Pseudacris nigrita and P. fouquettei across the Pearl River between Louisiana and Mississippi. This hybrid zone was initially characterized in 1980 as a narrow and steep “tension zone,” in which hybrid populations were inferior to parentals and were maintained through a balance between selection and dispersal. We reanalyzed historical tissue samples and compared them to samples of recently collected individuals using microsatellites. Clinal analyses indicate that the cline has not shifted in roughly 30 years but has widened significantly. Anthropogenic and natural changes may have affected selective pressure or dispersal, and our results suggest that the zone may no longer best be described as a tension zone. To the best of our knowledge, this study provides the first evidence of significant widening of a hybrid cline but stasis of its center. Continued empirical study of dynamic hybrid zones will provide insight into the forces shaping their structure and the evolutionary potential they possess for the elimination or generation of species.Although theory suggests that hybrid zones can move or change structure over time, studies supported by direct empirical evidence for these changes are relatively limited. We present a spatiotemporal genetic study of a hybrid zone between  Hybridization, the ability of individuals from different species to mate and produce viable offspring, has important consequences for the formation and stability of distinct species Hewitt . Many thHybrid zones create a natural laboratory in which researchers can study hybridization events and their effects on the speciation process Hewitt . The staTo understand the evolutionary trajectory of hybrid zones, research focused on the structure of hybrid zones must be conducted at multiple time points following secondary contact  are an excellent system to address questions about hybrid zones, because many closely related species in this genus occur in partial sympatry with potential for hybridization, character displacement, or other interactions. There are nine currently recognized Pseudacris species belonging to the “Trilling Frog” clade  or to the east (pure P. nigrita) of the contact zone.The North American trilling chorus frogs  were either toe clips or heart muscle. Tissues were either frozen in liquid nitrogen at collection or placed in 95% ethanol or tissue buffer and stored at −80°C until DNA extraction. The recent dataset includes 161 frogs from 29 collection sites across the southeastern United States, which we combined into 16 recent populations based on geographic proximity, designated alphabetically as A through P  using the E.Z.N.A. Tissue DNA Kit  following the manufacturer's protocols. Fifteen unlinked tetra‐ and dinucleotide microsatellite loci, previously identified and tested on P. nigrita and congeneric species P. feriarum, were chosen for amplification  was performed on an Applied Biosystems 3730 Genetic Analyzer at the Florida State University DNA Sequencing Facility. Fragment lengths were determined, and binning completed in Geneious v. 6.0.4  with manual confirmation of fragment lengths.Genomic DNA was extracted from all tissue samples , so 4 km was chosen as our cutoff to avoid combining genetically divergent populations . Mean dispersal distances of G‐test  can be assigned to each individual in a population of putative hybrids by comparing the individual's genotype to two distinct parental populations. A value of 0 or 1 is indicative of a pure parental individual, and any values between 0 and 1 indicate some degree of shared allele frequencies from each parental type. We used a maximum‐likelihood method (Buerkle h = 0) and an alternative (h = 1) parental population. In the historical dataset, population H1  found that F1 hybrids displayed h between 0.5 and 0.75 using the Buerkle , the individual is classified as a hybrid of undetermined class. These approaches were shown to successfully distinguish known hybrids from parental individuals by E. M. Lemmon and T. Juenger (unpubl. data), so we employ both methods here.Classifying interspecific hybrid classes using cross‐specific microsatellites can be difficult, due to variation in allele frequencies present in individual hybrids, as well as to variation in allele frequency estimates of parental populations Buerkle . A recen Buerkle  method  and δ (delta), that describe the shape of decay. These parameters were estimated for each side of the cline individually in the absence of a tail on the other side of the cline , constrained to a single τ and a single δ parameter for both sides of the cline , or were estimated independently for each side of the cline . For all analyses, pmin and pmax, the character values for the pure parental populations, were set to 0 and 1, respectively, in congruence with the h values for the pure reference and pure alternative populations. We first estimated covariances between parameters by running a Markov chain Monte Carlo (MCMC) analysis under each model for 106 generations and sampling every 100 generations, following a burn‐in period of 105 generations. Estimated covariances were used to improve MCMC proposals , we used the Akaike information criterion  and the best‐fit model based on the AIC values  to encompass the full geographic range of the dataset. We then ran 100 replicate cline analyses, in which we randomly selected one of the recent populations to match each historical population as grouped above. Individuals in each population were randomly subsampled, so both recent and historical datasets had the same sample size, reducing the sample size to seven localities and either 73 (using population L to match H7) or 77 (using population M to match H7) individuals for both recent and historical data. Distances between populations were similar between historical and subsampled recent data (within a few kilometers). For each replicate, we then re‐estimated the geographic cline using the subsampled data under the simplest model (no tails) for both the recent and historical data. Parameter estimates were considered significantly different if there was no overlap in the 95% credibility interval between the recent and historical data. This approach of using no overlap in 95% credibility intervals should be conservative and increase our confidence that any significant differences are due to shifts in the cline shape or position over time.Although these reciprocal constraint analyses can provide evidence of significant differences in parameter estimates among datasets, elucidating their cause is more difficult. Differences in parameter estimates could be due to differences in sampling between recent and historical datasets or due to actual changes in the hybrid zone. Therefore, to test for the effect of sampling strategy on the cline, a stratified subsampling approach was applied to minimize the differences in sampling strategy between the recent and historical data. Recent populations were grouped by the most geographically proximate historical population: H1 = A, H2 = E or F, H3 = G, H4 = H, H5 = I, H6 = J or K . These analyses confirm that these species are indeed genetically distinct in their allopatric ranges in both time periods and confirm our sampling strategy of parental and putative hybrid individuals.Results of the STRUCTURE analyses clearly indicated that FST values for all combined recent collection sites indicated no significant differences between collection sites pooled in the same population, after a sequential Bonferroni correction for multiple tests  private to P. nigrita. In the recent dataset, 291 alleles were recorded, with 45 alleles (range: 2–6 per locus) private to P. fouquettei and 102 (range: 3–21 per locus) to P. nigrita. The historical dataset, considering both pure and hybrid populations, contains 33 alleles not represented in the recent sampling, and the recent dataset includes 79 alleles not represented in historical sampling. These allelic results may be constrained by the small sample size of the P. fouquettei recent reference population, so we interpret them with caution. In nearly all populations across both time periods, observed heterozygosity was lower than expected heterozygosity, and GIS values  were positive , the original h values and hybrid indices were used with confidence in all downstream analysis. No evidence of LD was detected across loci in either the historical or recent dataset following standard Bonferroni correction.Some population–locus pairs showed departures from HWE following sequential Bonferroni correction for multiple tests, but patterns were not consistent across all loci Table . Each poh scores for putative hybrids were graphed from west to east to visualize geographic structure across the hybrid zone . Based on our microsatellite analysis of the historical dataset, no individuals in populations H2 or H6 were assigned a hybrid index between 0.25 and 0.75. This agrees with Gartside's h with microsatellite data. Gartside . In the historical dataset, 69.5% of individuals from populations H2 through H6 were classified as hybrids with the CI method, including individuals from both H2 and H6. Likewise, this method classified 93.1% of individuals from recent populations B through L as hybrids. Although the CI method may be overly liberal in classifying hybrids, both methods are consistent in suggesting that hybrids are much more common in the recent than in the historical dataset. This result suggests that hybridization has increased and expanded past the original boundary described by Gartside  with cleanly defined clusters, where putative hybrids clustered with one or the other parental species  with more uncertain placement across all populations, which could be indicative of increased gene flow across the range . To assess whether STRUCTURE Q‐scores (probability of belonging to one cluster vs. another) are comparable to h scores from hybrid analysis, we examined these values for known historical hybrids from Gartside's .Geographic cline models including only a western tail were the best fit to the historical data, while models including no tails were the best fit to the recent data, regardless of whether populations B, C, and D were included in the analysis Table , Fig. 3.Reciprocal constraints further support (1) the stability of the cline position over time and (2) a significant increase in the width of the hybrid zone from the historical to recent dataset. Analyses in which only the cline center was constrained were not significantly worse than unconstrained analyses across any tests Table . AnalyseK (Table P. nigrita population from Florida (M) was used as the recent equivalent of population H7, stratified subsampling analyses show no evidence of a shift in cline center position  was selected, stratified subsampling analyses supported a significant increase in cline width, with no overlap in credibility intervals between historical and recent datasets in 99% of the replicates  and propose a recent selective advantage for traits of one species over the other, driven by human modifications to the environment. In dispersal‐dependent (tension) zones, however, movement may be expected either when shifts occur in parental density and dispersal or when endogenous selection pressure on hybrids changes  relative to estimates of per‐generation dispersal distance  for P. fouquettei and P. nigrita is unknown, it is possible that this contact zone was relatively young when Gartside first described it in 1980. A young contact zone in this scenario could have two potential conclusions: (1) The species may be moving toward stable equilibrium, as predicted by the tension zone model  odel Key . This poodel Key . If the P. fouquettei and P. nigrita, over a 30 year period, including strong evidence for a dramatic increase in its width but stasis of its center. To our knowledge, this study provides the first evidence of this spatiotemporal pattern in a hybrid system and contributes important empirical evidence to the theory and understanding of hybrid zone movement. In the future, we hope to better understand the processes driving these patterns through estimating hybrid fitness via laboratory experiments, testing female discrimination via phonotaxis experiments, and evaluating recent changes in ecosystem conditions. Future time‐stratified sampling and genetic analysis of individuals throughout the contact region in the future will provide a more thorough understanding of the extended stability of this hybrid zone. This study exemplifies the critical need for consistent long‐term field studies to increase understanding of hybridization and speciation, as well as for studies of the anthropogenic and natural influences on these dynamic systems.We have characterized a dynamic hybrid zone between two trilling chorus frogs, www.datadryad.com).Dryad Data Repository (None declared.Figure S1. STRUCTURE Plots.Click here for additional data file.Figure S2. Hybrid Index score comparisons.Click here for additional data file.Figure S3. Posterior distributions from geographic cline analyses.Click here for additional data file.Table S1. Complete listing of specimens.Table S2. Microsatellite marker information.Table S3. FST and P‐values for combined collection site pairs.Click here for additional data file. Click here for additional data file.


