infer the mating system of potato early blight. Consistent with the expectation of asexual species, identical genotypes were recovered from different locations separated by hundreds of kilometers of geographic distance and spanned across many years. However, high genotype diversity, equal MAT1-1 and MAT1-2 frequencies within and among populations, no genetic differentiation and phylogenetic association between two mating types, combined with random association amongst neutral markers in some field populations, suggested that sexual reproduction may also play an important role in the epidemics and evolution of the pathogen in at least half of the populations assayed despite the fact that no teleomorphs have been observed yet naturally or artificially. Our results indicated that A. alternata may adopt an epidemic mode of reproduction by combining many cycles of asexual propagation with fewer cycles of sexual reproduction, facilitating its adaptation to changing environments and making the disease management on potato fields even more difficult.Reproductive mode can impact population genetic dynamics and evolutionary landscape of plant pathogens as well as on disease epidemiology and management. In this study, we monitored the spatial dynamics and mating type idiomorphs in ~700  Reproductive mode can have profound effects on epidemiological landscapes, population genetic dynamics, evolutionary trajectory and management of pathogens14711MAT1-1 and MAT1-2)171822Alternaria alternata, which is associated with the early blight disease on potatoes.Sexual reproduction in fungi is usually triggered by environmental stresses or nutrition deficiency15161718192122Potato early blight, characterized by the formation of dark-colored spots that are necrotic in the centre with a pattern of concentric rings on leaves, is among the most destructive emerging pathogen worldwideAlternaria alternata and A. solani can cause early blight on potatoes in the world26303132A. alternata produces densely turfy colonies in dark grey to black brown color on PDA plates. Its conidium is usually pale to light brown containing ~15 septa in length with numerous secondary and occasionally tertiary chains branching from apical and median cellsA. solani produces densely dark grey to black colonies with sparse aerial mycelium. Its conidia usually has 9–11 transverse septa and 1–2 longitudinal septa with one long to ovoid beak26A. alternata out-competed A. solani though in importance the relative proportions of these two species may vary in populationsBoth A. alternata is an opportunistic haploid pathogen affecting various important cropsMAT1 geneAscomycota) because teleomorph  has not been observed yet in nature. Conflicting with theoretical expectation for an asexual pathogen, previous population genetic surveys revealed high genetic variation in A. alternata populations2635A. alternata using the combination of highly polymorphic neutral markers such as SSR and functional genes such as the mating type gene is scarce, particularly for A. alternata from potato. Knowledge from this type of analysis is important for understanding the reproductive mode and adaptive potential of the pathogen, therefore, necessary in designing an effective and sustainable disease management program for controlling this emerging disease worldwide. It is reported that A. infectoria, a species which occupies a basal position in the phylogeny of Alternaria genus, connects to a Lewia teleomorph37Alternaria species may have a sexual ancestorA. alternate from citrus by sequence approachA. alternata; 2) to determine spatial distribution of the two mating types in A. alternata populations; and 3) to infer the occurrence of sexual reproduction in the field populations of A. alternata.Alternaria alternata isolates from different parts of China , no significant differences in mating type frequency were detected amongst field populations  were detected only once . The mosn of 3.7 .95 or U95 values in five out of nine field populations with a sample size >30 and the corresponding IA was not significantly different from theoretical expectation for random mating also in five of these populations  between the two mating type groups in the 17 field populations ranged from 0.01 to 0.31  was detected between two mating type groups. In three pathogen populations , moderate to high genetic differentiations were detected between the two mating type groups, but they were likely attributed to sampling error caused by small population sizes as only 5–8 genotypes were included in these populations for the analysis.Genetic differentiation between isolates from MAT1-1 and MAT1-2 genomes.Phylogenetic analyses for the relatedness between the two mating type groups of isolates using both partial  in North AmericaBoth meiotic recombination in sexual reproduction and mitotic recombination in parasexual reproduction could explain the observed patterns of population genetic structure in A. alternata in potatoes may have evolved to have an epidemic mode of reproductionA. alternata and its hosts to design a dynamic disease management program that combine all available approaches such as host resistance, cultivar rotation, disease sanitary and fungicide is important to manage potato early blight effectively and durably59In conclusion, our results indicate that, like many eukaryotic pathogens, A. alternata.Infected potato leaves with early blight were collected at random at 1–2 meter intervals from 17 farmer fields across China  between A. solaniA. dauciGenomic DNAs were amplified with the seven SSR markers previously developed for A. alternata isolates were determined by PCR amplification of genomic DNAs with two pairs of mating type-specific primers  downloaded from Genebank and synthesized by Ruiboxingke Biotech. Co. LTD. (Beijing). Primers A amplifies a part of the α-domain, generating a unique 661 bp fragment from the isolates carrying MAT1-1 idiomorph and Primers B amplifies a part of the HMG-box, generating a unique 494 bp fragment from the isolates carrying MAT1-2 idiomorph. PCR amplifications of mating types were performed using a mixture of two pairs of primers (multiplex PCR) and carried out in an 2720 thermal cycler  in 20 μL PCR reaction volume that consisted of 1 μL (10 μM) of each primer, 2 μL (2 mM) of dNTPs, 2 μL of 10 × reaction buffer, 1 unit of Taq polymerase , 2 μL of genomic DNA and 9 μL of sterilized distilled water. The program used for mating type amplification was: initially held at 95 °C for 5 min, followed by 33 cycles of 94 °C for 60 s, 53 °C for 30 s and 72 °C for 30 s; ended with a final extension at 72 °C for 10 min. PCR products were separated by electrophoresis on 1.2% agarose gel at 100 V for 60 min and visualized with a G8140 Golden View I .Mating types in  primers . The priAlleles were assigned based on the sizes of PCR amplifications that were generated by each pair of SSR primers using GeneMaker software version 1.31 with a binning procedure. PCR amplification with an identical size generated by the same pair of primers was considered as an allele. Multilocus haplotype for each isolate was formed by joining the alleles at each SSR locus in the same order. Isolates with the same multilocus haplotype and mating type were considered as an individual member of the same clone, the asexual progeny of a genotype. In the estimation of population genetic parameters except for genotype diversity, only one member of clone was included in each population. If the clone was detected in several populations, one representative of the clone was retained for each population. All isolates (clone-corrected) were used to estimate genotype diversity.2 test and heterogeneity in mating type frequency among populations was evaluated by a contingency χ2 testGSTA for each population was generated by 1000 resamples of original data and its difference from theoretical expectation or random mating was evaluated by a t-test. Clone-corrected SSR data were used for these analyses. A phylogenetic tree among 25 genotypes each of the two mating types randomly selected from the total collection was reconstructed using UPGMA and displayed with NTSYS . A further neighbor joining tree was reconstructed using SSR data of all 253 genotypes. Nei’s genetic distanceFungal isolates were hierarchically organized into “field” and “regional” populations. Isolates sampled from the same field were considered as the member of a field population and from different fields within the same cropping region were grouped into regional populations. There are four potato cropping regions in China: Northern Single-cropping Region (NSR), Central Double-cropping Region (CDR), Southwestern Multiple-cropping Region (SMR) and Southern Winter-cropping Region (SWR). The null hypothesis of 1:1 ratio between the two mating types within a population was evaluated by a simple χHow to cite this article: Meng, J.-W. et al. Population genetic analysis reveals cryptic sex in the phytopathogenic fungus Alternaria alternata. Sci. Rep.5, 18250; doi: 10.1038/srep18250 (2015).


Triatoma brasiliensis collected in distinct sites and ecotopes in Rio Grande do Norte State, Brazil. In the semi-arid zones of Brazil, this blood sucking bug is the most important vector of Trypanosoma cruzi—the parasite that causes Chagas disease. First, cytochrome b (cytb) and microsatellite markers were used for inferences on the genetic structure of five populations (108 bugs). Second, we determined the natural T. cruzi infection prevalence and parasite diversity in 126 bugs by amplifying a mini-exon gene from triatomine gut contents. Third, we identified the natural feeding sources of 60 T. brasiliensis by using the blood meal content via vertebrate cytb analysis. Demographic inferences based on cytb variation indicated expansion events in some sylvatic and domiciliary populations. Microsatellite results indicated gene flow between sylvatic and anthropic (domiciliary and peridomiciliary) populations, which threatens vector