Leishmania major are increasing and spreading from the south-west to new areas in the center. To improve the current knowledge on L. major evolution and population dynamics, we performed multi-locus microsatellite typing of human isolates from Tunisian governorates where the disease is endemic  and collected during two periods: 1991–1992 and 2008–2012. Analysis (F-statistics and Bayesian model-based approach) of the genotyping results of isolates collected in Sidi Bouzid in 1991–1992 and 2008–2012 shows that, over two decades, in the same area, Leishmania parasites evolved by generating genetically differentiated populations. The genetic patterns of 2008–2012 isolates from the three governorates indicate that L. major populations did not spread gradually from the south to the center of Tunisia, according to a geographical gradient, suggesting that human activities might be the source of the disease expansion. The genotype analysis also suggests previous (Bayesian model-based approach) and current (F-statistics) flows of genotypes between governorates and districts. Human activities as well as reservoir dynamics and the effects of environmental changes could explain how the disease progresses. This study provides new insights into the evolution and spread of L. major in Tunisia that might improve our understanding of the parasite flow between geographically and temporally distinct populations.In Tunisia, cases of zoonotic cutaneous leishmaniasis caused by  Leishmania major in Tunisia. Within the same area, in twenty years, parasite populations evolved by producing a genetically differentiated population, probably better adapted to the ecosystem. In agreement with the reported human cases of ZCL, the genetic data on samples from the three governorates shows that the disease did not spread according to a geographical gradient. Furthermore, L. major flows seem to still occur between governorates and neighboring districts. This study suggests that environmental changes, human activities and reservoir systems have influenced the spread and evolution of L. major populations. Our findings provide important knowledge on the epidemiology of L. major in Tunisia and might help understanding why the disease is still spreading from the south to the center, despite the control measures that have been put into place.In Tunisia, zoonotic cutaneous leishmaniasis (ZCL) constitutes a significant public health problem. Since 1884, the Gafsa, Kairouan and Sidi Bouzid governorates are the most endemic areas of ZCL. This study used a multi-locus microsatellite typing approach to study the evolution and the population dynamics of  Leishmania major [In Tunisia, zoonotic cutaneous leishmaniasis (ZCL), also known as “Le Bouton de Gafsa” (the pimple of Gafsa), was first described in 1884 by Déperet and Boinet in the Gafsa governorate (south-west of Tunisia) . ZCL cania major ,6. Moreoia major ,8. ZCL hia major . Then, tia major ,11. Sincia major .L. major is transmitted by the sand fly vector Phlebotomus papatasi [Psammomys obesus (fat sand rat) and Meriones shawi (Shaw’s jird) [L. major strains isolated in Tunisia belong to the MON-25 zymodeme [L. major populations, mainly because multi-locus enzyme electrophoresis (MLEE), which has been widely used for phylogenetic typing of parasites, is not discriminative enough. On the other hand, microsatellite markers in combination with recent statistical methods represent a powerful tool for studying the population structure and monitoring the dynamics of these pathogens in time and space because of their high discriminating power and their presumed neutrality [L. major in Tunisia with these powerful markers.papatasi  and rode’s jird) ,15. The zymodeme ,16,17. Izymodeme . Howeverutrality ,20. NeveL. major in the three main endemic areas of ZCL in Tunisia  by using a multi-locus microsatellite typing approach and population genetic statistical methods. Human isolates collected in the three governorates between 2008 and 2012 were used to assess the population dynamics of L. major according to the geographical distribution and the chronological emergence of leishmaniasis epidemics in human populations. Moreover, human isolates collected in Sidi Bouzid governorate in the 1991–1992 and 2008–2012 periods were compared to provide information on the evolution of L. major populations in the same area over twenty years.Therefore, the aim of this study was to analyze the spatio-temporal organization of Leishmania were isolated from patients after collection of the written informed consent at the time of the clinical examination. Human isolates were anonymized and the associated information coded for privacy-preserving data mining.Research included in this study was subject to ethical review by the International Review Board of the Pasteur Institute of Tunis and approved as part of a research project submitted in response to a “call” from the National Institutes of Health for funding Tropical Medicine Research Centers. The Pasteur Institute of Tunis, the study sponsor, took out an insurance policy in accordance with the Tunisian law concerning clinical research. All clinical or biological investigations were performed in the Health Centers of each governorate to guarantee the patients’ safety, confidentiality and respect. All human samples of L. major by sequence analysis of the gene encoding RNA polymerase II [A total of 172 parasite samples, taken from the swollen edge of cutaneous lesions, were collected from patients in different governorates of Tunisia. Isolates from Sidi Bouzid governorate were collected during the 1991–1992  and the 2008–2012 (recent isolates) periods, whereas isolates from Gafsa and Kairouan were only from the 2008–2012 period (recent isolates) . Isolateerase II  in combierase II  at the CPopulation dynamics were investigated at the: i) spatial scale, using recent isolates from Gafsa (GF2008-2012) (66 strains), Kairouan (KR2008-2012) (46 strains) and Sidi Bouzid (SBZ2008-2012) (25 strains) and ii) temporal scale, using the recent isolates  and the historical isolates  from the Sidi Bouzid governorate. Furthermore, the recent isolates from the Kairouan governorate  were subdivided in two groups based on the history of leishmaniasis infection in the area: 15 isolates from the historical focus of Mnara (MN2008-2012), where the first outbreak occurred in 1982, and 31 isolates from the recent focus of Chrarda (CH2008-2012), where the epidemic started only in 2005 .L. major [For all samples, total genomic DNA was extracted from parasite mass cultures (promastigotes) using DNA extraction kit, according to the manufacturer’s protocol. DNA quality was checked by agarose gel electrophoresis and the concentration measured with a NanoDrop spectrophotometer. Amplification was carried out using fluorescent-labeled primers for ten polymorphic microsatellite markers and the PCR conditions previously described for L. major . 1μl of A) and the Nei's unbiased estimate of genetic diversity within subsamples (Hs) [Ho) and expected heterozygosity (He) were also calculated. The Wright's F statistics [FIS measures the relative inbreeding of individuals due to the local non-random union of gametes in each subpopulation, and FST measures the relative inbreeding in subpopulations attributable to the subdivision of the total population into subpopulations of limited size. Therefore, FST also measures the genetic differentiation between subpopulations. The significant departure from 0 of these parameters was tested by 10,000 randomization procedures with FSTAT. The genetic differentiation between historical and recent populations, between governorates  and between the two sub-populations  in the Kairouan governorate was explored. The significance of these estimates was confirmed by p-values ≤0.05. The gene flow or migration rate between populations was also estimated using FSTAT, as Nm = 1- FST/4 FST [The FSTAT Version 2.9.3.2 software  updated les (Hs) . The obsatistics  were estatistics : FIS me/4 FST . A neigh/4 FST , based o/4 FST , was use/4 FST .L. major populations. STRUCTURE uses Bayesian Monte-Carlo Markov Chain (MCMC) sampling to identify the optimal number of clusters K for a given multi-locus dataset, without requiring the identification of the population subunits a priori. The parameters used were the admixture model with the length of burn-in period of 200,000 iterations, followed by 200,000 MCMC repeats after burn-in. Based on the multi-locus genotype data, isolates were divided into K subpopulations with K ranging from 1 to 10 and ten independent runs were performed for each value of K. The K optimal value  was calculated using STRUCTURE HARVESTER, web version [K. First, ΔK, which measures the second-order rate of change in the log likelihood of the data between successive values of K, was estimated [Finally, data were analyzed using a Bayesian model-based approach implemented in STRUCTURE, version 2.3.4 , to expl version . Two appstimated . Second,L. major genotypic diversity in these three areas where ZCL is endemic. Unique genotypes and original microsatellite profiles were identified in 18 GF2008-2012 isolates, in four SBZ2008-2012 isolates and in 11 KR2008-2012 samples (Hs) was highest in the Gafsa and lowest in the Sidi Bouzid isolates (A) and the mean observed heterozygosity (Ho) decreased progressively from Gafsa to Sidi Bouzid (The data obtained from the ten polymorphic microsatellite markers  were use samples . Compariisolates . Similari Bouzid .He) values were much higher than the Ho values in all three populations (FIS) values estimated for each locus and in each population, revealing a deficit in heterozygosity in all three populations  value was between the KR2008-2012 and SBZ2008-2012 populations, whereas the Nm values were much lower between the GF2008-2012 and KR2008-2012 and between the GF2008-2012 and SBZ2008-2012 populations   could be organized in four (maximum L(K)) or two (maximum ΔK) clusters (K) . For K =cond one . Based oclusters . For K =w group) , suggestThe low genetic differentiation , the recent migration events , the gene flow over the years  and the absence of clear separation  between governorates were also confirmed by the finding that the phenetic tree did not highlight any subdivision of the strains according to their geographical origin. .A) of 2.4 in the historical population and of 2 in the recent isolates (Hs) increased over time  showed an important genetic differentiation between historical and recent isolates and the migration rate Nm was very low (0.923)  and 25 recent (SBZ2008-2012) isolates from Sidi Bouzid governorate. Two of the microsatellites were monomorphic in the SBZ1991-1992 population, revealing 26 genotypes in the 35 isolates (0.74). Analysis of the SBZ2008-2012 isolates revealed three polymorphic microsatellites that generated 24 genotypes (0.96). The number of alleles per locus ranged from 1 to 8, with an allelic richness  .The Bayesian model-based clustering analysis indicated two clusters as the most probable genetic structure of these two populations. The first cluster included most of the historical samples (98.8%), which shared a common genetic background based on the genotyping results, and 19.3% of the recent isolates. The second cluster was mainly (80.7%) composed by recent isolates. STRUCTURE identified 0.42 and 4.82 misclassified samples for the