control efforts because sylvatic population are uncontrollable. A high natural T. cruzi infection prevalence (52–71%) and two parasite lineages were found for the sylvatic foci, in which 68% of bugs had fed on Kerodon rupestris (Rodentia: Caviidae), highlighting it as a potential reservoir. For peridomiciliary bugs, Galea spixii (Rodentia: Caviidae) was the main mammal feeding source, which may reinforce previous concerns about the potential of this animal to link the sylvatic and domiciliary T. cruzi cycles.We used an individual-based molecular multisource approach to assess the epidemiological importance of  Trypanosoma cruzi, which causes Chagas disease. Triatoma brasiliensis is the main Chagas disease vector in the Caatinga eco-region of northeastern Brazil. We showed that T. brasiliensis exhibits gene flow between sylvatic and anthropic (domiciliary and peridomiciliary) populations. This finding represents a challenge for vector control because sylvatic foci are not reached by control activities. Bugs from sylvatic foci harbor high T. cruzi prevalence (52–71%). The potentiality that the rodent Kerodon rupestris may act as a parasite reservoir was suggested because bugs infected by T. cruzi, were found in the same insects that fed on this animal. Peridomestic T. brasiliensis populations exhibited high prevalence for feeding on another sylvatic rodent, Galea spixii, which exhibit the potential to connect T. cruzi between ecotopes. The use of an individual-based approach directed to T. brasiliensis, linking vector population genetics to host and parasite identification, may help to better understand the eco-epidemiology of Chagas.Blood-sucking bugs are vectors of the parasite  Trypanosoma cruzi , which causes Chagas disease. More than five million people are infected, and approximately 70 million live at risk [Triatoma infestans—the vector responsible for most of cases of Chagas disease transmission in Brazil. As a result, in 2006, T. cruzi transmission by T. infestans, had been officially interrupted [Blood-sucking bugs  are vectors of the parasite  at risk . An inteT. cruzi was no longer actively transmitted by T. infestans, investment in entomological surveillance and control programs decreased. As a consequence, concerns are now high regarding the potential of native triatomines, such as Triatoma brasiliensis, for re-emerging hyperendemic Chagas disease foci in Brazil [Once it became clear that n Brazil .T. brasiliensis s.l. is a species complex that forms a monophyletic group [T. b. brasiliensis; T. b. macromelasoma) and three species . A taxonomic key has been published recently [Multidisciplinary studies have demonstrated that ic group –6. It inrecently .T. brasiliensis species complex are widespread in Brazil, occurring in 12 states, primarily within the Caatinga and Cerrado biomes [Members of the o biomes –10. Eacho biomes –18.Triatoma b. brasiliensis  is the best-studied member of the complex with respect to epidemiology and spatial distribution. It also has the highest prevalence of T. cruzi infection and a remarkable capacity to colonize homes. Therefore, T. brasiliensis is now the most important Chagas disease vector in the semi-arid zones of Brazil’s Caatinga ecoregion [coregion –18.T. brasiliensis is attributed to its capacity to occupy the domiciliary, peridomiciliary, and sylvatic environments [T. brasiliensis infestations are unknown: it was still unclear whether sylvatic foci are the major source of infestation or if bugs that invade domiciles are composed of domestic populations. To date, population genetic studies on T. brasiliensis have been based on a single mitochondrial gene, cytochrome b (cytb) [cytb are limited due to its maternal inheritance and limited variation for small geographic scales.The primary difficulty with controlling ronments  as it cab (cytb) ,19. But T. brasiliensis collected in distinct sites and ecotopes. Using this approach, we first explored the genetic structure of T. brasiliensis by using both cytb and microsatellites markers [T. cruzi infection and the parasite diversity in bugs and identified natural feeding sources for T. brasiliensis. Finally, we were able to make inferences about the potential T. cruzi reservoirs. We suggested that Kerodon rupestris (Rodentia: Caviidae) may be a potential parasite reservoir, because most sylvatic bugs had fed on this rodent and were also infected with T. cruzi, Moreover, population genetics results indicated that vector control efforts in domiciles are threatened by gene flow from the perennial sylvatic foci with high T. cruzi prevalence.The properties and behaviors of individuals may determine their function within the systems they compose, and therefore we used an individual-based approach directed at individuals of  markers . We thenCaatinga ecoregion. The Caatinga is a mosaic of xerophytic, deciduous, semi-arid, thorny scrub. The study area chosen for insect captures was determined based on unpublished reports of public health institutions in charge of vector control, which indicated a combination of high pressure of infestation by T. brasiliensis after control activities with records of bugs infected by T. cruzi captured in domiciliary units. All investigated sites had not been sprayed in the three years prior the captures (March). All captures were conducted during the rainy season.Triatomines were collected in the municipality of Caicó in northeastern Brazil  domiciles (Dom), or the indoor spaces of homes where triatomines are generally found in the crevices of mud walls, in furniture and under beds; 2) peridomiciliary areas (Per), or the areas outside but within approximately 100 m of homes where domesticated animals sleep or are maintained; and 3) sylvatic areas (Syl), or areas that are separate from peridomicilary areas and not occupied by humans. We maintain the traditional “sylvatic” terminology. However, we further divided sylvatic areas into “Syl-d”—degraded areas—where domesticated animals can be found  and domiciles  and manually captured with tweezers all bugs observed. In sylvatic environments, similar manual methods were employed; however, insects were searched with flashlights during the night in rocky outcrops. Domiciliary and peridomiciliary populations were defined according to the villages, in which bugs from three houses were sampled. Sylvatic populations were defined according to the rocky outcrop. In the study area, these formations were ~50 square meters. We obtained permission from house owners/residents to collect insects from all homes and properties.In domiciliary units, bug searches were conducted as routinely employed by local vector control-surveillance staff. We investigated all ecotopes usually colonized by cytochrome b marker (see below).We identified adult vectors to species according to the taxonomic key ,21. NympT. cruzi-like parasites, we began with the traditional method used by local control-surveillance systems : one fecal drop from each bug was obtained by abdominal compression, then diluted in saline solution (approximately 50 μl) and examined fresh by microscopy at 220-400X. Because starved insects do not eliminate enough fecal drops, the success of this approach depends on the insects’ nutritional status and 90 insects could be examined.To identify whether insects were carrying N ≥17 were used to ensure robust inferences. The resulting dataset for insect studies on population genetics was derived from 108 individuals distributed among five populations.The insects used for molecular studies were placed in absolute ethanol. Only adults and fifth instar nymphs (N5) were used for population genetic analyses to decrease sampling-strategic bias. Because older stages exhibit higher mobility, this strategy decreases the probability of sampling several relatives. Additionally, only populations with cytb gene with the CYTB7432F/CYTBR primers [cytb with the vertebrate-specific primers L14841/ H15149 [T. cruzi infection, by using a pool of three primers (TCI/TCII/TC) that amplify the non-transcribed intergenic region of T. cruzi mini-exon gene. TCI is specific to the T. cruzi I strain and TCII for T. cruzi II strain, generating a fragment of 300 and 350 bp, respectively [Rhodnius prolixus experimentally infected with T. cruzi (CL strain) was used as a positive control. The negative control was purified water. Given the putative epidemiologic threat from domiciliary vectors, the T. cruzi natural identification and characterization was also conducted on small sample sizes (N≤17).Following previous study , two set primers ,24,   . All ampN = 22), B (N = 17), C (N = 23), D (N = 27) and F (N = 19) populations. The available primers for seven T. brasiliensis loci [For microsatellite genotyping, 108 adults and N5 were used, belonging to A (sis loci  were tessis loci  were monPCR amplifications were performed using dye-labeled primer (Applied Biosystem) and for 40 cycles  method [http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html). Second, Bayesian inference (BI) was used to reconstruct phylogenetic relationships among unique haplotypes using MRBAYES v3.1.2 [T. brasiliensis species complex [Triatoma sherlocki was used as outgroup.We used two procedures to confirm the specific status of ) method , using KS v3.1.2 , with thS v3.1.2 . We ran  complex ,5,20. Trcytochrome b analysis, the following population genetic summary statistics were calculated for the same set of data (108 N5 and adults): number of haplotypes (Nh), haplotypic diversity (Hd) , nucleotide diversity (Pi), mean number of pairwise cytb differences (theta) and average number of nucleotide differences (k) [For nces (k) .D [FS statistics [iIS) were estimated using FSTAT [The evolutionary relationships among mitochondrial genotypes  were evaluated using Network version 4.6 . Tajima’D  and Fu’satistics –35 were atistics . Linkageatistics . Deviatiatistics , and theatistics . Inbreedng FSTAT .ST pairwise estimates computed with ARLEQUIN [ST/(1-FST)) [We compared populations