historical and recent populations, respectively. However, as the migration model used different prior and modeling assumptions for identifying migrants, three SBZ2008-2012 isolates showed contrasting patterns of assignment, suggesting high genetic connectivity over time. The Neighbor Joining tree revealed no strict partition between the historical and recent populations see . The treA) ranged from 2.2 (MN2008-2012 isolates) to 2.3 (CH2008-2012 isolates)  than in the MN2008-2012 population. As in all the populations assessed in this study, the mean Ho values in the two sub-populations were extremely low compared to the He values. The heterozygosity deficit (measured by FIS) was very high in both populations  came from the historical focus (1982) of Mnara  and from the emerging focus (2005) of Chrarda . Analysis of the genetic data of these two sub-populations showed that six of the ten tested microsatellite loci were polymorphic  in both populations. The remaining four markers  were monomorphic in one or the other population . The allsolates) . Hs, Hulations .FST value showed that the genetic divergence in the two foci was very low, but significant  and do not follow a south-north geographical gradient. It is worth noting that this genetic pattern  is classically observed for Leishmania species mainly because of the low dispersion capacity of sandflies and reservoirs [L. infantum and L. major in Tunisia do not seem to follow the same genetic and evolutionary pattern. Indeed, although L. infantum seems to have spread from the north to the center of the country [Despite these limitations, this study shows the presence of some genetic differentiation between Gafsa and Sidi Bouzid and Gafsa and Kairouan, but not between Kairouan and Sidi Bouzid. Furthermore, we obtained a gradient of diversity that progressively decreases from Gafsa to Kairouan and then to Sidi Bouzid. These results are in agreement with the chronology of servoirs . Moreoveservoirs , L. infa country ,39, moreP. obesus and M. shawi) also show a different distribution in the different governorates linked to the food availability [Leishmania species , we found a strong deficit of heterozygosity in all our populations, in agreement with a recently described mixed-mating system of reproduction  [L. infantum in Tunisia, high level of heterozygosity was observed in Kairouan with evidences of hybridization events [L. infantum compared to L. major could be explained by different proportions of the three reproduction mode of Leishmania parasites [Two hypotheses might explain the genetic differentiation and diversity patterns: 1) leishmaniasis spread first from Gafsa to Kairouan through human activities  and then from Kairouan to Sidi Bouzid; 2) parasites have evolved in different ways because of different ecosystems. Concerning the second hypothesis, rodent and sandfly ecology could influence the parasite transmission. Indeed, entomologic studies have demonstrated that in Tunisia sandfly populations vary in density and species composition according to the ecological conditions . Furtherlability ,41,42. Illogamy) ,45,46,47n events . These aarasites ,45,46,47arasites  was too L. major population in isolates from Sidi Bouzid governorate between 1991–1992 and 2008–2012. The results show that the recent population is more diverse than the historical one with a significant genetic differentiation over time. In twenty years, L. major evolved with a change in allelic frequencies. The increase of genetic diversity reflects the accumulation of genetic changes overtime in this population. The continuous presence of some genotypes over the two decades and the grouping in the same cluster of both the recent and historical populations   .L. major temporal/spatial dynamics in Tunisia, we also analyzed recent isolates (2008–2012) from two districts within the Kairouan governorate: Mnara  and Chrarda (emerging focus). Considering the long interval (23 years) between the human outbreaks in the two districts, we expected that the more recent focus would show a lower genetic diversity than the historical focus. However, the genetic data showed a slightly higher genetic diversity in the more recent focus (Chrarda), despite the low sample size. In parallel, the low differentiation suggests that these are not isolated populations. Indeed, the F-statistics and Bayesian methods estimated that the Mnara and Chrarda isolates are closely related, although the difference was sufficient to correctly assign most samples to their respective district. The migration analysis using both methods suggests the existence of historical migration events (F-statistics method), but not recent migration events (Bayesian method). These results support the hypothesis that the outbreak in Chrarda is the result of the spread of a population, rather than of a small set of genotypes, from Mnara several years ago. Based on the short distance between the Mnara and Chrarda districts (12 to 20 kilometers), the Leishmania population spread could be explained by human activities and also by the vector or reservoir dynamics. As sandflies are known to be bad flyers, Meriones shawi movements (the disease reservoir) and the human economic and social exchanges could be the main sources of the emergence of L. major in Chrarda.To further understand the L. major in Tunisia. Over two decades, the L. major population evolved into a new, genetically differentiated population, probably better adapted to the environment. This could explain the increase of parasite transmission to humans and the higher incidence of ZCL in these areas over the last years [This study brings new insights into the spatial and temporal evolution of st years ,8,48.L. major in new areas in Tunisia, it is now essential to identify the routes of spread. Our findings suggest that the parasite population dynamics do not follow a vertical south-north gradient. Indeed, the disease seems to have spread from Gafsa to Kairouan and then to Sidi Bouzid. Human activities and/or the disease reservoir dynamics might explain this geographically non-gradual spread.To control the emergence of L. major populations as it is largely demonstrated for Leishmania species [L. major evolution.When a disease settles in a new area, it normally sources a subset of the original pathogen population and, as a consequence, genetic diversity should be reduced in the new population. Analysis of the Mnara and Chrarda isolates indicates that the two populations are similar with very low differentiation and historical migration events. This genetic similarity suggests the occurrence of high flow of genotypes between these neighboring populations that would be at the origin of the outbreak in Mnara. The relationships between environmental changes, human activities and reservoir systems have doubtlessly influenced the spread and the evolution of the Tunisian  species . More woS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 TableThe microsatellite profiles are designed by three-digit numbers separated by a fraction bar. Each three-digit number assigns the detected allele, coded by the microsatellite size in base pairs .(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Populus alba and P. tremula hybrid zone along the Irtysh River system in the Altai region, northwest China, using both nuclear microsatellites and plastid DNA sequences. We found high interspecific differentiation, although the hybrid P. × canescens was prevalent. Bayesian inference classified most hybrids into F1, followed by a few back-crosses to P. alba, and fewer F2 hybrids and back-crosses to P. tremula, indicating a few introgressions but preference toward P. alba. When plastid haplotypes in parental species were distinct, P. × canescens carried the haplotypes of both parents, but showed significant linkage between intraspecific haplotype and nuclear genotypes at several microsatellite loci. Selection, rather than migration and assortative mating, might have contributed to such plastid-nuclear disequilibria. By removing later-generated hybrids carrying interspecific combinations of haplotype and nuclear genotypes, plastid-nuclear incompatibility has greatly limited the gene exchange between P. alba and P. tremula via backcrossing with hybrids, demonstrating a significant association between plastid haplotype and the proportion of nuclear admixture.In plants, the maintenance of species integrity despite hybridization has often been explained by the co-adaption of nuclear gene complexes. However, the interaction between plastid and nuclear sub-genomes has been underestimated. Here, we analyzed the genetic structure of a  In nature, species often maintain their integrity in the absence of complete geographical or prezygotic isolation12314679et al.131712Cellular sub-genomes in the nucleus, mitochondria, and plastids communicate in complex ways and co-evolveHybrid zones have often been considered as “natural laboratories for evolutionary studies”Populus alba L. (white poplar) and P. tremula L. (European aspen) are ecologically and morphologically well-differentiated species2425P. alba is widely distributed in river basins over northern Africa, southern Europe, and central Asia, while the P. tremula habitat is found in the mountainous regions of the boreal and temperate deciduous parts of the Eurasian continent. The distributions of these two species overlap considerably along European river systems, and can form large mosaic hybrid zones24P. × canescens (gray poplar), is frequently found close to flood-plain forests of P. alba2428Populus hybrid zones in Europe have shown that, even in sympatric settings, parental species and their hybrids form three distinct ancestry groups27P. tremula into P. alba across marker loci2527P. × canescens31Populus hybrids act as strong genotypic filters2728Populus. Thus, an alternative possibility is that incompatibility between the maternally-inherited plastid genomeP. × canescens are as fit or even fitter than their parental species due to epistatic interactions within genomesP. alba and P. tremula also occurs along the Irtysh River system in the Altai region of Northern Xinjiang, ChinaP. alba population is distributed in the lowland flood-plains of the Irtysh River, P. tremula populations are found in nearby mountainous regions, and their hybrids, P. × canescens, are prevalent along the tributaries of the Irtysh River, such as the Haba, Buerjin, and Buliezi Rivers. In this study, we described the genetic structure of individuals in the P. alba and P. tremula hybrid zone along the Irtysh River system, using both nuclear microsatellite and plastid DNA  trnL-trnF sequences to test whether plastid-nuclear incompatibility contributes to maintaining the integrity of Populus species in the face of gene flow. More specifically, we aimed to answer the following questions, 1) Do P. alba and P. tremula maintain high nuclear and/or plastid divergence in the Irtysh River hybrid zone? 2) What is the genomic composition of hybrids in this zone? 3) Does significant linkage disequilibrium (LD) occur between plastid trnL-trnF variation and the nuclear microsatellite genotype? 4) What evolutionary processes may lead to the CND?A natural hybrid zone between A), variance in allele size (Var), expected heterozygosity (HE), and observed heterozygosity (HO) varied among 11 nuclear microsatellite loci  were higher than those for P. tremula (0.586 and 0.524) and P. alba (0.360 and 0.430). Estimates of diversity  did not vary greatly across populations within each species, but P. tremula populations generally had higher diversity than P. alba populations  . After excluding this population, significant LD was detected at only one pairwise microsatellite locus comparison in P. tremula. Significant LD was detected at 52 of 55 pairwise microsatellite locus comparisons in P. × canescens.In each taxon, the number of alleles (loci see . Across ulations . In one um (HWE) . After sFST  and RST (0.564). Many loci exhibited very high differentiation, with seven of the 11 loci displaying both FST and RST values >0.3, only one having an FST < 0.1, and two having an RST < 0.1  than from P. alba , consistent with a stronger affinity of hybrids for P. alba than for P. tremula in the neighbor-joining tree based on the population allelic frequencies of the 11 microsatellite loci (see P. tremula (FST = 0.105 and RST = 0.151) than that in P. alba (FST = 0.049 and RST = 0.089).The differentiation between the two parental species was high across all microsatellite loci, as estimated by both ST < 0.1 . The genloci see . Within P. alba and P. tremula, resulting in a high statistic for interspecific genetic divergence (FCT = 0.423). The proportion of variation among populations within each species was small , while the proportion of variation within populations followed a complementary pattern  showed that 42.3% of the nuclear microsatellite variation occurred between P. alba, P. tremula, and P. × canescens were mostly grouped separately in line with morphological classification in the field, except for several mismatches between hybrids and each parental species; the hybrid species were located between the two parental species, but had a stronger affinity to P. alba than to P. tremula. From the plot, compared with the two parental species, P. × canescens showed the largest genotypic variation among individuals, while variation among P. alba individuals was greater than that among P. tremula individuals, mostly coming from P. alba individuals in mixed locations.Principal coordinate analysis (PCoA) of the pairwise individual genetic distances is presented in K = 2, P. alba and P. tremula individuals from pure populations were classified into their respective clusters with high admixture coefficients (Q > 0.95), and only six P. alba and four P. tremula were classified as hybrids based on their Q values  giving values >0.95. In contrast, a wide range of admixture proportions was found for P. × canescens hybrid morphotypes , indicating the presence of a wide range of hybrid generations and back-crossing to both parental species.STRUCTURE analysis classified all individuals from the Irtysh River hybrid zones into two clusters K = 2, . The majQ values . The admP. alba and P. tremula individuals to their respective clusters with high probabilities . Whereas individuals identified as hybrids in STRUCTURE analysis, including several P. alba and P. tremula and the majority of P. × canescens, were mostly assigned to hybrids. Using 0.75 as a threshold, 107 of the 136 P. × canescens individuals and one individual from a P. tremula population were identified as F1, eleven P. × canescens individuals and two P. alba individuals were identified as back-crossing to P. alba, and none were identified as F2 and back-crossing to P. tremula. Only two individuals were assigned to F2 and one was assigned to back-crossing to P. tremula with probabilities ~0.5.NewHybrids analysis also assigned most s >0.75; . In additrnL-trnF were excluded from analysis because of a potentially high rate of reverse mutation. A matrix of 1004-bp sequences from 325 individuals yielded four nucleotide substitutions and four indel variations, combining into seven haplotypes (H1–H7). Haplotype descriptions are listed in P. tremula, and the other five (H3–H7) in P. alba  were detected in hybrid P. × canescens  of  P. alba . Five ofanescens . Median-anescens .trnL-trnF for each Populus population varied from 0  to 0.680  was much higher than that in P. tremula (hT = 0.094), but slightly lower than in the hybrids (hT = 0.692). AMOVA analysis showed that more than half of the trnL-trnF sequence variations (55.75%) occurred between P. alba and P. tremula, resulting in an FCT reaching 0.558; only 8.50% of the variation occurred among populations within species (FCS = 0.192), and 35.5% of the variation occurred within populations  were significantly associated with the haplotypes of P. tremula (Ht), showing significant disequilibria involving homozygous genotypes (D1 and D3); the heterozygous genotypes were also positively associated with the haplotypes of P. tremula (D2) at seven of the eight examined nuclear loci. In the group of hybrids, significant positive CND was also found between plastid haplotype and nuclear alleles in three microsatellite loci (D), resulting in significant negative values of D3; a positive value of D1 was found to be significant at one locus and the values were very high at the other four loci  yielded significant results both for all individuals  and after excluding P. alba and P. tremula populations . The distribution of admixture proportion (Q) in each haplotype showed that the range of Q varied greatly among haplotypes FST = 0.418 and RST = 0.564), reflecting their long divergence time and limited interspecific gene flowP. × canescens has not yet formed a completely distinct gene pool. Notably, we found that population differentiation within parental species was relatively higher in P. tremula (FST = 0.105 and RST = 0.151) than that in P. alba (FST = 0.049 and RST = 0.089), which is contrary to the results from the European populations of these species, where much lower differentiation was always found among populations of P. tremulaP. tremula in our current analysis was mainly caused by population Pt3, which was located at a lower altitude than other two P. tremula populations ; P. alba haplotypes only occurred in hybrids with a reduced genomic composition of P. tremula (Q > 0.4), demonstrating a pattern of interspecific plastid-nuclear incompatibility  coexisted with P. alba  and P. tremula (European aspen), and the hybrid P. × canescens (gray poplar) were sampled from the Irtysh River system from 85°E to 88°E, including the main Irtysh River and three tributaries: the Haba, Buerjin, and Buliezi Rivers. In this region, pure P. alba populations were mainly found along the main Irtysh River; pure P. tremula populations were located in mountainous regions near tributaries; and P. × canescens prevailed along the tributaries with a few coexisting P. alba.The two parental species, P. tremula individuals were sampled from three pure populations (Pt1–3) and 90 P. alba individuals were sampled from four pure populations (Pa1–4). In addition, 136 P. × canescens individuals and 49 P. alba individuals (PaM) that coexisted along the three river tributaries were sampled from 28 sites , see et al.The samples were screened for variation at 11 nuclear microsatellite loci supplied by the International Populus Genome Consortium , making use of the different fluorescent dyes and size differences between loci for multiplexing. Molecular sizes in base pairs were determined using the GENESCAN-500 ROX size standard. The microsatellite genotyping was subsequently analyzed using GENEMAPPER software version 3.7 (Life Technologies) and visually checked twice. Each set of 48 reactions included a positive (known genotype) and a negative (water) control carried from PCR through to the final automated sequencer analysis of microsatellites.trnL-trnF region was amplified using the primers trnLF-f (5′-ATTTGAACTGGTGACACGAG-3′) and trnLF-r (5′-CGAAATCGGTAGACGCTACG-3′), which were improved from the original of Taberlet et al.P. trichocarpa chloroplast , and 1 μl (10–50 ng) genomic DNA. PCRs were performed in an Eppendorf Mastercycler, programmed for initial denaturing at 94 °C for 4 min, followed by 30 cycles of denaturing for 45 s at 94 °C, annealing at 60 °C for 45 s, and extension for 1 min and 20 s at 72 °C. A final extension followed at 72 °C for 8 min. PCR products were purified with a Quick PCR Purification Kit , and then all DNA sequencing was performed using an ABI Prism BigdyeTM terminator cycle sequencing ready reaction kit . The reaction mixtures were analyzed on an ABI 3130xl automated sequencer (Life Technologies).The plastid intergenic spacer A), variance in allele size (Var), expected heterozygosity (HE), and observed heterozygosity (HO) were calculated for each locus. In each population, Var, allelic richness (Ar), HE, and HO were calculated across all microsatellite loci. All analyses were performed using the MSA programFIS was calculated using FSTATFIS was tested for departures from HWE for each population after Bonferroni correction. LD among microsatellite loci was analyzed for each species using the log-likelihood ratio statistic (G-test) implemented in Genepop 4.3In order to characterize the microsatellite loci in the two study species and their hybrids, the number of alleles , and deltaK (ΔK), where the modal value of the distribution is located at the real KTo identify the possible genetic structure and admixture of individuals in the hybrid zone, we used a model-based Bayesian approach implemented in STRUCTURE ver. 2.3.352Q) of each sampled individual to one of six genotype frequency classes: P. tremula, P. alba, F1 (first generation hybrids), F2 , B × tremula (back-cross of F1 hybrids towards pure P. tremula) and B × alba . The program was run with the nuclear microsatellite data using 50 000 burn-in iterations followed by 500 000 Markov chain Monte Carlo iterations using default priors for allele frequencies and mixing proportions.The method developed by Anderson and ThompsontrnL-trnF were first aligned with Clustal Xwww.fluxus-engineering.com).The sequences of P. alba and P. tremula populations.Gene diversity, the probability that two randomly-chosen homologous sites are different, was analyzed for both populations and species using ARLEQUIN ver. 3.1trnL-trnF locus by testing departures from random cytonuclear associationsP. tremula; A, alleles typical of P. alba) and by encoding plastid DNA alleles as synthetic haplotypes . Species-specific nuclear alleles were defined based on both clear frequency differences and diagnostic alleles. The loci GCPM1063, GCPM1065, and GCPM1353, whose common alleles did not show clear frequency differences between the two species, were excluded from analysis.Plastid-nuclear LD was estimated between each nuclear microsatellite locus and the plastid P. × canescens only, following Asmussen and BastenThe normalized CND was calculated for the whole dataset and for the hybrid Q) estimated by the STRUCTURE program at K = 2 and plastid haplotype data were compared in a simple way: nonparametric Spearman rank correlations were used to test for a possible association between nuclear and plastid genomic composition using SPSS . The test was done both for all individuals and for the hybrid P. × canescens only. The distribution of nuclear admixture proportions was demonstrated for each trnL-trnF haplotype with Origin 7.5 .To test for a possible role of plastid-nuclear incompatibilities in determining the patterns of introgression across the hybrid zone, the proportion of nuclear admixture .