using FARLEQUIN . BonferrARLEQUIN . Isolati(1-FST))  and pair(1-FST)) .s) for heterozygosity excess and for pw Wilcoxon test (two-tailed for H excess or deficiency) were calculated. Finally, Geneclass2 was used to select or exclude populations as the origins of individuals [The distributions of the expected heterozygosity for each locus and population under both the Infinite Allele Model (IAM) and the Stepwise Mutation Model (SMM) were calculated using BOTTLENECK . The proividuals .T. pseudomaculata (N = 14) and T. petrochii (N = 3) adults were collected and excluded from further analysis, after identification by morphology. We identified 81 adult insects as T. brasiliensis by morphology, and the remaining 216 immature insects were identified as T. brasiliensis based on our molecular analysis (see below).Fifteen sites  were sampled: four in sylvatic areas, four in peridomiciliary and seven in domiciliary environments . A few TT. brasiliensis complex analyzed. This was true for all substitution models used . All of our samples were clustered in the T. brasiliensis main group with 100% Bayesian posterior probability (We used 108 insects that were composed of N5 and adults in this approach. Using the ABGD (Automatic Barecode Gap Discovery) species delimitation tool, the distribution of genetic distances display two modes separated by a “barcode gap” between 0.04 and 0.07 that leads to partitions with 5 groups for all taxa of the bability .Cytochrome b sequences showed 30 variable sites, including 13 singletons, resulting in 28 haplotypes for the 106 adults and N5 (N = 7), while the sylvatic population F had the lowest (N = 3). The three sylvatic populations had similar haplotype diversities . Overall nucleotide diversity was 0.0054, ranging from 0.0051  to 0.0069 (population B). To explore demographic population events, the Fu’s FS statistics were estimated , suggesting expansion events. No significant departure from the standard neutral model was observed by the Tajima’s D values.s and N5 . Overallstimated . Three pEleven haplotypes were shared by at least two populations; two haplotypes had the highest frequencies  and occurred in almost all sites. Four haplotypes were shared by the sylvatic and peridomiciliary populations, while the domiciliary population shared only two haplotypes with the sylvatic populations and three with the peridomiciliary populations. The sylvatic population C shared the most haplotypes with the others; namely, H_6 and H_5 with sylvatic populations A and F, respectively. The peridomiciliary population D and domiciliary population B showed the most unique haplotypes . The haplotype network  was relaIS) was observed, except for population A. Under the SMM, three populations  showed a significant heterozygote excess with the two tests used, but this was not the case using the IAM  were significantly different  and the sylvatic population A , suggesting genetic flow. The lack of differentiation between populations A and F  likely indicates that sylvatic populations belong to the same panmictic unit whereas sylvatic populations A and C are geographically related but genetically differentiated . The Mantel test revealed no significant isolation by distance  among populations (See Gene flow among populations was estimated using Fifferent . The FSTions See .Genetic assignment (GeneClass2) was used to select or exclude populations as origins of individuals . The rescytb sequencing from the DNA gut content allowed us to identify the sources of blood for approximately half of the specimens , 73% of bugs fed on mammals, 20% on birds, 5% on reptiles and 2% on amphibians. Mammals were distributed in four orders: Rodentia, Artiodactyla, Carnivora and Primate. Half of the genera were native species and half were domesticated animals or humans. The type of feeding sources varied according to the ecotope. Wild animals such as lizards, frogs and native rodents were feeding sources in both peridomiciliary and domiciliary environments. The opposite was true in sylvatic environments, where we found evidence of T. brasiliensis feeding on domestic animals such as goats  and cats . Overall, in sylvatic environments, up to 97% of the feeding sources were mammals, and of those, 53% were rodents. The rodent feeding sources were mostly composed of the native Caviidae: K. rupestris  and Galea spixii .Overall, vertebrates identified as feeding sources of Amphibia . Of the Gallus gallus) were the most prevalent (47%) feeding source in the peridomestic environment, representing 25% of feedings in domiciles. In the domiciliary ecotope, goats were the principal vertebrate feeding sources (50%), consistent with field observations that these animals circulate indoors. Eight (42%) blood sources detected in peridomiciliary areas were associated with the rodent G. spixii.Chickens  and humans .Areas A and C are located in the environmental conserved area protected by military armed forces where hunting is forbidden and used for training combats. Of