India is home to many ethnically and linguistically diverse populations. It is hypothesized that history of invasions by people from Persia and Central Asia, who are referred as Aryans in Hindu Holy Scriptures, had a defining role in shaping the Indian population canvas. A shift in spoken languages from Dravidian languages to Indo-European languages around 1500 B.C. is central to the Aryan Invasion Theory. Here we investigate the genetic differences between two sub-populations of India consisting of: (1) The Indo-European language speaking Gujarati Indians with genome-wide data from the International HapMap Project; and (2) the Dravidian language speaking Tamil Indians with genome-wide data from the Singapore Genome Variation Project.SLC24A5 gene that has been functionally shown to affect skin pigmentation, with a higher degree of genetic sharing between Gujarati Indians and Europeans.We implemented three population genetics measures to identify genomic regions that are significantly differentiated between the two Indian populations originating from the north and south of India. These measures singled out genomic regions with: (i) SNPs exhibiting significant variation in allele frequencies in the two Indian populations; and (ii) differential signals of positive natural selection as quantified by the integrated haplotype score (iHS) and cross-population extended haplotype homozygosity (XP-EHH). One of the regions that emerged spans the Our finding points to a gene-flow from Europe to north India that provides an explanation for the lighter skin tones present in North Indians in comparison to South Indians. India is one of the most populous countries and spans a significant amount of land area in south Asia. As a country, India is ethnically and linguistically diverse, and several studies have studied the genetic aspect of this diversity in Indian populations-10. A stThe presence of the caste system along with two major distinct language families has altered the mating pattern in Indian societies, and this has magnified the diversity of the gene pools that are present in the Indian subcontinent. One of the most apparent differences between north Indians and south Indians is in skin complexion, where north Indians are much fairer compared to the darker south Indians. In thisReich and colleagues were amoST provides a measure of the variation in allele frequencies between populations[Here, we use three population genetics metrics for quantifying genomic diversity between the north Indians and south Indians: (i) the Wright’s Fulations; (ii) thulations, which wulations. These mulations, and we ST metric; (ii) regions under pressure of positive selection in only one of the two populations as quantified by the iHS metric; (iii) regions where XP-EHH exhibited significant evidence of differential selection between GIH and INS. To minimize the chance of false positive findings, we require any regions that have been identified by any one of the three criteria to be validated in at least one of the remaining two criteria, although the validation criteria were less stringent  and the 83 samples from Singapore that are predominantly Tamil Indians (INS) from the SGVP (INS). A total of 1,362,474 autosomal SNPs that are present in both databases and phased haplotypes from the two public resources were used. Our interrogation for genetic evidence of north–south differences focused on three aspects of population genetics: (i) searching for genomic stretches that contained an excess of SNPs where the allele frequencies are markedly different between GIH and INS as quantified by the FST); and (iii) the program structure that aims to assign population membership of each individual to a pre-specified number of populations.We investigated the extent of population structure between the GIH and INS samples with three approaches: (i) principal components analysis (PCA); (ii) Wright’s fixation index  were found to be larger than the distances between northern and southern Han Chinese populations in HapMap and SGVP respectively , and was less than the distances between any two African populations .We quantified the genetic distance between populations with the average F = 0.20%), but wastructure analyses were performed with the two Indian populations and four populations from another three ancestry groups  at three settings where the number of populations K was set to 4, 5 and 6. At K = 4, the Europeans, East Asian and African samples were assigned almost homogeneously to unique populations whereas the Indian samples, while clearly distinguishable from the other three ancestry groups, showed evidence of admixture with the Europeans  from the Italian Toscans (TSI)  the region corrected for nominal SNP density contains an excess of SNPs with significant differences in allele frequencies between GIH and INS; (ii) there are differential evidence from iHS such that one population exhibits evidence from iHS of positive selection while the other population does not; and (iii) there is evidence from XP-EHH of differential haplotype lengths between GIH and INS. The details of the discovery and validation criteria with these three metrics can be found in Table The population structure analyses with PCA, FSLC24A5) gene that has been associated with skin pigmentation[SLC24A5 to be associated with lighter skin pigmentation, and this allele was present at a frequency of 4.7% in INS compared to 40.2% in GIH (FST = 18.1%), indicating that the differential evidence in this region concurs with the significant difference in the frequency of an allele that has been linked to skin pigmentation.A total of eight regions were identified from our analyses, of which six regions encompassed at least one gene  have previously been implicated with variation in intracranial volume[MAPT) gene has been consistently reported to be associated with Parkinson’s disease in Europeans[Another region that emerged with consistent evidence from regional Fl volume and the uropeans-24. The ST criterion and was further corroborated by evidence from iHS in the top 1% in GIH but not in INS  to comparing haplotype structures and the decay of haplotypes (XP-EHH and iHS). A priori, we expected the MHC to emerge as one of the differentiated regions, but there were no evidence even at the SNP-level to indicate that the allelic spectrum was significantly different between the two populations. This differed from the observations made in the Han Chinese, where segments of the MHC emerged as one of the differentiated regions between north and south Chinese[One striking omission in the differentiated regions is the Major Histocompatibility Complex (MHC) that has often been reported to be differentiated even between closely related populations. In all three metrics, we did not observe any significant evidence of genomic dissimilarity between the two populations at this region on chromosome 6. Although the three metrics are not strictly independent, they survey different features of the genomic architecture, from measuring differences at the allelic level  across 1,389,511 autosomal SNPs, where tST[To identify SNPs where the frequencies of the alleles differ significantly between GIH and INS, we calculated Wright’s FST for eachp1 and p2 denote the allele frequencies of a specific allele at a SNP in GIH and INS respectively. In addition, we calculated the empirical p-value for each FST value by counting the proportion of SNPs out of 1,362,474 SNPs that displayed FST values that are at least as large as that observed. This empirical p-value is meant to indicate whether the observed FST value is significantly different from bulk of the SNPs in the rest of the genome. As we chose to discount individual SNPs that possess large FST values due to the possibility that such one-off differences are artefacts attributed to genotyping errors, we adopted a region-based approach and searched for contiguous stretches of the genome that carried an excess of SNPs with extreme FST values. Each chromosome was divided into non-overlapping segments of 100 kb, and a binomial test was performed for each segment to calculate whether the number of SNPs that were present with empirical p-values < 0.01 were higher than expected by chance. For assessing the robustness of the findings, a similar analysis was performed with a window size of 500 kb at an empirical p-value threshold of 0.001. The regions across all 22 autosomal chromosomes were subsequently pooled together and ranked, and regions found in the top 0.1% of the respective genome-wide distributions for the 100 kb and 500 kb analyses were considered to be significantly different between GIH and INS.where pca option that is available as part of the eigenstrat software[We used the software to perfosoftware across aSTRUCTURE program (version 2.3.4) to determine the level of admixture present in the GIH and INS samples. We used the following four populations from HapMap 3 as a baseline for calibration: (i) 112 Utah residents with northern and western European ancestry (CEU); (ii) 89 Toscans in Italy (TSI); (iii) 85 Han Chinese in Beijing, China (CHB); and (iv) 113 Yoruba from the Ibadan region of Nigeria (YRI). The analysis was performed with five different sets of 10,000 randomly selected SNPs across the genome. The admixture model was selected as the ancestral model that assumed the genome of each individual is a mosaic of the content from K populations, where the K parameter was set to 4, 5 and 6. No prior population information was provided in the analysis, and we run the analysis with a burn-in of 10,000 iterations and for 20,000 samplings, where the posterior mean estimates across the 20,000 samplings were used to calculate the admixture proportion from the K populations for each individual.We used the http://hgdp.uchicago.edu/Software/ for iHS and XP-EHH to perform the analyses[The iHS and XP-Eanalyses on the pTo evaluate the extent of similarity between GIH haplotypes and those from southern Europe (TSI) and south India (INS), we divided each chromosome into non-overlapping windows of 100 kb and calculate a haplotype similarity score between GIH and TSI, and between GIH and INS. In eachThe authors declare that they have no competing interests.YYT conceived, designed and directed the experiment; MA, XL. ENP and PC analzyed the data; YYT, CCK and RTHO wrote the paper. All authors read and approved the final manuscript.Figures for rest of the significantly differentiated regions between INS and GIH.Click here for file


Triatoma sordida is a species that transmits Trypanosomacruzi to humans. In Brazil, T. sordida currentlydeserves special attention because of its wide distribution, tendency to invadedomestic environments and vectorial competence. For the planning and execution ofcontrol protocols to be effective against Triatominae, they must consider itspopulation structure. In this context, this study aimed to characterise the geneticvariability of T. sordida populations collected in areas withpersistent infestations from Minas Gerais, Brazil. Levels of genetic variation andpopulation structure were determined in peridomestic T. sordida bysequencing a polymorphic region of the mitochondrial cytochrome bgene. Low nucleotide and haplotype diversity were observed for all 14 sampled areas;π values ranged from 0.002-0.006. Most obtained haplotypes occurred at lowfrequencies, and some were exclusive to only one of the studied populations.Interpopulation genetic diversity analysis revealed strong genetic structuring.Furthermore, the genetic variability of Brazilian populations is small compared tothat of Argentinean and Bolivian specimens. The possible factors related to thereduced genetic variability and strong genetic structuring obtained for studiedpopulations are discussed in this paper. Trypanosomacruzi transmission to humans: Triatoma brasiliensis,Panstrongylus megistus, Triatoma pseudomaculata andTriatoma sordida  is endemic in the Cerrado, the mainbiome of Central Brazil from which dispersion towards the southwest took place, and it isnow widely distributed throughout Argentina, Bolivia, Paraguay and Uruguay . The dispersal pattern of thistriatomine is linked to increased infestations of dwellings closest to sylvaticenvironments and suggests that recolonisation flows to artificial environments from naturalecotopes  cytochrome b gene (cytb). To our knowledge, this is the first study to characterise thegenetic diversity of insects from areas with reports of persistent triatominereinfestations despite the chemical control activities existence in accordance with theBrazilian Ministry of Health.Therefore, this study aimed to characterise the genetic variability of Insects’ origins - The studied populations were manually collected in2007 with the assistance of technicians from Gerência Regional de Saúde de Montes Clarosand Sete Lagoas, Minas Gerais (MG), Brazil, without using a dislodging agent. Theinsects came from peridomiciles in the central  and northern  areas of MG state,Brazil  were performed using the protocol described by b gene:CYT BF-5` GGACAAATATCATGAGGAGCAACAG 3` and CYT BR-5` ATTACTCCTCCTAGCTTATTAGGAATTG 3` using the following reaction conditions: 95ºC for 5 min, 30 cyclesat 95ºC for 45 s, annealing at 50ºC for 45 s, 72ºC for 1 min, and a final extension stepat 72ºC for 10 min. The amplified PCR products were purified using a GFX-96 PCR kit and directly sequenced with specific primers(CYT BF and CYT BR) using a DYEnamicTM ET dye terminator kit . The products were analysed on a MegaBace 500automated DNA sequencer (Amersham). The isolate sequences were sequenced at least twicefor each strand from independent PCR amplifications.AATTG 3`. BrieflySequence analysis - Sequence alignment was performed using the MUSCLEmultiple alignment program (ST) (ST) and geographicaldistances was estimated by nonparametric Spearman’s rank correlation. Phylogenetic treeswere reconstructed by the maximum likelihood method in PhyML 3.0 (b sequences of P. megistus (GenBank accessionnumber: AF045722.1) and Rhodnius prolixus (EF011726.1) as outgroups. Asequence of Bolivian isolate (AF045730.1) was used as a reference in this study ( program . Thenum program . BetweenNucleotide and haplotype diversity of cyt b among Brazilian isolates -We sequenced a fragment consisting of 233 bp of the cyt b gene from 126isolates of T. sordida originating from 14 different areas of MG statein southwestern Brazil. The sequenced region (from nt 86-318) corresponds to the mostpolymorphic portion of the gene. 13 polymorphic sites (six synonymous substitutions andseven nonsynonymous substitutions) were identified, with an overall nucleotide diversityof 0.004  are distinctlydifferent from all other studied populations . Moreover,the FST values did not correlate with the geographic distance betweenpopulations .ulations . The FSTPhylogenetic analysis - The phylogenetic relationships among T.sordida isolates from different areas including Brazil, Argentina andBolivia were inferred from the cyt b sequences  by analysing 28allozyme loci. None of these loci presented fixed differences between any pair ofpopulations, and only two revealed polymorphisms, accounting for extremely low levels ofheterozygosity (He = 0.027). Regardless of the low levels of polymorphism obtained, theresults indicated the existence of genetic structure among the populations analysed(FST = 0.214). In turn, the studies using isoenzymes , RAPD  was obtained in 60% of samples. Most of the 15identified haplotypes were observed at low frequency, and some were exclusive to onlyone of the study populations. It is noteworthy that we obtained a very differenthaplotype profile for the three populations from Coração de Jesus municipality. The interpopulation genetic diversity analysis alsorevealed strong genetic structuring - particularly in populations from Coração de Jesus- compared with other study populations of this insect vector (FST >0.3).Corroborating T. sordida (T. sordida(RR50 2.5-7.2). Of the 14 studied populations, all populations presentedequal or higher slopes compared to the Susceptibility Reference Lineage (SRL),suggesting low genetic variation when compared to SRL. Moreover, different deltamethrinsusceptibility profiles were identified in populations from distinct locations that,nevertheless, belong to the same municipalities ,reinforcing the complexity of the resistance phenotype, not only at themacrogeographical level, but at the microgeographical level. It should be noted that theinsecticide used in the field does not appear to have homogeneous effects over differentpopulations; consequently, it applies different selection pressures to differentpopulations, which is a reflection of the genetic variability among those samepopulations. The complexity of the peridomicile itself cannot be neglected, either. Thelarge variety of ecotopes that exist in peridomiciles makes spraying these ecotopes anexhaustive job. Separating all the material accumulated there for spraying isoperationally impossible for the responsible health agent. Consequently, T.sordida  remain even after the application of theinsecticide, hidden deep in piles of firewood, under barn roofs and in a variety ofother nearly inaccessible places, staying free from contact with the active chemicaland/or in contact only with sublethal doses, which favours their multiplication in theseecotopes possible geographical isolation due to an obstacle to triatomine flow betweenneighbouring localities ; (ii) fo sordida ; and recolonisation flow to artificial environments from natural ecotopes  and two nuclear markers (18S and 28S), showing that theT. sordida from group two were restricted to Chaco, while those fromgroup one were restricted to Bolivia and Brazil (The phylogenetic analysis performed in this study showed that ntiation . Inaddid Brazil .