the 28 feeding sources detected, 68% of bugs had fed on the native rodent T. cruzi-like parasites. The infection prevalence was highest in the sylvatic environment .Using light microscopy, we were able to successfully analyze fecal drops from a total of 90 specimens from three populations . Forty-six percent of insects were infected with T. cruzi parasites in population D that we did not detect with the direct method. Consistent with direct fecal laboratory observations, none of the bugs from domiciliary population B was found to be infected using the molecular approach. However, one adult female insect from domiciliary population P was positive for T. cruzi, which was not included in the cytb sequencing was also infected by T. cruzi, and so each should be considered potential a T. cruzi reservoir. Almost all T. cruzi were TCII; however, two samples from sylvatic population C were classified as mixed TCI and TCII lineages, exhibiting both the 300 and 350bp bands . However, we were able to detect the presence of bp bands . NotablyT. cruzi vector, T. infestans. However, Rio Grande do Norte is one of the few Brazilian states where T. infestans had never been detected, even in places where Chagas disease was hyperendemic. These observations suggest that T. brasiliensis was always the main T. cruzi vector in this region due to its synanthropic nature [T. infestans, domiciliary captures of some native triatomines—such as T. brasiliensis—had also decreased dramatically after PCDCh [T. brasiliensis re-infestations, the natural feeding sources for triatomines and the natural infection prevalence for T. cruzi. Here, we provide a multisource approach that takes advantage of a combination of high resolution molecular tools to investigate the role of T. brasiliensis in the contemporary Chagas disease eco-epidemiology.Intensive control measures that involved treating houses with insecticide ultimately controlled Brazil’s major c nature ,17. Desper PCDCh . To undeT. brasiliensis using the taxonomic keys [T. brasiliensis [T. pseudomaculata and T. petrochii—occur in sympatry. Triatoma pseudomaculata is commonly found concomitantly with T. brasiliensis in peridomicilary and domicilary areas [T. petrochii is sometimes found in rocky outcrops in the sylvatic environment [T. petrochii and T. brasiliensis co-occurred in some sylvatic environments and on the same rock outcrop spot. Even the adults of T. petrochii are morphologically similar to T. brasiliensis, as they were once in synonymy [brasiliensis subcomplex of the infestans complex [T. brasiliensis, especially for nymphs, for which there are no available taxonomic keys.All adult samples identified as mic keys ,21 were ry areas , whereas complex . This cocytb haplotypes and other low-frequent haplotypes (differing in one or two nucleotide substitutions) formed a star-like haplotype network, which suggests population expansion. Even though D Tajima indicators were not significant, Fu’s FS showed that domiciliary population B and sylvatic populations C and F have more likely experienced a recent expansion event. Fu’s FS may be a more sensitive indicator for this event [D indicator, explaining this discrepancy. At most, four mutational steps from the central haplotype may be discerned in the network. Little structure using cytb haplotypes was observed, consistent with previous studies [Domiciliary population B had the highest haplotype diversity, revealing high genetic variability. We found that two central is event –35 than  studies  for micrST to infer the gene flow between populations. A recent study [ST by microsatellites in diploid organisms, results are robust even with minimal sample size (N = 20). The authors stated that improving the sampling design may show better results than genotyping more than 20 samples. With our sampling design, two populations did not meet the minimum sample size . Microsatellites applied to T. brasiliensis revealed low but significant genetic differentiation  between some neighboring populations, such as between sylvatic populations A and C, which are separated by less than 1 km. This differentiation may be associated with demographic events—low heterozygosity in population C might be related to bottlenecks. According to Luikart and Cornuet [IS may be a result of the Walhund effect, reflecting the sampling of subpopulations with distinct allelic frequencies. It is worth noting that this sylvatic population A exhibited high indices of assignment outside of its own cluster, also suggesting that such subpopulations exist. The low variability in sylvatic population F, with both few alleles and low observed heterozygosity, may be a result of ecologic factors because it was collected in a degraded sylvatic area.We used Fnt study  has show Cornuet , the SSMT. infestans were used to suggest that adjacent sylvatic foci are not the source for re-colonization, but rather, survivors of insecticide treated houses [T. infestans populations from communities under sustained vector control were highly structured; however, populations from communities with sporadic vector control did not show such structure. Despite the great versatility in re-infestation profiles exhibited by T. dimidiata on the Yucatan Peninsula a variety of foci for re-infestations were found, which were depending on the site and the season [T. brasiliensis also come from distinct sources: peridomiciliary and sylvatic areas. This finding is worrisome from a vector control perspective because sylvatic populations represent perennial and uncontrollable foci for re-infestation. Additionally, domiciliary population B and peridomiciliary population D—which came from distinct villages—were genetically related, being probably in gene flow. The direction of re-colonization of homes after insecticide spraying is likely to be from peridomiciliary to domiciliary environments [In the Bolivian Andes, observations of the genetic differentiation between sylvatic and domiciliary populations of d houses . In Arged houses  a previoe season . Each ofronments .K. rupestris—a species of rodent also called mocó by locals—was the main feeding source for T. brasiliensis in the conserved sylvatic environment. However, no evidences of feeding on K. rupestris in other environments in non-preserved areas were found. This might be because K. rupestris is targeted for hunting by locals due to its relatively large size and appreciated meat, whereas G. spixii seems to be a synanthropic rodent less appreciated. A recent study [K. rupestris and G. spixii as T. brasiliensis feeding sources in Ceara State. Indeed, the prevalence of rodent-dominant feeding sources we found supported the earlier suspicions that T. brasiliensis is primarily associated with rodents that also occur in rocky complexes [T. brasiliensis feeds on cats within this conserved area. Because this area is used for basic combat training, the identification of human blood within these vectors highlights the risk of Chagas transmission to soldiers, particularly in the light of the high T. cruzi prevalence in these sylvatic (A and C) T. brasiliensis populations, as discussed below.Populations A and C are located in the environmental conserved area surrounded by huge walls where hunting is strictly forbidden and domesticated animals  are removed by military guards when they eventually enter the area. The rock cavy, nt study  also detomplexes , DomestiT. brasiliensis collected from the non-conserved sylvatic environment had sufficient material in their gut contents for amplification and sequencing. However, blood sources were identified as G. spixii, goats and cats in population F, similar to species of feeding sources for peridomiciliary areas (except for the absence of chickens). It is not surprising that chicken was the major feeding source in peridomiciliary areas, as they are known to be responsible for maintaining high densities of T. brasiliensis colonies [G. spixii composed half of the T. brasiliensis feeding source in peridomiciliary areas is worrisome because it indicates this rodent is no longer only sylvatic. but that it has become a highly synantropic animal that presents the potential to connect T. cruzi between the sylvatic and domiciliary cycles. Neither the frogs, Proceratophrys sp., nor the lizard, Mabuya sp., had ever been predicted as feeding sources for T. brasiliensis. Indeed, frogs and lizards are often observed in the same peridomiciliary areas as T. brasiliensis during field captures. Even though they are T. cruzi refractory [Bugs from domiciles fed on goats, chickens and frogs, all of which were observed within domiciles during captures. The absence of human blood may be a result of movement of the bug across ecotopes: according to some authors ,12,53,54fractory , they maT. cruzi-like) and 52–70% of T. cruzi prevalence via traditional and molecular methods for sylvatic populations of T. brasiliensis, respectively. The State Health Department in Rio Grande do Norte (RN) uses direct microscopic observation of fecal drops in Chagas disease control surveillance campaigns to identify T. cruzi. Using this method, we detected higher prevalence of T. cruzi infection in sylvatic populations than using molecular methods that might be a result of misidentifying T. cruzi-like parasites for T. cruzi during direct microscopic examination. Such misidentification might be also generating some proportion of false-positive results for local control-surveillance. There is also the possibility of false negative results using molecular analyses, due to PCR inhibition and/or annealing troubles. To prevent such results, a combination of other markers should be used. However, for domiciliary and peridomiciliary areas, infected bugs were only detected via molecular methods. False negatives acquired using traditional methods of direct fecal observations might be a result of low parasitic loads in insects.We reported 83–95%  [T, cruzi intraspecific nomenclature [T. cruzi infection and to provide clues about the diversity in the area, according to these two major parasite lineages (TCI and TCII). The TCII affiliation was confirmed by the similarity of the sequences with the Tu18 TCII strain. We found TCII prevalence (94%) similar to that found previously [T. cruzi nomenclature. [T. brasiliensis population may harbor a mixture of the two major T. cruzi lineages.The division of TCI and TCII proposed by authors  in 1996 ts (DTU) , our prots (DTU)  can be anclature . Indeed,nclature , we aimeeviously  for thiseviously  had the clature. . Our anaclature.  