NAT2 gene. The environmental causative factor (if any) driving its evolution is as yet unknown, but significant differences in prevalence of acetylation phenotypes are found between hunter-gatherer and food-producing populations, both in sub-Saharan Africa and worldwide, and between agriculturalists and pastoralists in Central Asia. These two subsistence strategies also prevail among sympatric populations of the African Sahel, but knowledge on NAT2 variation among African pastoral nomads was up to now very scarce. Here we addressed the hypothesis of different selective pressures associated to the agriculturalist or pastoralist lifestyles having acted on the evolution of NAT2 by sequencing the gene in 287 individuals from five pastoralist and one agriculturalist Sahelian populations.Dietary changes associated to shifts in subsistence strategies during human evolution may have induced new selective pressures on phenotypes, as currently held for lactase persistence. Similar hypotheses exist for arylamine N-acetyltransferase 2 (NAT2) mediated acetylation capacity, a well-known pharmacogenetic trait with wide inter-individual variation explained by polymorphisms in the NAT2 genetic structure of African populations is mainly due to frequency differences of three major haplotypes, two of which are categorized as decreased function alleles (NAT2*5B and NAT2*6A), particularly common in populations living in arid environments, and one fast allele (NAT2*12A), more frequently detected in populations living in tropical humid environments. This genetic structure does associate more strongly with a classification of populations according to ecoregions than to subsistence strategies, mainly because most Sahelian and East African populations display little to no genetic differentiation between them, although both regions hold nomadic or semi-nomadic pastoralist and sedentary agriculturalist communities. Furthermore, we found significantly higher predicted proportions of slow acetylators in pastoralists than in agriculturalists, but also among food-producing populations living in the Sahelian and dry savanna zones than in those living in humid environments, irrespective of their mode of subsistence.We show that the significant NAT2 diversity in sub-Saharan African populations.Our results suggest a possible independent influence of both the dietary habits associated with subsistence modes and the chemical environment associated with climatic zones and biomes on the evolution of The online version of this article (doi:10.1186/s12862-015-0543-6) contains supplementary material, which is available to authorized users. All along human evolution and continuing in present times, populations have developed a wide variety of cultural innovations. Many of these may have affected the interactions between humans and their environment in such a way as to leave gene-culture coevolution imprints in the genome, due to both demographic and selective processes –5. AmongIt is currently held that these differential developments in subsistence strategies also influenced the selective regimes to which past populations were subjected, including through the action of new population-specific  selective pressures induced by dietary changes –15. LactN-acetyltransferase 2 (NAT2) activity is another trait whose evolution was probably also shaped by differential, population-specific selective pressures [N-acetylation activity is currently one of the best known pharmacogenetic traits [N-acetyltransferase 2 (NAT2) enzyme. This cytosolic enzyme, encoded by the gene NAT2 of chromosome 8, is mainly expressed in the liver, small intestine and colon [NAT2 gene. Mutations in NAT2 result indeed in diverse acetylation phenotypes that explain inter-individual variation in response to standard drug dose administration (which can vary from lack of therapeutic efficacy to adverse drug reactions) [NAT2 may also act as risk factors for different types of cancers [NAT2 alleles  of the coding exon [Reduced arylamine ressures –26. Due c traits . The phend colon  where itnd colon . Hydrazind colon , 31. Thund colon . Acetylaactions) , 33, 34. cancers , 36. The.gr, see ). Inter-ing exon   modal . A simplulations , 42–44, proposed .NAT2 genetic variation among African pastoral nomads was up to now very scarce, thus hindering a meaningful statistical analysis of the likely differences between food-producing subsistence modes in sub-Saharan Africa .NAT2 sequence variation among six Sahelian populations relying either on nomadic pastoralism or on sedentary agriculture sampled in an area extending from western Burkina Faso to northeastern Chad, in order to explicitly address the hypothesis that different selective pressures associated to these lifestyles acted on the evolution of NAT2. Moreover, by combining our dataset with published African samples of NAT2 sequences  and agriculture (Kanembou in Chad). We completed this new dataset of NAT2*4a, NAT2*5Ba, NAT2*6Aa, and NAT2*14Ba), while their predicted effect on the enzyme’s activity was considered unchanged from the defining haplotype . The EM algorithm also led to the inference of three new haplotypes, and the following names were attributed by the official NAT2 nomenclature committee (nat.mbg.duth.gr): NAT2*12N, NAT2*13D and NAT2*14K. All three are defined by a new combination of recognized signature SNPs from the official NAT2 gene nomenclature with other SNPs, so that enzymatic activity could not be predicted for any of these three haplotypes.From the 15 single nucleotide polymorphisms (SNPs) observed, a total of 21 haplotypes were inferred with the expectation-maximisation (EM) algorithm of Arlequin for the six Sahelian populations , also a low-activity haplotype. Thus, these two slow haplotypes together account for 67.4 % of the gene copies in the total sample of our study. Further low-activity haplotypes detected in most of our samples include NAT2*5C, *14A, and *14B, as well as *5A, *7B and 6C. In turn, only three different fast haplotypes were detected in the Sahelian samples , and these represent on average less than 18 % of the gene copies. Finally, six haplotypes could not be classified according to their effect on acetylation status. Their total frequency is of 3 % or less in the Fulani samples, whereas it is of 8.2 % and 6.1 % in the Kanembou and Daza, respectively.Frequency distributions of the 21 Fs tests) was found . On another hand, a significant departure from the neutral equilibrium model in favor of overdominant selection was found with Tajima’s D test for the Fulani nomads from the Banfora area  values ranging from 0.72 to 0.78, and nucleotide diversity (π) values ranging from 0.0018 to 0.0019 , and from 0.0022 to 0.0033 for nucleotide diversity (π) .Analysis of estimated diversity levels for the 870 bp NAT2 haplotypes was observed among African populations, particularly so for slow haplotypes NAT2*5B and *6A, and the fast one NAT2*12A , whereas its frequency is lower than 14 % in most Sahelian and East African populations. It is also rather frequent in those two Yoruba samples of small size , but not in the larger sized ones .Substantial variation in frequency distributions of NAT2 coding exon, a positive and significant Tajima’s D value was found again for the Fulani from Banfora, as well as for the Fulani from Ader, the Fulani tested by Mortensen et al. [D values ranging from 1.92 to 2.65). For the Mbuti Pygmies, a significant excess of observed heterozygosity was detected also with the Ewens-Watterson test (P = 0.0463), thus leading to the rejection of selective neutrality and demographic equilibrium. However, none of these results remained significant after Benjamini and Hochberg false discovery rate (FDR) adjustment for multiple testing.When considering only the 870 bp of the n et al. , the MbuNAT2 sequences (in terms of the number of pairwise differences between NAT2 haplotypes), the global level of population structure estimated for the African continent is of 3.3% , which means that 3.3% of the total genetic variation is attributed to differentiation among populations, while 96.7% is due to differences among individuals within populations. In the AFR dataset, 33.4 % of the pairwise genetic distances between populations were found to be significant at the 5 % level. This proportion drops to 19.5 % in the FP dataset, thus suggesting substantial NAT2 genetic differentiation both among hunter-gatherer populations and between hunter-gatherer and food-producing societies. Consistently, the proportion of genetic variance explained by differences among populations drops to 2.2% in the FP dataset, but it is still significant . Thus, while differentiation of hunter-gatherer populations contributes to NAT2 population structure in the African continent, population structure among food-producing communities is also significant. Among the non-significant pairwise genetic distances, a rather large proportion could actually be due to lack of power in significance testing because of small sample sizes. Indeed, the proportion of significant distances increases again to 34.6% in the FPLS dataset, i.e., in the dataset that excludes food-producing population samples of less than 20 individuals. In line with this, the proportion of genetic variance explained by differences among populations also raises to 2.6% , thereby suggesting that the inclusion of samples of small size in the AFR and FP analyses leads to a lowering of the power to differentiate populations.To investigate for the presence of a genetic structure differentiating populations with distinct food-producing subsistence modes , analyses of population structure were performed on three subsets of the population data Table , namely:Most of the significant pairwise genetic distances in the FPLS dataset differentiate the Yoruba and Akele populations from the others , whereas the Mantel test with the FPLS dataset led to a significant negative correlation . This somewhat surprising result stems from both a set of large genetic distances between populations located in close geographic proximity and a set of small genetic distances between populations geographically far apart  and greater than ΦSC indices in all cases . Significant genetic structure was also detected with a classification according to subsistence strategy, but under this categorization criterion and for each of the three datasets, the ΦCT index is lower than the ΦSC index, thereby meaning that more differentiation is found among populations in groups with matching subsistence mode than between those groups, even if only slightly more so. Finally, the results indicate that neither differentiation among geographic groups nor among linguistic groups does associate with the genetic structure of populations displayed by NAT2 sequences, since ΦCT indices were never found significant for any of the AFR, FP, or FPLS datasets  were carried out to gain further insight into a possible association of the genetic structure of populations with factors related to their demographic and cultural history. Four categorization criteria were tested Table : geograpThe two-dimensional plots resulting from the nonmetric multidimensional scaling (MDS) analyses of genetic distances among populations are shown in Fig. NAT2 genotypes . Predicted proportions of slow acetylators in African populations . This difference remains both when hunter-gatherers and when small samples are excluded from the analysis . When hunter-gatherers are excluded (FP dataset), only three subsistence modes are represented in the data, namely agriculturalists, agro-pastoralists, and pastoralists  corroborated the results of the Kruskal-Wallis rank sum tests in that predicted slow acetylation frequency is observed to differ significantly between populations classified both by ecoregion (Clim) and subsistence mode (Subsist), but not by geography (Geo) or linguistic classification (Lang) , consistent with recent genomic results that inferred smaller expansion rates for pastoral populations than for those having adopted agriculture [FS tests on NAT2 variation rejected the null hypothesis of selective neutrality and demographic equilibrium, Tajima’s D tests were found significantly positive for three Fulani groups, although not anymore