in the sclature. –59 conduT. cruzi infection in the same T. brasiliensis individual, we can infer potential reservoirs. Except for the peridomiciliary population, T. brasiliensis fed mainly on mammals, which might be both the vector feeding sources and the T. cruzi reservoir. Differences in natural T. cruzi infection prevalence according to the ecotope can also be explained by the differences in feeding sources. Because birds are refractory to T. cruzi infection [By detecting feeding sources and natural nfection , the natK. rupestris. This rodent probably plays an important role in the epidemiological T. cruzi cycle, as we found that it was the prevalent feeding source in the same T. brasiliensis individuals that were infected by T. cruzi. This suggestion should to be critically investigated, because the infected insects may have acquired T. cruzi via any other mammal before subjected to the test.In the sylvatic environment, no birds were identified as feeding sources. Instead, rodents were the major blood source—especially T. cruzi cycles. Goats were identified recently as feeding sources for T. brasiliensis [T. cruzi prevalence. In Argentina [T. cruzi populations. Indeed, cats are a common feeding source of triatomines almost everywhere [T. cruzi transmission in northeastern Brazil.Because cats and goats also served as feeding sources for insect vectors in sylvatic and domiciliary ecotopes, they may also link sylvatic and domiciliary iliensis ; howeverrgentina , cats arerywhere . TherefoT. cruzi strains in a sylvatic T. brasiliensis population is also consistent with a link between sylvatic and domiciliary cycles. As G. spixii circulates between distinct ecotopes, the finding of feedings on this mammal reinforces [T. cruzi from sylvatic to domiciliary environments. The distances between sylvatic and peridomestic spots where feedings were detected on G. spixii were less than 1 km. Therefore, the identification of this rodent as vector feeding source is even more relevant. Recently some authors [a priori hypotheses about the drivers of site-occupancy by T. brasiliensis—showed that T. brasiliensis was strongly associated with key hosts, including rodents in peridomiciliary environments. In these findings the ecotope structure had small to negligible effects.Using an individual-based multisource approach, we identified potential key-links for Chagas disease eco-epidemiology between the ecotopes examined. Microsatellites showed triatomine gene flow between ecotopic populations and highlighted varied sources of domiciliary and peridomiliciary infestations, which threaten vector control efforts. The co-occurrence of two inforces  the need authors —testing T. brasiliensis because the rate of determination via direct sequencing was limited. In order to have a better representation of the host diversity, metagenomics or metabarcoding approaches using high throughput sequencing (HTS) technology is currently being developed. Additionally, to improve host assignation, the set of sequences deposited in GenBank, including for the fauna of neglected and remote sites, must likewise be improved. This limitation may have resulted in the low BLAST identity for some taxa not well studied. To exemplify this limitation, a sequence matched (with 79% of identity) an endemic lizard species  from Cape Verde Islands (Africa) [Mabuya sp. in northeastern Brazil [Our study did not characterize completely the dietary habits of (Africa) . This lon Brazil . TherefoG. spixx in peridomiciles [see T. cruzi cycles.Because Chagas disease may be also transmitted through contact with hunted animals, as is suggested for the Amazon region in Brazil , cautioniles see  because Chagas disease control is complex by nature due to an overlap of sylvatic, peridomestic and domestic cycles. However, the use of integrative approaches, such as population genetics to estimate gene flow between habitats combined with ecological data in a landscape genetics perspective –68 as weS1 FileTriatoma sherlocki was defined as the outgroup. Values in the nodes indicate Bayesian posterior probabilities. All of our samples fit in the T. brasiliensis cluster defined by Costa et al. (2013).Additional sequences used for the phylogenetic reconstruction were downloaded from GenBank (GB) and were identified by their GB code after the (sub)specific taxonomic definition. (JPG)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(JPG)Click here for additional data file.