after correction for multiple testing. Nevertheless, for one of those Fulani groups (FBAN), Tajima’s D was positive when tested on the entire sequenced segment of 1,396 bp, and it remained significant after correction  loci [D values can also result from the presence, in the populations’ gene pools, of several molecularly rather distant haplotypes at frequencies higher than expected under neutrality. Such frequency distributions can develop if several standing variants become submitted to positive directional selection [NAT2 locus sequence (ca. 10 kb) in 14 world populations from the 1000 Genomes Project [Twenty-one haplotypes were parsimoniously inferred for the six Sahelian samples, of which three haplotypes not yet described to the best of our knowledge  loci . As alreelection . This in Project , but no NAT2 in Africa (ΦST = 3.3 % for the AFR dataset of 38 populations). Here we further showed that this structure is not only attributable to the genetic differentiation of hunter-gatherer populations but is also found among African food-producing communities, as attested by the significant ΦST value of 2.6 % for the FPLS dataset of 13 populations. This significant genetic structure is mainly due to the variation in frequencies of three major haplotypes, two of which are categorized as decreased function alleles (NAT2*5B and NAT2*6A), which are particularly frequent in populations living in the dry savanna biome, and one fast allele (NAT2*12A), more frequently detected outside this region  than within it. Accordingly, both the AMOVA . Kruskal-Wallis tests of homogeneity in proportions of slow acetylators among groups of populations did not evidence significant variation between geographic groups, nor between linguistic groups. In turn, significant difference of slow acetylators were found between subsistence strategies, and specifically between pastoralist and agriculturalist populations . Multi-factor ANOVAs further showed that no significant interaction between subsistence mode and ecoregion explained the observed differences in predicted mean slow acetylation frequency .Similarly to the finding of a significant difference in proportions of acetylation phenotypes between sedentary farmers and nomadic pastoralists in Central Asia , these rhydrates , 74, or hydrates , 76, thuoralists . FurtherNAT2 molecular variation [NAT2 haplotypes in specific dietary and environmental conditions has been acting on the genetic evolution of populations, such selective pressures might have acted on multiple standing genetic variation rather than on specific new NAT2 mutations [D and Fu’s FS tests used here) are known to have little power to detect a signal of selection on standing genetic variation [NAT2 molecular diversity, which differentiates populations from distinct climatic zones, could result from differentiated selective pressures on standing variation related to the xenobiotic environment of distinct climate and biome zones, and explain the close genetic proximity of Sahelian populations with East African populations independently of their lifestyle.Consistent with previous investigations on ariation  , or a r Project , as well Project , 79–81.NAT2, notably if the function of the enzyme encoded by this gene is redundant with other enzymes such as NAT1. In this case, the low genetic differentiation observed between Sahelian and East African populations could be due to a recent common origin. Under this last hypothesis, we would expect a consistent pattern of variation of NAT2 with the genetic structure inferred from other independent genetic markers in the genome, thus pointing to the demographic history of these populations rather than to specific selective pressures having acted on NAT2 evolution. This last alternative is not favored at present, given the patterns of variation known for other genomic segments, as discussed above, but it calls for further investigation because it would allow disentangling the effects of demographic and selective processes in the evolution of NAT2.Finally, as discussed in , it is aNAT2*5 and NAT2*6, with NAT2*5 homozygotes being faster acetylators than NAT2*5/NAT2*6 heterozygotes, and the latter displaying faster acetylation than NAT2*6 homozygotes. In addition, the existence of variation in acetylation capacity due to polymorphisms outside the coding-exon of the NAT2 gene is not considered here, although we know that regulatory polymorphisms could alter levels of protein expression and thereby acetylation activity. Besides, the existence of epistatic relationships between the expression of NAT2 and its ortholog NAT1 has been described [Our analysis suffers of course of some drawbacks. Firstly, as already stated, because it makes the simplistic assumption of a discrete categorization model of acetylation phenotypes  on the basis of genotypes, although heterogeneity in these categories has been recently demonstrated , 34. Forescribed , thus fuNAT2 in human populations is probably very often underestimated with the samples sizes currently in use, as shown by the high and significant correlation of the latter with the number of alleles detected, at least for African populations  and variants with unknown effect on phenotype as well , but not for fast acetylation alleles  are rather gross approximations of much more complex subsistence strategies. Extant cultures are not mono-subsistent as their diets also rely on various complementary modes of food supply, such as fishing and gathering, and complex networks of food exchange are concurrently used , 84. For peoples . At pres2 but it is estimated that it was ~350,000 km2 some ten thousand years ago [Finally, the ecological zones considered in our analyses have been shifting in the past, so that populations living in one zone today might have coped with a different environment several generations ago. Extreme environmental changes are documented specifically in the Lake Chad basin; the current extension of the lake is ~20,000 kmears ago . In factears ago .NAT2 in sub-Saharan African food-producing populations differs from that expected under a model of isolation-by-distance or from those observed with other genetic systems, but it is compatible with the hypothesis that it was shaped by selective pressures linked to the chemical environment in which populations evolved. We have shown that it is possible that differences in xenobiotic environments associate with climates and biomes that oppose arid, seasonally dry regions, such as the Sahel to tropical humid regions, such as around the Gulf of Guinea, hence explaining NAT2 genetic and phenotypic differentiation across the southern Sahelian limit. However, the possibility exists that differences in xenobiotic environments interacting with NAT2 could also result from differential dietary habits linked to subsistence modes. Under this hypothesis, the genetic similarity between eastern and central-western African populations from the Sahel would then be explained by two non-mutually exclusive processes, namely significant gene flow across the Sahel, or secondary shift from pastoralism to agriculturalism in those Sahelian and East African populations that practice agriculture nowadays. Future studies including measurements of phenotypes and more sampling of populations (with large sample sizes) will certainly shed a new light on these conjectures.The pattern of variation of Biological samples  were collected during several missions in 2003, 2004, 2005 and 2010 in Niger, Burkina Faso and Chad, from unrelated anonymous volunteers, including only individuals with four grand-parents born to the population. Variability of DNA extracted from these samples was analysed in numerous former studies, such as in , 89–91.n=49, and Tindangou area, n=50). More eastern Fulani groups were sampled in southern Niger  and western Chad . As Fulani groups move frequently, the samples were secured in the temporary camps during the dry seasons, when the nomads rested at the southern extremity of their migration routes or transhumance corridors. The semi-nomadic Daza people (n=41) were collected in the northernmost regions of Lake Chad basin [n=49) from the northern fringes of Lake Chad represent agriculturalists in our dataset [Fulani nomads, representing the nomadic pastoral lifestyle, were sampled at different places within their geographic range across the Sahelian zone , 89. Thead basin . The Daz dataset . InformeNAT2 gene was amplified and subsequently sequenced in both forward and reverse directions. Two partially overlapping pairs of primers were used (number-named according to the NAT2 exon): −128F/+914R [DNA was extracted from buccal swabs using the method described in . Daza sa8F/+914R  and +7028F/+914R . Sequenc8F/+914R ).The sequence haplotypes and their associated maximum likelihood (ML) frequencies were inferred separately for each population sample, using the Bayesian approach based on an approximate coalescent model implemented in the software PHASE v.2.1 , 94, andNAT2 alleles [NAT2*4 (GenBank accession X14672) was used as a reference for the coding exon. However, because of known variation in the flanking region of the coding exon of NAT2*4 alleles [http://genome.ucsc.edu/) as a reference for the upstream and downstream flanking regions, and created a NAT2*4/hg19 construct. The sequences generated in this study were thus aligned with this construct, using ClustalW [NAT2*4/hg19 construct, and (2) classify inferred phased haplotypes according to the official NAT2 gene nomenclature.Following the standards of the official nomenclature of human  alleles , haploty alleles , 42, 44,ClustalW . Two in-NAT2 sequences from sampled African populations obtained through a comprehensive search of publications. Only populations represented by samples including at least 10 individuals  were considered. Sequence genotypes from [NAT2 sequences reconstructed from the phased variant call file extracted from the 1000 Genomes Project , the complete dataset assembled includes 38 African population samples and one sample of African Americans, totalizing 1,192 individuals , which NAT2 haplotypes in the six Sahelian population samples and published frequency distributions from other African samples were used to estimate molecular diversity indices (gene and nucleotide diversity), and to test for possible deviation from Hardy Weinberg equilibrium and selective neutrality  with the Arlequin software.The ML frequency distributions of P-values. The R environment was also used to calculate Pearson's product–moment correlation coefficient between samples sizes and observed levels of diversity .For all tests that revealed at least one significant departure from the null hypothesis in one population, we used R  to applyST values , test their statistical significance  and compute Reynold’s genetic distances, as well as to perform AMOVA analyses to test the significance of population groups’ structures  according to geographic location, linguistic affiliation, subsistence mode or climatic zone and biome . Under ollowing .NAT2 gene nomenclature. All other combinations (including those made of a low-activity haplotype and an unknown-activity haplotype or a newly described haplotype) were parsimoniously pooled together in a “fast/unknown” phenotype category, thus ensuring conservative estimation of slow acetylation prevalence in each population sample. Ninety-five percent non-parametric confidence intervals of slow-acetylation frequencies were generated by bootstrapping over individuals in each sample with the boot package of R . Homogeneity of inferred phenotype frequencies  among geographic regions, linguistic groups, subsistence modes, and climatic zones was tested with R by performing Kruskal-Wallis rank sum tests and single/multiple factors ANOVAs, and differences in average slow acetylation frequencies among subsistence modes or between ecoregions were tested with pairwise Wilcoxon rank sum and Tukey’s HSD tests.Following previous reports , 63 (andNAT2 sequences generated in this study are available under accessions KR231046 to KR231619 in the GenBank database at http://www.ncbi.nlm.nih.gov/genbank/.The data sets supporting the results of this article are available within the article and its additional files. The 574